La Matriz Extracelular en Mecanorreceptores de Vertebrados

UNIVERSIDAD DE OVIEDO

Programa de Doctorado en Ciencias de la Salud

La Matriz Extracelular en

Mecanorreceptores de Vertebrados

Jorge García Piqueras

Tesis doctoral, 2019

UNIVERSIDAD DE OVIEDO

Programa de Doctorado en Ciencias de la Salud

La Matriz Extracelular en

Mecanorreceptores de Vertebrados

Jorge García Piqueras

Tesis doctoral, 2019

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RESUMEN DEL CONTENIDO DE TESIS DOCTORAL

1.- Título de la Tesis

Español: LA MATRIZ EXTRACELULAR EN MECANORRECEPTORES DE VERTEBRADOS

Inglés: THE EXTRACELLULAR MATRIX IN VERTEBRATE MECHANORECEPTORS

2.- Autor

Nombre: JORGE GARCÍA PIQUERAS

DNI/Pasaporte/NIE:

Programa de Doctorado: CIENCIAS DE LA SALUD

Órgano responsable: CENTRO INTERNACIONAL DE POSTGRADO

RESUMEN (en español)

Los mecanorreceptores son estructuras nerviosas especializadas que se encargan de percibir los estímulos táctiles. Se encuentran distribuidos por todos los tejidos de los vertebrados, en el inicio de las vías aferentes sensitivas. En la piel, los corpúsculos de Meissner y Pacini son mecanorreceptores de adaptación rápida y bajo umbral encargados del tacto ligero y tacto profundo respectivamente. Ambos están formados por un axón rodeado por células de Schwann modificadas, y en el caso de los corpúsculos de Pacini, también por tejido conectivo organizado formando una cápsula. Los complejos célula de Merkel-axón son mecanorreceptores de adaptación lenta y bajo umbral responsables de diferentes sensaciones del tacto fino; están formados por una asociación sinapsis-like entre un axón y una célula de Merkel epitelial que se localiza en la base de la epidermis. Dos de los aspectos menos conocidos sobre los corpúsculos sensitivos cutáneos son, por un lado, la matriz extracelular y, por otro, los cambios en estas estructuras durante el proceso de envejecimiento. La primera parte de la tesis se compone de cuatro publicaciones distintas relacionadas con la matriz extracelular. En la primera se ha investigado el origen endoneural o perineural de los distintos compartimentos de los corpúsculos de Pacini humanos; el principal resultado ha sido la identificación de una lámina intermedia CD34-positiva alrededor del núcleo interno, de origen endoneural. En la segunda se ha analizado la expresión del glicosaminoglicano condroitín sulfato en los corpúsculos sensitivos: mientras que los corpúsculos de Meissner no expresan proteoglicanos de condroitin sulfato, en los corpúsculos de Pacini se expresan asociados a la lámina intermedia endoneural CD34-positiva. En el tercer trabajo se ha estudiado el patrón de expresión de otro glicosaminoglicano, el heparán sulfato: tanto en los corpúsculos de Meissner como en los de Pacini, los proteoglicanos de heparan sulfato están asociados a la membrana basal, la cual es especialmente prominente en las primeras lamelas del núcleo externo de los corpúsculos de Pacini. En el cuarto estudio se ha determinado la expresión de los Pequeños Proteoglicanos Ricos en Leucina (SLRPs) de clase I y clase II, ambas de localización extracelular, en los corpúsculos de Pacini: los proteoglicanos biglicano, decorina, lumicano, osteomodulina y fibromodulina se expresan en el núcleo interno y en el núcleo externo; todos ellos excepto la osteomodulina también están presentes en la cápsula externa; contrariamente, la osteomodulina sí se expresa en la lámina intermedia, donde los demás están ausentes. La segunda parte de la tesis tiene un capítulo metodológico, para determinar datos cuantitativos y cualitativos sobre los mecanorreceptores cutáneos, que se ha utilizado en el estudio del envejecimiento y establece las bases para su utilización para la validación de la biopsia cutánea en el diagnóstico de neuropatías periféricas. En el quinto y último trabajo se ha realizado un análisis de los cambios cualitativos y cuantitativos que aparecen en los corpúsculos de Pacini y de Meissner y en los complejos célula de Merkel-axón como consecuencia del envejecimiento. Además, se ha evaluado la expresión de la mecanoproteína Piezo2 (principal canal iónico implicado en la función mecanorreptora) y la expresión del sistema neurotrófico TrkB-BDNF (responsable del desarrollo y mantenimiento de los mecanorreceptores cutáneos) en sujetos de edad avanzada.

Carmen

Rectángulo

RESUMEN (en Inglés)

Mechanoreceptors are specialized nervous structures involved in sensing tactile stimuli. They are localized throughout all the vertebrate tissues at the beginning of the sensory afferent nerve fibres. In the skin, Meissner’s and Pacinian corpuscles are rapidly adapting low-threshold mechanoreceptors, responsible for fine and deep touch respectively. Both of them consist of an axon surrounded by modified Schwann cells, and in the case of Pacinian corpuscles, also by organized connective tissue forming a capsule. Merkel’s cells-axon complexes are slowly adapting low-threshold mechanoreceptors responsible for several light-touch sensations; they consist of a synapsis-like association between an axon and an epithelial Merkel’s cell, which is localized in the basal epidermis. Two of the least known topics about cutaneous sensory corpuscles are, on the one hand, the extracellular matrix, and on the other hand, the changes on these structures during the aging process. The first part of the thesis is compound by four different publications related to the extracellular matrix. In the first one, it has been investigated the endoneural or perineural origin of the different human Pacinian corpuscle compartments; the main result has been the identification of an endoneurial intermediate CD34-positive layer around the inner core. In the second one, it has been analysed the expression of chondroitin sulfate glycosaminoglycan: whereas Meissner’s corpuscles do not express chondroitin sulfate proteoglycans, in Pacinian corpuscles they are expressed in association to the endoneural CD34-positive intermediate layer. In the third work it has been studied the expression pattern of another glycosaminoglycan, the heparan sulfate: in both Meissner and Pacinian corpuscles, the heparan sulfate proteoglycans are associated to the basement membrane, which is specially prominent in the first lamellae of the outer core of the Pacinian corpuscles. In the fourth study, it has been established the expression of extracellular classes I and II Small Leucine-Rich Proteoglycans (SLRPs) in Pacinian corpuscles: biglycan, decorin, lumican, osteomodulina and fibromodulin are expressed in both the inner and outer core; all of them except osteomodulina are present in the external capsule; contrary, osteomodulina is expressed in the intermediate layer, where the others are absent. The second part of the thesis includes a methodological chapter, in order to define quantitative and qualitative data about cutaneous mechanoreceptors, which has been used in the study of aging and it sets the basis for its utilization for the validation of cutaneous biopsy in the diagnosis of peripheral neuropathies. In the fifth and last work, it has been analysed the qualitative and quantitative changes occurring in Pacinian and Meissner’s corpuscles and Merkel’s cells-axon complexes with the aging process. Furthermore, it has been evaluated the expression of Piezo2 mechanoprotein (main ionic canal involved in the mechanoreceptive function) and the expression of TrkB-BDNF neurotrophic system (responsible for the development and maintenance of cutaneous mechanoreceptors) in elderly subjects.

SR. PRESIDENTE DE LA COMISIÓN ACADÉMICA DEL PROGRAMA DE DOCTORADO EN CIENCIAS DE LA SALUD

ÍNDICE

1.- Introducción 2.- Estado actual del problema 2.1. Sistema somatosensorial del tacto 2.2. Formaciones nerviosas sensitivas: corpúsculos sensitivos

2.2.1. Corpúsculos de Meissner 2.2.2. Corpúsculos de Pacini 2.2.3. Complejos célula de Merkel-axón

2.3. Matriz extracelular: glicosaminoglicanos y proteoglicanos 2.3.1. Conceptos generales de la matriz extracelular 2.3.2. Proteoglicanos y glicosaminoglicanos de la matriz extracelular 2.3.3. Localización de los proteoglicanos en el sistema nervioso periférico 2.3.4 Función de los proteoglicanos en el sistema nervioso periférico

2.4. Edad y tacto 2.4.1. Envejecimiento del sistema somatosensorial del tacto 2.4.2. Cambios edad-dependientes en los mecanorreceptores cutáneos de mamíferos

3.- Hipótesis de trabajo y objetivos 4.- Material y técnicas 4.1. Material 4.2. Técnicas 4.2.1. Tinción hematoxilina-eosina 4.2.2. Inmunohistoquímica simple indirecta 4.2.3. Doble inmunohistoquímica indirecta

4.2.4. Doble inmunofluorescencia indirecta con microscopía confocal 4.2.5. Análisis cuantitativo 4.2.6. Análisis estadístico

5.- Resultados 5.1. CD34 5.2. Condroitín sulfato 5.3. Heparán sulfato 5.4. SLRPs 5.5 Capítulo libro 5.6. Envejecimiento

6.- Discusión 7.- Conclusiones 8.- Bibliografía

1. INTRODUCCIÓN

El grupo de investigación en el que se ha realizado el presente trabajo de tesis

doctoral, denominado SINPOS (Sistema Nervioso Periférico y Órganos de los Sentidos)

perteneciente al Cluster de Biomedicina del Campus de Excelencia Internacional de la

Universidad de Oviedo, tiene entre sus líneas de investigación prioritarias el estudio de

los corpúsculos sensitivos cutáneos. Los análisis incluyen la estructura, perfil

inmunohistoquímico, desarrollo y envejecimiento, así como diferentes patologías del

sistema nervioso en las que se ven alterados. El resultado de 30 años de investigación

ha dado lugar a numerosas publicaciones científicas en prestigiosas revistas

internacionales (ver revisiones Vega y Cols., 1996; 2009; 2012; Montaño y Cols., 2010;

del Valle y Cols., 2012).

Los conocimientos acerca de los corpúsculos sensitivos están en continua expansión. A

lo largo de los años, se ha tratado de descubrir la naturaleza de las células que forman

los distintos compartimentos de los corpúsculos sensitivos cutáneos, así como de

determinar marcadores específicos para cada uno de ellos. Un área de especial interés

ha sido el estudio del patrón de expresión proteico en los constituyentes celulares de

los corpúsculos sensitivos. El grupo SINPOS ha contribuido notablemente en este

campo de investigación, demostrando la expresión de numerosas proteínas en los

distintos tipos celulares de los corpúsculos: proteínas ligantes del calcio, filamentos

intermedios, receptores de factores de crecimiento, neurotrofinas o canales iónicos.

Teniendo en cuenta que los corpúsculos sensitivos son funcionalmente

mecanorreceptores, la presencia de canales iónicos en ellos da apoyo molecular a la

mecanorrecepción y mecanotransducción (García-Añoveros y Cols., 2001; Cabo y Cols.,

2012; 2015; Alonso-González y Cols., 2017; García-Mesa y Cols., 2017).

Sin embargo, tras varias décadas estudiando los corpúsculos sensitivos en nuestro

grupo de trabajo todavía existen vacíos de conocimiento sobre estas estructuras

sensitivas, y con esta tesis doctoral se pretende llenar o completar alguno de ellos.

En primer lugar, se tratará de establecer la naturaleza del núcleo externo de los

corpúsculos de Pacini humanos, desconocida hasta el momento, mediante

inmunohistoquímica para marcadores de células endoneural o perineural.

En segundo término, estudiar la matriz extracelular de los corpúsculos de Meissner y

de Pacini humanos, la cual ha sido ignorada casi por completo en los estudios sobre

estas estructuras, en lo que concierne a uno de sus componentes principales: los

proteoglicanos.

Y por último, las variaciones inmunohistoquímicas en algunas proteínas presentes en

los corpúsculos sensitivos asociadas con el envejecimiento, ya que se ha demostrado

que con la edad se produce un deterioro de la sensibilidad táctil que podría deberse, al

menos en parte, a alteraciones en los corpúsculos sensitivos. Actualmente, tan solo se

han publicado datos aislados y poco congruentes sobre los cambios edad-

dependientes del aparato sensitivo de la piel. En el presente trabajo de tesis doctoral

se estudian en detalle los cambios cuantitativos, cualitativos y morfológicos que

aparecen en los corpúsculos sensitivos cutáneos humanos con la edad. La consecución

de los objetivos planteados, los cuales se detallarán más adelante, contribuirá a

ampliar el conocimiento sobre los corpúsculos sensitivos. El trabajo permitirá

determinar el origen celular del núcleo externo de los corpúsculos de Pacini, con

especial interés de la lámina intermedia (referida como zona de crecimiento) y de

forma indirecta, identificar marcadores específicos para ella.

Por otra parte, tomando como referencia las funciones de los proteoglicanos en el

sistema nervioso periférico (regeneración/degeneración axonal, mantenimiento

estructural, mecanotransducción, etc.; ver para una revisión Chen y Birck, 2013), los

resultados que se aporten sobre la expresión y distribución de estas moléculas dentro

de los corpúsculos sensitivos permitirán hipotetizar sobre el papel que desempeñan en

estas formaciones sensitivas. Finalmente, el análisis de los corpúsculos sensitivos

humanos a diferentes edades, proporcionará datos más fiables que los existentes

hasta la fecha para establecer el papel de la porción más periférica del sistema

nervioso periférico en la disminución de la sensibilidad táctil que ocurre con el

envejecimiento.

En la última década, los corpúsculos sensitivos se han convertido en un elemento clave

para el diagnóstico clínico mediante biopsia cutánea de algunas neuropatías periféricas

y enfermedades neurodegenerativas (Herrmann y Cols., 2007; Nolano y Cols., 2008;

Almodovar y cols., 2012; Nolano y Cols., 2017). Este estudio constituirá una base

fundamental para establecer qué variaciones en estas formaciones sensitivas son

propias de la edad y no están asociadas a patologías, lo cual permitirá incluir de forma

sistemática a los corpúsculos sensitivos en la biopsia cutánea con fines diagnósticos

2. ESTADO ACTUAL DEL PROBLEMA

El eje central de esta tesis doctoral está constituido por los corpúsculos sensitivos

cutáneos, considerados funcionalmente mecanorreceptores. Con el fin de poder

abordar los objetivos del trabajo, realizar una adecuada interpretación y discusión de

los resultados es necesario realizar una revisión detallada sobre la estructura y

características funcionales de estas formaciones sensitivas.

2.1. Sistema somatosensorial del tacto

De los cinco sistemas sensoriales clásicos (auditivo, olfatorio, somatosensorial,

gustativo, y visual) solo el sistema somatosensorial es multimodal, es decir, es el único

capaz de detectar diferentes tipos de estímulos específicos tales como la posición de

las articulaciones (propiocepción), dolor, temperatura y tacto. Este último, a su vez,

incluye una gran variedad de componentes entre los que se encuentran la detección

de la curvatura, dureza, forma textura y presión.

La piel humana posee una rica inervación sensitiva a expensas de las prolongaciones

periféricas de las neuronas sensitivas localizadas a nivel de los ganglios sensitivos de

los pares craneales y de los ganglios raquídeos. Los extremos terminales de dichas

prolongaciones finalizan mayoritariamente a nivel de la dermis, formando los

denominados corpúsculos sensitivos o formaciones nerviosas sensitivas (Malinovský,

1990; 1996) (Figura 1).

Algunos de estos órganos especializados funcionan como mecanorreceptores ya que

tienen la capacidad de detectar, discriminar y transducir los estímulos mecánicos y,

posteriormente, transmitir la información al sistema nervioso central.

Figura 1. Esquema de la organización anatomofuncional de las neuronas periféricas de la sensibilidad

general del dolor y el tacto (tomado de SG Waxman, Clinical Neuroanatomy).

Los corpúsculos sensitivos están formados por las mismas estructuras básicas que

constituyen las fibras nerviosas:

a) Axón central (zona dendrítica): formado a partir del extremo terminal de la

prolongación periférica del axón de las neuronas sensitivas periféricas. Puede

pertenecer a cualquier subtipo de fibra nerviosa (Lawson, 1992), aunque en el caso

de los corpúsculos sensitivos cutáneos de los mamíferos, suelen ser fibras Aα, Aβ y

Aδ (Dalsgaard, 1988; Perl, 1992).

b) Células de Schwann modificadas (células gliales periaxónicas): constituyen el

núcleo interno de los corpúsculos de Pacini y las células lamelares de los

corpúsculos de Meissner, y la glia asociada a los demás tipos de corpúsculos

sensitivos (Vega y Cols., 1992, 1993). Se originan a partir de células de la cresta

neural (Renehan y Munger, 1990; Saxod, 1996; Feito y Cols., 2018).

c) Estructuras relacionadas con el endoneuro: corresponden al estrato intermedio de

los corpúsculos de Pacini del mesenterio del gato descrito por Ide y Hayashi (1987)

y se originan a partir de fibroblastos endoneurales (Munger e Ide, 1988).

d) Estructuras relacionadas con el perineuro: corresponden a la cápsula de los

corpúsculos (en el caso de estar presente) y las lamelas del núcleo externo de los

corpúsculos de Pacini (Munger e Ide, 1988). Se originan por condensación a partir

del mesénquima local (Halata y Cols., 1990).

Debido a la continuidad estructural de los elementos que los forman, los corpúsculos

sensitivos y las fibras nerviosas comparten la mayoría de sus características

inmunohistoquímicas (Vega y Cols., 1994; 1996) (Figura 2).

En los mamíferos, los corpúsculos de Meissner, los corpúsculos de Pacini y los

complejos célula de Merkel-axón representan tres tipos de formaciones nerviosas

sensitivas cutáneas diferentes en base a su estructura y organización tisular. Los

corpúsculos de Pacini son el prototipo de corpúsculos capsulados mientras que los

corpúsculos de Meissner representan a los no capsulados o parcialmente capsulados.

Funcionalmente, ambos son receptores de bajo umbral y adaptación rápida y

dependen de fibras nerviosas sensitivas Aα y Aβ originadas de neuronas sensitivas de

tamaño grande o intermedio (Perl, 1992; Zelená, 1994; Johnson, 2001). Los complejos

célula de Merkel-axón son una asociación, a modo de sinapsis, entre una célula

epitelial (célula de Merkel) y axones de neuronas sensitivas de tipo Aβ. También son de

bajo umbral pero de adaptación lenta (Johnson, 2001; Zimmerman y Cols., 2014)

(Figuras 1 y 2).

Figura 2. Tipos y fisiología de los corpúsculos sensitivos cutáneos (tomado de Delmas y Cols., 2011).

2.2. Formaciones nerviosas sensitivas: corpúsculos sensitivos

2.2.1. Corpúsculos de Meissner

Los corpúsculos de Meissner son estructuras sensitivas complejas localizadas en el

interior de las papilas dérmicas de la piel glabra con su eje mayor dispuesto

perpendicularmente a la superficie epidérmica. Suelen tener forma ovoide y su

tamaño varía entre 30-200 micras de longitud y 25-70 micras de diámetro (Guinard y

Cols., 2000; Vega y Cols., 2012) (Figura 3).

Están formados por terminales axónicos y células no nerviosas de naturaleza glial,

denominadas células lamelares o laminares, dispuestas a modo de pila de monedas

(lamelas) con su eje mayor paralelo a la superficie cutánea y núcleos de disposición

periférica. Pueden presentar una cápsula incompleta, sobre todo en la parte basal del

corpúsculo, formada por fibroblastos.

Una de las características morfológicas de los corpúsculos de Meissner es el curso

serpenteante del axón entre las lamelas. Este axón contiene numerosos filamentos

intermedios (neurofilamentos) en su parte central y abundantes mitocondrias

dispuestas preferentemente bajo el axolema. Cerca de la membrana hay vesículas de

transcitosis que recuerdan a las sinápticas. Además del axón principal, los corpúsculos

de Meissner pueden contener axones accesorios, en número variable, considerados de

naturaleza nociceptiva en base a los marcadores que expresan (Paré y Cols., 2001).

Normalmente, las fibras nerviosas que forman los corpúsculos de Meissner pierden la

vaina de mielina cuando entran en el corpúsculo, en el que pueden penetrar por la

base, por un lado o por la parte distal (Figuras 3 y 4). No obstante, se ha demostrado

que el axón del 25% de los corpúsculos de Meissner de la piel digital humana presenta

segmentos de mielina en su interior (García-Suárez y Cols., 2009).

Figura 3. Estructura de un corpúsculo de Meissner. Izquierda: hematoxilina-eosina (e: epidermis).

Derecha: representación tridimensional (tomado de http://images.wellcome.ac.uk).

Las células lamelares son células de Schwann modificadas (Idé, 1982) y comparten con

ellas la mayoría de sus características inmunohistoquímicas (ver Vega y Cols., 1996)

(Figura 4). Tienen una morfología acintada o laminar y se disponen orientadas hacia el

axón, con los núcleos en la periferia. Sus cuerpos están rodeados por una membrana

basal que se pierde en la superficie de contacto con la fibra nerviosa (Vega y Cols.,

1995). Las lamelas contienen filamentos intermedios (de vimentina y no proteína

gliofibrilar ácida) y mitocondrias, así como vesículas de transcitosis (Munger e Idé,

1988). Entre ellas, hay un espacio interlamelar casi virtual que se encuentra ocupado

principalmente por fibras de colágeno (Takahashi-Iwanaga y Shimoda, 2003).

Figura 4. Perfil inmunohistoquímico de los corpúsculos de Meissner de la piel digital humana. Axón

reactivo para NFP (a) y células lamelares para S100 (b,c) y vimentina (d) (e: epidermis) (cortesía de J.A.

Vega).

http://images.wellcome.ac.uk/

Ocasionalmente, los corpúsculos de Meissner poseen una cápsula incompleta formada

por fibroblastos y fibras de colágeno. La cápsula está ausente a nivel superior del

corpúsculo, mientras que en la base inferior está compuesta por 2-4 estratos de

células capsulares aplanadas con fibrillas elásticas de colágeno dispuestas en los

espacios intercelulares. Estas células capsulares de la base pueden estar cubiertas por

láminas basales que estarían ausentes del resto del corpúsculo (Zelená, 1994).

Funcionalmente, los corpúsculos de Meissner son los responsables principales del

tacto fino. Se trata de mecanorreceptores de adaptación rápida, que responden a

estímulos comprendidos entre los 10 y 400 Hz, con una sensibilidad máxima para 100-

200 Hz (Johnson, 2001).

Por lo que se refiere a su composición proteica, la figura 5 resume las principales

proteínas descritas en los corpúsculos de Meissner según su localización.

El axón presenta inmunorreacción para marcadores neuronales generales como la

enolasa neuronal específica y la proteína PGP9.5 (Vega y Cols., 1996; Pawson y Cols.,

2000), además de para la subunidad de 200 kDa del neurofilamento, lo cual subraya el

carácter mecánico del mismo (Vega y Cols., 1996). También contiene proteínas ligantes

de calcio (calbindina D-28K, parvalbúmina, calretinina, y neurocalcina), las cuales

regulan presumiblemente la homeostasis del calcio axónico en la transducción

mecanoeléctrica (Vega y Cols., 1996; Albuerne y cols., 1998; Galeano y cols., 2000). La

β-arrestin-1 axónica juega un papel importante en la desensibilización e internalización

de los receptores asociados a proteínas G. Esta molécula emigra desde las neuronas de

los ganglios espinales a los corpúsculos que las suplen (Komori y cols., 2003). Otras

moléculas relacionadas con el crecimiento axónico, como la proteína GAP-43, también

están presentes en los corpúsculos cutáneos de Meissner (Verzé y cols., 2003).

Figura 5. Representación de las proteínas que se expresan en los corpúsculos de Meissner según su

localización (azul: células lamelares; rojo: axones accesorios; amarillo: axón principal).

Desde principios de este siglo, diferentes familias de canales iónicos, especialmente los

pertenecientes a las superfamilias de degenerina/epitelial- Na+ (DEG/ENaC) y Transient

Receptor Potential (TRP) han sido foco de interés en el estudio de los corpúsculos

sensitivos debido a su implicación en el proceso de mecanotransducción. En este

sentido, nuestro grupo de investigación ha centrado sus esfuerzos en la localización de

diferentes potenciales mecanoproteínas en los corpúsculos sensitivos, ampliando

considerablemente los conocimientos sobre este tema. A nivel proteico, se han

localizado BNaC1α, ASIC2, TRPV4 y TRPC6 en el axón principal de los corpúsculos de

Meissner de la piel digital humana (García-Añoveros y cols., 2001; Cabo y cols., 2012;

2015; Alonso-González y cols., 2017). Recientemente, hemos detectado en esta

situación el canal PIEZO2, perteneciente a una familia de canales iónicos reciente

identificada y caracterizada, relacionado de forma directa con la mecanotransducción

del tacto fino (García-Mesa y cols., 2017).

Respecto a los axones accesorios que ocasionalmente suplen los corpúsculos, se han

detectado en ellos diferentes neuropéptidos como la sustancia P, CGRP, hormona

estimulante de los melanocitos, galanina o somatostatina, todos ellos relacionados con

la nocicepción. Por supuesto estos axones también presentan inmunorreactividad para

anticuerpos neuronales específicos, especialmente la PGP9.5 (Johansson y Cols., 1999;

Paré y Cols., 2001).

En cuanto a las células lamelares, la vimentina es el principal filamento intermedio de

su citoesqueleto, y no la proteína gliofibrilar ácida (GFAP) como cabría esperar dada la

naturaleza glial de estas células (Vega y Cols., 1996). Además, Calavia y Cols. (2012)

demostraron la presencia de otro filamento intermedio, la nestina, en una pequeña

población de las células lamelares vimentina positivas, relacionándola con un posible

nicho de células neurales pluripotenciales presentes en los corpúsculos sensitivos. La

proteína ligante de calcio S100 es específica de las células lamelares y se co-localiza

con la parvalbúmina o calbindina D-28k (Vega y Cols., 1996). Por otro lado, los

receptores para las neurotrofinas p75, TrkA y TrkB también se detectan en este tipo

celular (Vega y Cols., 1993; Vega y Cols., 1994a; Bronzetti y Cols., 1995; Calavia y Cols.,

2010), así como el receptor para el factor de crecimiento epidérmico (Vega y Cols.,

1994b). Además, la membrana basal de las células lamelares contiene laminina y

colágeno tipo IV (Pawson y cols., 2000).

Existen algunas proteínas que no son específicas de ninguno de los componentes de

los corpúsculos y aparecen tanto en el axón como en las células lamelares. Es el caso

del canal iónico ASIC2 y el de algunas proteínas ligantes de calcio, que como se ha

mencionado previamente, aparecen en el axón pero también en una pequeña

población de las células lamelares (Vega y Cols., 2012; Cabo y Cols., 2015).

2.2.2. Corpúsculos de Pacini

Los corpúsculos de Pacini son los corpúsculos sensitivos de mayor tamaño (hasta 4 mm

su eje mayor y 2 mm su eje menor), tienen forma ovoide y detectan la presión y la

vibración. Se distribuyen por la mayoría de los órganos y tejidos, como páncreas,

mesenterio, mesocolon, adventicia de vasos y anastomosis arteriovenosas, piel,

tendones y ligamentos. En la piel, los corpúsculos de Pacini se sitúan en la dermis

profunda (Zelená, 1994).

Al microscopio óptico muestran un aspecto típico en “bulbo de cebolla”, debido a una

serie de formaciones laminares, más o menos concéntricas, dispuestas en torno al

axón. Se diferencian en ellos dos compartimentos denominados núcleo interno y

núcleo externo, ambos rodeados por una cápsula densa constituida por un número

variable de láminas (Figura 6 y 7).

Figura 6. Estructura de un corpúsculo de Pacini. Izquierda: hematoxilina-eosina (c: cápsula; ne: núcleo externo; ni: núcleo interno). Derecha: representación tridimensional (modificado de http://images.wellcome.ac.uk).

En la parte central del núcleo interno se dispone el axón, generalmente único, aunque

ocasionalmente puede contener hasta cinco o más (García-Suárez y Cols., 2010) (Figura

7). De forma característica, un axón mielínico de tipo Aα o Aβ entra por uno de los

polos del corpúsculo y termina en un engrosamiento con espículas denominado zona

dendrítica. El axón contiene neurofilamentos en su parte central, microtúbulos

dispersos y mitocondrias en grupos bajo el axolema. La fibra nerviosa que suple el

corpúsculo, en su trayecto intracorpuscular, mantiene uno o dos segmentos de mielina

y luego se rodea por las prolongaciones de las células laminares que forman el núcleo

interno (Bell y Cols., 1994). Además del axón principal, mecánico, los corpúsculos de

Pacini pueden tener fibras nerviosas accesorias, finas y amielínicas (fibras C o fibras

postganglionares simpáticas) que siguen un curso serpenteante en contraposición al

trayecto longitudinal de la fibra principal (Malinovský y Pác, 1982).

http://images.wellcome.ac.uk/

Las láminas o lamelas que forman el núcleo interno son células de Schwann

modificadas (Figura 7). Tienen una disposición muy compacta ya que están muy

próximas entre ellas. Permanecen separadas únicamente por espacios casi virtuales

que contienen fibrillas de colágeno muy finas (Idé y Cols., 1988; Malinovský y Cols.,

1990) y diferentes componentes de la matriz extracelular (Dubový y Bednárová, 1999;

Pawson y Cols., 2000; Sames y Cols., 2001). Las lamelas del núcleo interno carecen de

lámina basal, salvo las más externas (Munger e Idé, 1988).

Figura 7. Perfil inmunohistoquímico de los corpúsculos de Pacini de la piel digital humana. Axones

reactivos para el neurofilamento (NFP) y células del núcleo interno para S100P (cortesía de J.A. Vega).

El núcleo externo está formado por células aplanadas que rodean por completo al

núcleo interno, formando capas separadas entre sí por un compartimento con

diferentes componentes de la matriz extracelular (Dubový y Bednárová, 1999; Pawson

y Cols., 2000; Sames y Cols., 2001). Las células del núcleo interno muestran uniones

tipo “gap” o comunicantes, mientras que las lamelas del núcleo externo tienen

uniones intercelulares de tipo “tigh junction” o estrechas (Munger e Ide, 1988).

Entre los núcleos interno y externo se dispone un estrato celular intermedio, que ha

sido demostrado de manera fehaciente en los corpúsculos de Pacini del mesenterio del

gato (Idé y Cols., 1988; Munger e Idé, 1988; Pawson y Cols., 2000) y, recientemente, en

los corpúsculos de Pacini humanos por nuestro grupo de investigación (García-

Piqueras y Cols., 2017).

La cápsula es continuación del perineuro del tronco nervioso que inerva el corpúsculo

(Zelená, 1994). Su función podría ser la de aportar cierta tensión al núcleo externo

(Munger e Idé, 1988). En ella se localizan vasos sanguíneos y macrófagos. Se ha

sugerido que la cápsula y sus lamelas no solo tienen importancia en el procesamiento

de los estímulos mecánicos, sino que constituyen una parte del sistema metabólico del

corpúsculo (Malinovský y Cols., 1990).

La estructura de un corpúsculo de Pacini no es uniforme a lo largo del recorrido del

axón, sino que se distinguen tres zonas de acuerdo a las relaciones entre él y las células

periaxónicas (Malinovský y Cols., 1986) (Figura 8):

a) segmento preterminal: situado dentro de la cápsula y en la que el axón aún

permanece envuelto por la vaina de mielina y las células de Schwann;

b) segmento terminal: las láminas se disponen formando dos mitades simétricas,

separadas entre sí por las denominadas “clefts” o hendiduras. En esta zona, el axón

se caracteriza por emitir pequeñas prolongaciones, espículas, que se introducen en

dichas hendiduras. Las espículas siempre encaran las dos hendiduras del núcleo

interno en la porción terminal del corpúsculo (Munger e Idé, 1988); y

c) segmento ultraterminal: se corresponde con la parte final del axón, que está

engrosado y presenta numerosas espículas en su superficie. A este nivel, las células

del núcleo interno pierden la simetría bilateral y se disponen de forma irregular.

Figura 8. Componentes estructurales de un corpúsculo de Pacini, representado en cortes transversales a

nivel de la zona preterminal (A), terminal (B) y ultraterminal (C) (modificado de Malinovský y Cols.,

1986).

Funcionalmente, los corpúsculos de Pacini son mecanorreceptores de adaptación

rápida que responden a estímulos vibratorios comprendidos entre los 20 y los 1500 Hz,

con sensibilidad máxima en los 200-400 Hz, y por lo tanto, detectan presión y

vibración.

Lo mismo que en el caso de los corpúsculos de Meissner, los corpúsculos de Pacini

tienen un patrón de expresión proteico muy amplio, que varía en función de los tipo

celulares que los forman (Figura 9).

Figura 9. Representación de las proteínas expresadas en los corpúsculos de Pacini según su localización

(azul: núcleo interno; verde: lámina intermedia; rojo: núcleo externo y cápsula; amarillo: axón).

El axón central es identificable por la expresión de enolasa neuronal específica,

neurofilamento y PGP9.5 (Vega y Cols., 1996a). También se han detectado en él

calbindina, parvalbúmina y calretinina, implicadas en la homeostasis para la

transducción mecanoeléctrica (Vega y Cols., 1996a). En los últimos años se han

localizado además canales iónicos relacionados con la mecanotransducción como

ASIC2 y ASC1 en el axón de los corpúsculos de Pacini de Macaca fascicularis (Cabo y

Cols., 2012) y humanos respectivamente (Calavia y Cols., 2010). Por otro lado, se ha

descrito la proteína Bcl-2 en el axón principal (González-Martínez y Cols., 2006).

El resto de elementos que constituyen los corpúsculos de Pacini son el núcleo interno,

el núcleo externo y la cápsula. Una contribución importante de nuestro grupo de

investigación ha sido la identificación de antígenos que son específicos de

determinados compartimentos del corpúsculo de Pacini, como la proteína S100 para el

núcleo interno (Vega y cols., 1996b) y el CD34 para la lámina intermedia (García-

Piqueras y Cols., 2017). Hasta el momento actual, sólo se ha encontrado expresión de

la mecanoproteína ASIC2 en el núcleo interno de los corpúsculos de Pacini humanos

(Calavia y Cols., 2010; Cabo y Cols., 2015), al igual que la proteína básica de la mielina,

cuya expresión se restringe al segmento preterminal del núcleo interno (García-Suárez

y Cols., 2009).

Sin embargo, la mayoría de las proteínas suelen expresarse en varios elementos

corpusculares: 1) los filamentos intermedios vimentina y nestina aparecen en el

núcleo interno y núcleo externo (la vimentina también en la cápsula) (Calavia y Cols.,

2012); 2) las proteínas de membrana basal colágeno IV y laminina se localizan en

todos los elementos no neurales del corpúsculo (Vega y Cols., 1995); 3) los receptores

de neurotrofinas TrkA, p75 y EGFR se encuentran en todos los compartimentos (Vega

y Cols., 1994a; 1994b), TrkB en el axón y ocasionalmente en las células lamelares del

núcleo interno de los corpúsculos de Pacini de Macaca fascicularis (Cabo y Cols., 2015),

y su ligando BDNF en el núcleo interno y núcleo externo (Cabo y Cols., 2015).

No obstante, también se dan situaciones contradictorias. Mientras que para Britsch

(2007) el axón central y las células de Schwann presentan inmunorreactividad para los

receptores de neorregulinas erb2, erb3, erb4, nuestro grupo de investigación ha

demostrado que erb4 exclusivamente aparece en la cápsula y en el núcleo externo de

los corpúsculos de Pacini (González-Martínez y Cols., 2007).

2.2.3. Complejos Célula de Merkel-axón

Los complejos célula de Merkel-axón o discos de Merkel son estructuras

mecanorreceptoras que consisten en una asociación sinapsis-like entre axones de

neuronas sensitivas Aβ y células epiteliales especiales conocidas como células de

Merkel (Fleming y Lou, 2013; Zimmerman y Cols., 2014) (Figura 10). Se localizan

fundamentalmente en la epidermis de la piel, tanto glabra como pilosa (Moll y Cols.,

1986), aunque también pueden encontrarse en mucosas derivadas del ectodermo

(Rettig y Halata, 1990).

Figura 10. Complejo célula de Merkel-neurita (modificado de Biology Stack Exchange).

Las células de Merkel tienen forma ovalada, con un eje mayor de 10-15 µm, aunque

también pueden presentar morfología de medialuna (Halata y Cols., 2003) (Figura 10 y

11). Normalmente, se localizan en la capa basal de la epidermis de la mayoría de los

vertebrados (Moll y Cols., 1996), bien de forma aislada, bien formando grupos

(“clusters”). Se anclan a la epidermis mediante finas protuberancias citoplasmáticas y

desmosomas proyectados hacia los queratinocitos circundantes del polo opuesto al

terminal nervioso (Iggo y Muir, 1969). El citoplasma contiene filamentos intermedios

de citoqueratina y gránulos osmófilos densos con neuropéptidos que se acumulan en

el polo sináptico (Iggo y Muir, 1969; Halata y Cols., 2003). En la mayoría de las células,

la membrana citoplasmática se encuentra estrechamente unida a la membrana del

terminal axónico (Chen y Cols., 1973).

La fibra nerviosa de tipo Aβ que contacta con las células de Merkel es el axón de una

neurona mecanosensitiva de bajo umbral y adaptación lenta de tipo I, cuyas

terminaciones forman una ampliación discoide en su parte distal para hacer conexión

sinapsis-like con el polo basal de las células de Merkel. Esta asociación es la que

permite que el complejo sea capaz de iniciar la respuesta para la discriminación táctil

(Maksimovic y Cols., 2013; Zimmerman y Cols., 2014).

Figura 11. Perfil inmunohistoquímico del complejo célula de Merkel-Neurita. Izquierda: células de

Merkel inmunorreactivas para CK20. Derecha: arriba, célula de Merkel marcada con fluorescencia para

CK20 (rojo); abajo, célula de Merkel marcada con fluorescencia para PIEZO2 (verde) y enolasa neuro-

específica (rojo; las flechas blancas denotan la fibra nerviosa) (tomado de García-Mesa y Cols., 2017).

Respecto al origen embrionario de las células de Merkel se han barajado dos hipótesis

diferentes. La primera apostaba por un origen de la cresta neural, dado que son células

excitables con capacidad de sintetizar neuropéptidos y expresar moléculas

presinápticas (Grim y Halata, 2000; Szeder y Cols., 2003). La segunda hipótesis afirma

que las células de Merkel son de origen ectodérmico debido a que expresan

citoqueratinas de bajo peso molecular, no tienen capacidad mitótica y establecen

contactos mediante desmosomas con los queratinocitos (Munger, 1965; Compton y

Cols., 1990; Moll y Cols., 1990; Kim y Holbrook, 1995). Actualmente se acepta esta

segunda teoría, ya que se ha demostrado que las células de Merkel provienen de

células madre epidérmicas (Morrison y Cols., 2009; Van Keymeulen y Cols., 2009).

Además, en la epidermis se ha identificado una población celular con fenotipo distinto

al de los queratinocitos que residen junto a las células de Merkel y con capacidad de

regenerar este tipo celular (Woo y Cols., 2010).

La función principal del complejo célula de Merkel-axón es la mecanosensación, pero

existe una gran controversia sobre el papel que desempeñan las células de Merkel en

este proceso. Algunos autores detallan que la fibra nerviosa de tipo Aβ es la

responsable de la mecanotransducción en el complejo, mientras que la célula de

Merkel ocupa un papel modulador (Kinkelin y Cols., 1999). Otros autores, por el

contrario, establecen que la célula de Merkel es el componente indispensable del

complejo para la transducción del estímulo mecánico en la señal química que

posteriormente activará la fibra nerviosa a la que se encuentra unida (Maricich y Cols.,

2009). Una tercera alternativa defiende que ambos son imprescindibles y necesarios,

es decir, que tanto la célula de Merkel como la fibra nerviosa están implicadas en el

proceso de mecanotransducción (Yamashita y Ogawa, 1991; Fleming y Luo, 2013;

Maksimovic y Cols., 2014).

En estudios con ratones que carecen de células de Merkel (knock-out para Atoh1),

pero que conservan las terminaciones nerviosas de tipo Aβ, no se produce reacción

ante estímulos de presión sostenida, resultando la sensación del tacto ligero

completamente alterada (Maricich y Cols., 2009). También existen evidencias de que

las células de Merkel producen neurotransmisores capaces de actuar sobre la

terminación nerviosa a modo de sinapsis-like (Hartschuh y Weihe, 1980; Tachibana y

Nawa, 2002; Haeberle y Cols., 2004; Hitchcock y Cols., 2004). Recientemente, se ha

demostrado la presencia del canal iónico mecanosensitivo PIEZO2 (Ranade y Cols.,

2014; Xiao y Cols., 2014) en las células de Merkel de piel glabra digital humana, pero

no en el terminal axónico (García-Mesa y Cols., 2017). Todos estos resultados avalan la

segunda de las hipótesis mencionadas anteriormente, según la cual, las células de

Merkel transducen los estímulos mecánicos en señales eléctricas mediante PIEZO2 y,

en consecuencia, se inducen los potenciales de acción en las fibras aferentes mediante

la activación de canales de calcio dependientes de voltaje. Aunque a día de hoy,

tampoco puede descartarse la tercera alternativa, en la que ambos componentes del

complejo serían mecanosensibles y, por tanto, ambos participarían en la

mecanotransducción (Maksimovic y Cols., 2013).

En la figura 12 se recogen las principales proteinas descritas en las células de Merkel,

así como los marcadores inmunohistoquímicos utilizados para su identificación.

Figura 12. Esquema de las proteínas que se expresan en el complejo célula de Merkel-neurita.

En el citoplasma de las células de Merkel se localizan citoqueratinas características de

las células epiteliales (Moll y Cols., 1995), además de marcadores neuronales como

neurofilamentos, enolasa neuronal específica y PGP9.5 (Leff y Cols., 1985; Dalsgaard y

Cols., 1989) y marcadores neuroendocrinos como cromogranina A y sinaptofisina (Moll

y Cols., 1995; Ortonne y Cols., 1998). En la membrana hay glicoproteínas como EpCAM

y CD200 (Kurzen y Cols., 2003; Woo y Cols., 2010), transportadores de glucosa como

GLUT1 y GLUT2 (Hitchcock y Cols., 2004) y receptores como NMDA (Cahusac y Cols.,

2005). Dado que tienen la capacidad de transmitir la señal, expresan diversos

neuropéptidos y neurotransmisores como VIP, CGRP, sustancia P, serotonina, Met-

encefalina, somatostatina, bombesina, pancreastatina y CCK8 (Gould y Cols., 1985;

Hartschuh y Weihe; 1989). Maksimovic y Cols., 2003). La prueba fehaciente de su

carácter mecanotransductor es la expresión de PIEZO2 (García-Mesa y Cols., 2017).

La fibra nerviosa de tipo Aβ presenta inmunorreacción positiva para marcadores

neuronales generales como cabría esperar (Dalsgaard y Cols., 1989; Gould y Cols.,

1985; Narisawa y Cols., 1994). Se ha demostrado también la expresión de la proteína

mecanotransductora ASIC2 (Cabo y Cols., 2015).

2.3. Matriz extracelular: glicosaminoglicanos y proteoglicanos

2.3.1. Conceptos generales sobre la matriz extracelular

Los tejidos de los organismos pluricelulares están formados, no solo por un conjunto

organizado de células, sino también por componentes no celulares que se localizan en

el espacio que rodea a las mismas, donde forman una estructura ordenada

denominada matriz extracelular (Alberts y Cols., 2002; Theocharis y Cols., 2016).

La matriz extracelular es una red compleja formada por moléculas de diferente

naturaleza que pueden ser clasificadas en tres grupos (Alberts y Cols., 2002; Bosman y

Stamenkovic, 2003; Theocharis y Cols., 2016) (Figura 13):

a) Proteínas fibrosas: diferentes tipos de colágenos y elastinas;

b) Proteínas de adhesión: lamininas, fibronectinas, tenascinas, fibulinas y nidógeno; y

c) Proteoglicanos constituidos por diferentes tipos de glicosaminoglicanos.

No obstante, esta composición es diferente en cada tejido, en cada situación

fisiológica/patológica y en cada especie (Frantz y Cols., 2010; Theocharis y Cols., 2016).

Dentro de la matriz extracelular de los tejidos, aparecen frecuentemente dos motivos

estructurales diferentes (Figura 13):

a) Membrana basal: estructura bien definida que se encuentra en el exterior celular,

pero inmediatamente a continuación de la membrana citoplasmática (LeBleu y

Cols., 2007). Sus componentes más conocidos son colágeno IV y laminina, pero

también los proteoglicanos agrina, perlecano y endostatina, y otras glicoproteínas

como nidógeno y fibulinas (Sasaki y Cols., 2004).

b) Matriz intersticial: correspondiente al resto de moléculas de matriz que rellenan el

espacio extracelular sin relación con la membrana basal (Alberts y Cols., 2002;

Bosman y Stamenkovic, 2003). Su composición tiene mayor variabilidad según el

tejido en el que se encuentra (Couchman y Pataki, 2012).

Una de las peculiaridades de la matriz extracelular es su alto grado de dinamismo, es

decir, las enzimas son capaces de producir, modificar y degradar la matriz extracelular

permanentemente (Page-McCaw y Cols., 2007; Pengfei y Cols., 2011; Bonnans y Cols.,

2014).

Figura 13. Representación general de la matriz extracelular que conforma los tejidos (tomado de

https://basicmedicalkey.com/cellular-biology/).

La función principal de la matriz extracelular es actuar como un andamio o armazón

(“scaffold”) sobre el que se sustentan y mantienen las células que constituyen los

diferentes tejidos (Alberts y Cols., 2002; Theocharis y Cols., 2016). Gracias a las

características físicas y bioquímicas de las moléculas que la forman, la matriz

extracelular proporciona a los tejidos y órganos propiedades como elasticidad,

resistencia, tensión, retención de agua, etc. (Frantz y Cols., 2010). Inicialmente se creía

que este era su único cometido, pero con el paso de los años, se ha demostrado que la

matriz extracelular también está implicada en importantes procesos celulares como el

crecimiento, la morfogénesis, la diferenciación y la homeostasis, mediante la unión de

factores de crecimiento y la interacción con receptores celulares (Frantz y Cols., 2010;

Clause y Barker, 2013).

Tal es la diversidad funcional que un gran número de patologías y enfermedades se

deben a alteraciones en los componentes de la matriz extracelular o en las enzimas

que los producen, modifican o degradan, por lo que tarde o temprano se convertirá en

una importante diana farmacológica (Järveläinen y Cols., 2009).

https://basicmedicalkey.com/cellular-biology/

2.3.2. Proteoglicanos y glicosaminoglicanos de la matriz extracelular

Los glicosaminoglicanos son polisacáridos lineares constituidos por una unidad

disacárida que se repite a lo largo de su estructura molecular. Según el azúcar del

disacárido, existen varios tipos de glicosaminoglicanos: hialuronano, heparán sulfato,

heparina, condroitín sulfato, dermatán sulfato y keratan sulfato (Schaefer y Schaefer,

2010) (Figura 14). Los glicosaminoglicanos se encuentran unidos a una proteína

principal, formando moléculas denominadas proteoglicanos; todos, excepto el

hialuronano, que es sintetizado en la superficie celular y liberado directamente en el

medio extracelular (Schaefer y Schaefer, 2010).

Los proteoglicanos forman parte de la matriz extracelular de todos los tejidos de los

mamíferos, ya que todas las células son capaces de sintetizarlos y secretarlos al

exterior, insertarlos en la membrana plasmática o empaquetarlos intracelularmente

(Gandhi y Mancera, 2008). Pero no todas las matrices extracelulares contienen los

mismos proteoglicanos (Couchman y Pataki, 2012).

En la matriz extracelular de los mamíferos, se han identificado más de 40

proteoglicanos con una gran variabilidad a nivel genético y proteico, lo cual ha

dificultado su clasificación. Según se ha propuesto recientemente, los proteoglicanos

pueden agruparse en base a la localización celular y subcelular, la homología de

secuencia genética/proteica y la presencia de motivos proteicos particulares (Iozzo y

Schaefer, 2015) (Figura 15).

Figura 14. Esquema molecular de los glicosaminoglicanos (tomado de Papakonstantinou y Karakiulakis,

2009).

En general, los proteoglicanos de heparán sulfato, como por ejemplo las familias de

los sindecanos y de los glipicanos, localizados en la superficie celular, actúan como

receptores moleculares y participan en diferentes vías de transducción de la señal

relacionadas con el desarrollo y la proliferación celular (Carey, 1997; Filmus y Cols.,

2008; Lambaerts y Cols., 2009; Filmus y Capurro, 2014). Otro grupo de proteoglicanos

de heparán sulfato son los que forman parte de las membranas basales: agrina,

perlecano, colágeno XVIII (endostatina) y colágeno XV (Yurchenco y Patton, 2009).

Como ya se ha expuesto anteriormente, la membrana basal es una diferenciación de la

matriz extracelular que se encuentra en el exterior de la mayoría de células

(epiteliales, musculares, adipocitos, células de Schwann, etc.) (Bosman y Stamenkovic,

2003) y que cumple funciones específicas en cada tejido (LeBleu y Cols., 2007).

Figura 15. Clasificación de los proteoglicanos en mamíferos (tomado de Iozzo y Schaefer, 2015).

Los proteoglicanos de condroitín sulfato son importantes elementos estructurales de

la matriz extracelular ya que organizan la correcta disposición de las fibras de

colágeno, aportan propiedades viscoelásticas, mantienen la presión osmótica y tienen

capacidad de retener agua (Nishimura y Cols., 1998; Heinegård, 2009; Iozzo y Schaefer,

2015). Pero también actúan como correceptores o moduladores de la señal,

intervienen en procesos de diferenciación o regeneración celular y están implicados en

la plasticidad neuronal (Mikami y Kitagawa, 2013). La familia de hialectanos o

lecticanos es un ejemplo de proteoglicanos de condroitín sulfato de localización

extracelular, especialmente relevantes en el sistema nervioso central (Crespo-

Santiago, 2004; Howell y Gottschall, 2012). Sin embargo, fosfacano, NG2 y β-glicano

son proteoglicanos de condroitín sulfato insertados en la membrana citoplasmática y,

por tanto, asociados a la superficie celular (López-Casillas y Cols., 1991; Nishiyama y

Cols., 1991; Maurel y Cols., 1994).

La familia más grande de proteoglicanos la constituyen los SLRP (“small leucine-rich

proteoglycans”), también de localización extracelular, dentro de la cual se distinguen

cinco clases. Su similar estructura proteica hace que tengan características y funciones

comunes, como por ejemplo, regular la fibrilogénesis mediante la unión a colágenos

(Kalamajski y Oldberg, 2010; Chen y Birk, 2013) o influenciar la actividad celular

mediante la unión a receptores de superficie celular, factores de crecimiento,

citoquinas u otros componentes de la matriz extracelular (Schaefer y Iozzo, 2008).

Participan en importantes vías de señalización celular como la del factor de

crecimiento transformante β (TGF-β) y la proteína morfogénica ósea (BMP)

(Hildebrand y Cols., 1994). Sin embargo, cada uno de ellos también puede desempeñar

funciones únicas en tejidos específicos gracias a la variabilidad del extremo N-terminal

y a la unión de los diferentes glicosaminoglicanos (Iozzo y Schaefer, 2015).

2.3.3. Localización de los proteoglicanos en el sistema nervioso periférico

En el sistema nervioso periférico, alrededor de las células de Schwann se dispone una

membrana basal sobre la que se cimientan el resto de los constituyentes de la matriz

extracelular (Feltri y Wrabetz, 2005), entre ellos, los proteoglicanos.

Como ya se ha mencionado, algunos de los proteoglicanos de heparan sulfato son

constituyentes importantes de la estructura de las membranas basales. Mediante

cultivos celulares se ha demostrado que las células de Schwann y las neuronas

sensitivas expresan proteoglicanos de heparán sulfato en su membrana basal (Mehta y

Cols., 1985); en este sentido, se han detectado tres de ellos (perlecano, agrina y

endostatina) en las membranas basales de las células de Schwann de nervios y ganglios

periféricos (Halfter y Cols., 1998; Ma y Cols., 1994; Halfter y Cols., 1997; Chernousov y

Cols., 1998). Además, se ha localizado perlecano en los nódulos de Ranvier de

neuronas periféricas (Bangratz y Cols., 2012). Los otros dos grandes grupos de

proteoglicanos de heparán sulfato (sindecanos y glipicanos) también se han descrito en

el sistema nervioso periférico. Los glipicanos 1 y 3 se expresan en las neuronas de los

ganglios raquídeos (Litwack y Cols. 1994; Bloechlinger y Cols., 2004; Iglesias y Cols.,

2008), mientras que los sindecanos 3 y 4 en la periferia de las células de Schwann

(Carey y Cols., 1992; Erdman y Cols., 2002; Goutebroze y Cols., 2003) y en los nódulos

de Ranvier (Melendez-Vasquez y Cols., 2005). El receptor del factor de crecimiento

transformante β3, también conocido como betaglicano, el cual está formado por

cadenas de heparan sulfato y de condroitín sulfato, también se expresa en las células

de Schwann (Thomas y De Vries, 2007).

Los proteoglicanos de condroitín sulfato son mayoritarios en el sistema nervioso

central (Bandtlow y Zimmermann, 2000), mientras que en el periférico su distribución

es menor o no ha sido tan estudiada hasta la fecha. Mediante microscopia electrónica

e inmunohistoquímica, se ha demostrado en nervios periféricos su expresión en la

lámina basal de las células de Schwann (Aquino y Cols., 1984) y en el endoneuro

alrededor del axón (Tona y Cols., 1993; Morrison y Cols., 1994); concretamente son

versicano, agrecano y NG2 (Neural/glial antigen 2; Bode-Lesniewska y Cols., 1996;

Rezajooi y Cols., 2004; Ali y Cols., 2011). Además, también se ha encontrado versicano

en los nódulos de Ranvier (Melendez-Vasquez y Cols., 2005).

Todavía es más desconocido el patrón de expresión de los SLRP en el sistema nervioso

periférico y, en la literatura científica, son escasas las menciones a estos

proteoglicanos. Estudios de hibridación in situ han demostrado que las células de

Schwann expresan decorina (Hanemann y Cols., 1993). También se han detectado

decorina y lumicano en ganglios raquídeos al final de la segunda semana de desarrollo

del ratón (Wilda y Cols., 2000) y biglicano en el nervio óptico adulto (Ali y Cols., 2011).

En relación a los corpúsculos sensitivos, los proteoglicanos se encuentran embebidos

en la matriz extracelular correspondiente a cada compartimento corpuscular, aunque a

día de hoy, la información existente sobre la misma es escasa. Actualmente, se conoce

que tanto los corpúsculos de Pacini como los corpúsculos de Meissner expresan

laminina y colágeno tipo IV (componentes de lámina basal) en las células periaxónicas,

así como en la cápsula (Halata 1975; Zelená 1994; Vega y Cols., 1995). Los estudios de

Dubový y Bednárová (1999a) y Chouchkov y Cols. (2003) confirmaron la presencia de

distintas lamininas en las zonas de los corpúsculos de Pacini en las que ya se había

descrito la lámina basal (próxima a las células perineurales y lamelas), pero también en

las células de Schwann modificadas de los corpúsculos de Pacini (Dubový y Bednárová,

1999a; 1999b). Por otro lado, en los espacios interlamelares del núcleo externo y de la

cápsula de los corpúsculos de Pacini del mesenterio del gato predomina el colágeno

tipo II, mientras que la lámina intermedia contiene colágeno tipo IV (Pawson y Cols.,

2000). Los espacios equivalentes de los corpúsculos de Meissner de Macaca fuscata

fueron estudiados por Takahashi-Iwanaga y Shimoda (2003), demostrando de igual

forma la existencia de colágeno. Todavía resultan más desconocidos los proteoglicanos

que componen dicha matriz extracelular en los corpúsculos sensitivos. Los primeros

estudios en los que se documenta la presencia de proteoglicanos son los realizados por

Dubový y Svízenská (1993) y Dubový y Bednárová (1999). Tan sólo existe otro estudio

en corpúsculos de Pacini del mesenterio del gato en el que fueron localizados decorina

y biglicano en las lamelas perineurales (Sames y Cols., 2001). No obstante, el contenido

de proteoglicanos de la matriz extracelular es complejo y varía en cada zona del

corpúsculo (Dubový y Bednárová, 1999).

2.3.4. Funciones de los proteoglicanos en el sistema nervioso periférico

En el sistema nervioso periférico, la matriz extracelular proporciona el entorno físico-

químico adecuado para el desarrollo y la supervivencia de las neuronas y de las células

de la glía ya que está implicada en múltiples actividades y procesos celulares (Barros y

Cols., 2001; Gardiner, 2011):

a) Desarrollo embrionario: el sistema nervioso periférico se forma tras una serie de

procesos migratorios coordinados que experimentan las células de cresta neural en

los que los proteoglicanos juegan un papel fundamental. La expresión de los

proteoglicanos de condroitín sulfato en determinadas zonas, se relaciona

directamente con la ausencia de células de cresta neural durante el desarrollo

(Perris y Cols., 1991). Por otra parte, los SLRP están prácticamente ausentes

durante el movimiento de las células de cresta neural, pero muestran un aumento

de expresión en la unión dermoepidérmica, lo que les relaciona presumiblemente

con la guía de las terminaciones nerviosas sensitivas que inervan la piel (Perris,

1997). Además, los sindecanos podrían estar implicados en la proliferación de las

células de Schwann antes de la formación del nódulo de Ranvier (Melendez-

Vasquez y Cols., 2005), así como en las interacciones axogliales en el desarrollo

(Goutebroze y Cols., 2003).

b) Crecimiento axonal y mielinización: La lámina basal de las células de Schwann, de

la que forman parte diferentes proteoglicanos de heparán sulfato, interviene en la

migración y diferenciación de las células de Schwann, en su disposición alrededor

del axón y, al mismo tiempo junto a integrinas, en la mielinización (McGarvey y

Cols., 1984; Anton y Cols., 1994; Shorer y Cols., 1995; Feltri y Cols., 2002). Se ha

sugerido que la agrina participa en el proceso de crecimiento axonal mediante la

unión a factores de crecimiento y a proteínas de adhesión (Halfter y Cols., 1997).

c) Regeneración axonal: En un nervio adulto intacto predominan los componentes de

matriz inhibitorios para el crecimiento axonal, de forma que se controla la

proliferación neurítica. Sin embargo, en un nervio lesionado, la matriz extracelular

cambia su composición para crear un ambiente favorable a la regeneración. Tras

una lesión en un nervio periférico, se produce un aumento de proteoglicanos de

condroitín sulfato, que impiden la regeneración axonal al inhibir el efecto positivo

de las lamininas de las células de Schwann sobre el crecimiento neurítico (Muir y

Cols., 1989; Zuo y Cols., 1998). En este mismo sentido, se ha demostrado que la

degradación de proteoglicanos de condroitín sulfato en neuronas sensitivas,

favorece la regeneración axónica tras una lesión nerviosa (Udina y Cols., 2010). Por

el contrario, la expresión de proteoglicanos de heparan sulfato está relacionada

con la permisividad para el crecimiento y prolongación axonal, como se ha sugerido

con los glipicanos en el proceso de regeneración (Litwack y Cols. 1994; Bloechlinger

y Cols., 2004).

Actualmente, poco se conoce sobre la matriz extracelular de los corpúsculos sensitivos

de la piel glabra digital humana. La existencia de lámina basal (estructura derivada de

la matriz extracelular) en los componentes perineurales de los corpúsculos sensitivos

(Vega y Cols., 1995; Dubový y Bednárová, 1999; Chouchkov y Cols., 2003), podría estar

relacionada con la organización celular (Beck y Cols., 1990) o con la interacción celular

mediante receptores de superficie (Edgar, 1989). Tampoco puede descartarse su

implicación en el crecimiento axonal (Baron van Evercooren y Cols., 1982; Wang y

Cols., 1992; Feltri y Cols., 2002). Los distintos tipos de colágenos caracterizados en los

espacios interlamelares de los corpúsculos de Pacini del mesenterio del gato (Pawson y

Cols., 2000) y en los espacios equivalentes de los corpúsculos de Meissner de Macaca

fuscata (Takahashi-Iwanaga y Shimoda, 2003) pueden desempeñar una función

estructural o de regulación como se ha demostrado en las células de Schwann de

nervios periféricos (Koopmans y Cols., 2009). Sin embargo, todavía se conoce menos

sobre los proteoglicanos que conforman la matriz extracelular de los corpúsculos

sensitivos (Dubový y Svízenská, 1993; Dubový y Bednárová, 1999; Sames y Cols., 2001).

Estas moléculas son especialmente importantes para las capacidades físico-químicas

de los corpúsculos sensitivos, como son la retención de agua, viscoelasticidad,

transmisión de la presión, etc. (Sames y Cols., 2001), aunque tampoco pueden

descartarse otras funciones.

2.4. Edad y tacto

2.4.1. Envejecimiento en el sistema somatosensorial del tacto

En la piel, la pérdida de sensibilidad es una consecuencia inevitable asociada a la edad,

debida fundamentalmente a un proceso de denervación y deterioro de las estructuras

nerviosas periféricas (Kelly y Cols., 2005; Chang y Cols., 2004; Panoutsopoulou y Cols.,

2009). Los estudios realizados hasta la fecha se han centrado preferentemente en las

terminaciones nerviosas libres (Wickremaratchi y Llewelyn, 2006; Taguchi y Cols.,

2010; Decorps y Cols., 2014). Sin embargo, poco se conoce acerca de los cambios que

tienen lugar en los corpúsculos sensitivos, principales estructuras encargadas del tacto,

como consecuencia de la edad.

En el sistema nervioso periférico, con el envejecimiento, se producen cambios

estructurales en las fibras nerviosas, menor densidad de las mismas, afectación de los

umbrales de detección y disminución de la velocidad de conducción nerviosa (Cerimele

y Cols., 1990; Stevens y Patterson, 1995; Verrillo y Cols., 2002; Perry, 2006). Los

cambios estructurales guardan relación con la reducción del diámetro del axón,

degeneración axónica, aumento de formas irregulares y aumento del número de

fibrillas de colágeno en el endoneuro y el perineuro (Chase y Cols., 1992). La

disminución en la velocidad de conducción nerviosa se debe a una mayor

vulnerabilidad de la vaina de mielina (Bouche y Cols., 1993; Verdú y Cols., 2000; Di

Iorio y Cols., 2006).

Los estudios funcionales han demostrado que con la edad se produce una alteración

en las habilidades para detectar vibraciones, lo que implica que se requiera mayor

amplitud de vibración para percibir la misma magnitud de sensación (Verrillo, 1979;

Gescheider y Cols., 1994; Goble y Cols., 1996; Verrillo y Cols., 2002; Perry, 2006).

Además, se ha comprobado que con el envejecimiento se deterioran la agudeza y la

discriminación espacial táctil (Stevens y Patterson, 1995; Leveque y Cols., 2000),

mientras que aumenta el umbral para el tacto fino (Bruce, 1980; Thornbury y

Mistretta, 1981).

2.4.2. Cambios edad-dependientes en los morfotipos de mecanorreceptores

cutáneos

La variabilidad en la metodología empleada así como la utilización de modelos

animales muy dispares hacen que los escasos resultados existentes sobre los cambios

cuantitativos y cualitativos de los corpúsculos sensitivos como consecuencia de la edad

sean imprecisos y dispares.

Respecto a los corpúsculos de Meissner, hay grandes discrepancias en cuanto a la

densidad normal (número de corpúsculos de Meissner/mm2 de piel, “CM/mm2”) ya

que no hay un valor standard aceptado como único. Haciendo referencia a una zona

concreta de la mano, en la tabla 1 se recogen valores obtenidos en las dos últimas

décadas por diferentes grupos de investigación:

Tabla 1. Valores de densidad de corpúsculos de Meissner/mm2 según diferentes autores.

Localización Densidad (CM/mm2) Edad (años) Referencia

Yemas dedos 1.5 - Johnson y Cols., 20001

Yemas dedos 2.70 CM/mm 30.3 Kelly y Cols., 20052

Yemas dedos 2.53 CM/mm 68.3 Kelly y Cols., 20052

Yemas dedos 25 61.2 Nolano y Cols., 2008

Yema 5o dedo 12 34.3 Herrmann y Cols., 2007

Yema 5o dedo 6.77 43.2 Herrmann y Cols., 2011

Yema 5o dedo 45 - Nolano y Cols., 2003

Yema 3er dedo 33 33.7 Nolano y Cols., 2003

Palma proximal 1.33 CM/mm 30.3 Kelly y Cols., 20052

Palma proximal 1.35 CM/mm 68.3 Kelly y Cols., 20052

Eminencia tenar 5.1 34.3 Herrmann y Cols., 2007

Eminencia tenar 2.53 43.2 Herrmann y Cols., 2011 1 Humanos y otros primates; 2no tiene en cuenta el grosor de la sección (mm = longitud epidermis).

Como se observa en la tabla, hay grandes diferencias en los valores de densidad que

ponen de manifiesto la necesidad de establecer un protocolo adecuado.

En relación al envejecimiento, algunos autores afirman que con la edad se produce una

reducción de la densidad de corpúsculos de Meissner, la cual ha sido demostrada tanto

en humanos (Bolton y Cols., 1966; Bruce, 1980a; 1980b; Schimrigk y Rüttinger, 1980;

Thornbury y Mistretta, 1981; Halata y Munger, 1983; Iwasaki y Cols., 2003) como en

animales (Mathewson y Nava, 1985; Nava y Mathewson, 1996; Hoffman y Cols., 2004).

Los estudios clásicos establecen que la densidad es de 40-50 corpúsculos de Meissner

por mm2 al final de la infancia (Thornbury y Mistretta, 1981), la cual disminuye

progresivamente hasta los 50 años llegando a 10-25 corpúsculos de Meissner por mm2

(Bolton y Cols., 1966; Bruce 1980a; Thornbury y Mistretta, 1981; Halata y Munger,

1983) y alcanza valores mínimos de 5-8 corpúsculos de Meissner por mm2 a partir de

los 60 años (Bolton y Cols., 1966; Bruce, 1980b).

Contrariamente, otros grupos de investigación no constatan un cambio edad-

dependiente en la densidad de corpúsculos sensitivos (Nolano y Cols., 2003; Kelly y

Cols., 2005).

Además de los cambios cuantitativos, se han descrito en detalle, principalmente en

modelos murinos, ciertos cambios morfológicos que afectan a la capacidad funcional

de los corpúsculos sensitivos: los corpúsculos de Meissner de menor tamaño

corresponden a los ratones más jóvenes, los ratones de mediana edad presentan los

más grandes y los ratones de edad más avanzada tienen corpúsculos desorganizados y

lobulados que decrecen en tamaño hasta atrofiarse por completo. En relación a los

componentes corpusculares, las lamelas de los corpúsculos se atenúan y disminuyen

en número mientras que el material de la lámina basal se duplica al aumentar las

fibrillas de colágeno; el axón se vuelve más complejo con ramificaciones hasta la

mediana edad, mientras que en la vejez se pierde la forma característica serpenteante.

Además se ha contabilizado un mayor número de papilas dérmicas desocupadas en la

piel de los ratones de mayor edad (Nava y Mathewson, 1996; Mathewson y Nava,

1985).

Muchas de estas variaciones cualitativas también se han encontrado en los

corpúsculos de Meissner humanos (Bolton y Cols., 1966; Bruce, 1980; Matsuoka y

Cols., 1983; Iwasaki y Cols., 2003): distribución más irregular, con formas lobuladas u

alargadas y alejados de la epidermis u orientados oblicuamente en la papila, mayor

tamaño con la edad pero reducción a partir de los 70 años debido a cambios en el

tamaño de las células lamelares y formas retorcidas a partir de los 60 años.

Los corpúsculos de Pacini de la piel digital humana también sufren un proceso de

degeneración y atrofia con la edad, así como una disminución en número (Cauna y

Mannan, 1958; Gescheider y Cols., 1994). El tamaño del corpúsculo de Pacini es de

500-700 µm al nacer y se incrementa hasta alcanzar los 3-4 mm; pero a partir de los 70

años, el corpúsculo disminuye en dimensión y se vuelve más irregular (Cauna y

Mannan, 1958). Además, se ha demostrado que la sensibilidad vibrotáctil que implica a

los corpúsculos de Pacini se reduce con la edad (Verrillo, 1979).

En el caso de las células de Merkel, también se produce un descenso en número con el

envejecimiento tanto en humanos como en modelos murinos (Moll y Cols., 1984;

Fundin y Cols., 1997). Nolano y Cols. (2003) determinan que hay una densidad de 4

células de Merkel por mm2 de piel en individuos con edad media de 33.7 años, y

además, especifica que el número de corpúsculos de Meissner es 5 veces mayor que el

de células de Merkel en la piel digital humana. Otros resultados muy diferentes

establecen que hay una densidad media de 1700 células de Merkel por mm2 en crestas

glandulares de piel plantar de fetos humanos de 18-24 semanas, la cual se reduce

progresivamente en recién nacido y en adulto (Moll y Cols., 1984). De igual forma, Kim

y Holbrook (1995) establecen que en la epidermis palmar de fetos humanos, la

densidad llega hasta 1400 células de Merkel por mm2 de piel a las 8 semanas de edad

gestacional, momento en el cual el número empieza a caer.

3. HIPÓTESIS DE TRABAJO Y OBJETIVOS

En la presente tesis doctoral se han planteado los siguientes objetivos:

El objetivo general es contribuir al conocimiento de los corpúsculos sensitivos de la

piel glabra humana aportando nuevos datos sobre el origen de las células que los

componen, estudiando los proteoglicanos y glicosaminoglicanos de su matriz

extracelular y analizando las variaciones morfológicas, cuantitativas y cualitativas que

tienen lugar como consecuencia de proceso de envejecimiento.

Los objetivos específicos son los siguientes:

1. Investigar la presencia y distribución de antígenos específicos de células

endoneurales (CD34) o perineurales (Glut1 y EMA) en los corpúsculos de Pacini de

la piel digital humana con el fin de determinar el origen de las células del núcleo

externo y de la cápsula de los mismos.

2. Analizar la presencia de condroitín sulfato en la matriz extracelular de los

corpúsculos sensitivos humanos determinando su localización en los corpúsculos

sensitivos mediante doble inmunofluorescencia.

3. Analizar la presencia de heparán sulfato en la matriz extracelular de los

corpúsculos sensitivos humanos determinando su localización en los corpúsculos

sensitivos mediante doble inmunofluorescencia.

4. Identificar qué miembros de la familia SLRP se encuentran presentes en los

corpúsculos de Pacini humanos y analizar mediante inmunofluorescencia asociada

a microscopía confocal su distribución en los mismos.

5. Determinar los cambios cuantitativos, cualitativos y morfológicos que aparecen en

los mecanorreceptores cutáneos (corpúsculos de Meissner y células de Merkel) de

la piel digital humana como consecuencia del envejecimiento.

4. MATERIAL Y TÉCNICAS

4.1. Material

El material humano utilizado en el estudio procede de 11 sujetos con edades

comprendidas entre 12 y 90 años y sin enfermedad neurológica demostrable. La piel

digital se obtuvo de la cara palmar de la falange distal de los dos primeros dedos de las

manos (Tabla 2).

Tabla 2. Casos incluidos en el estudio.

Caso Edad (años) Sexo Mano Dedo

1 12 Mujer Derecha 1o y 2o

2 23 Varón Izquierda 1o y 2o

3 30 Mujer Izquierda 1o y 2o




7 55 Varón Derecha 1o y 2o

8 72 Varón Derecha 1o y 2o



11 90 Mujer Izquierda 1o y 2o

Las muestras fueron recogidas en el Servicio de Anatomía Patológica del Hospital

Central de Asturias (Oviedo) y del Complejo Hospitalario Universitario de Salamanca, a

partir de amputaciones, necropsias y donaciones, bajo cumplimiento de la legislación

española (RD 1301/2006; Ley 14/2007; RD 1716/2011; Orden ECC/1404/2013) y de

acuerdo a las directrices de la declaración de Helsinki II.

Todos los tejidos fueron fijados con orientación perpendicular a la superficie cutánea

en una solución de formaldehido al 4% PBS 1M pH7.6 (neutral buffered, Sigma-

Aldrich™) durante 24-72 horas y procesadas para su inclusión rutinaria en parafina

(Leica™ ASP6025, Leica Biosystems™, Wetzlar, Germany). Los bloques de parafina se

cortaron en secciones de entre 3-10 µm de espesor y fueron colocadas en

portaobjetos gelatinizados.

4.2. Técnicas

4.2.1. Tinción hematoxilina-eosina

Se realizaron manualmente tinciones con hematoxilina-eosina para identificar las

secciones de tejido que contenían los corpúsculos sensitivos de interés. El protocolo

empleado fue el siguiente: desparafinado en xilol y rehidratación con alcoholes de

concentración decreciente hasta llegar a agua corriente; baño en hematoxilina durante

10 segundos y lavado en agua corriente; baño en eosina durante 60 segundos y lavado

con agua destilada; deshidratación con alcoholes de concentración creciente,

diafanización en xilol y montaje del cubreobjetos con Entellan®.

4.2.2. Inmunohistoquímica simple indirecta

Las secciones de tejido se desparafinaron con xilol y se rehidrataron mediante una

batería de alcoholes de concentración decreciente hasta llegar al agua. A continuación,

para bloquear la actividad peroxidasa endógena, se trataron las muestras con H2O2 al

3% durante 10 minutos, seguido de un lavado en tampón PBS 1M a pH 7.6 con Tween-

20 al 0.5% (PBS-T) para permeabilizar las membranas celulares. Después de ello, se

bloquearon las uniones inespecíficas con albumina bovina (BSA) al 3% durante 30

minutos. Posteriormente, las muestras se incubaron toda la noche a 4oC y en cámara

húmeda con los anticuerpos primarios según cada estudio (Tabla 3).

Tras la incubación, las secciones se lavaron en PBS-T durante 15 minutos y se

incubaron a temperatura ambiente con el anticuerpo secundario correspondiente

conjugado con peroxidasa (Dako EnVision labelled polymer-HRP anti-conejo IgG o de

IgG anti-ratón) durante 90 minutos. Tras un lavado en PBS-T, se reveló la

inmunorreacción con una solución de 3-3’ diaminobencidina (Leica Bond™ Polymer

Refine Detection Kit, Leica Biosystems™). Las secciones se contrastaron con

hematoxilina, se lavaron en agua, se deshidrataron con alcoholes de concentración

creciente, se diafanizaron en xilol y se montaron con Entellan®. Por último, se

fotografiaron en un microscopio óptico Nikon Eclipse® 80i acoplado a una cámara

Nokia® DS-5M.

Como control, algunas secciones fueron procesadas del mismo modo descrito

anteriormente pero utilizando suero de ratón o conejo en lugar del anticuerpo

primario, o bien omitiendo la incubación con los anticuerpos primarios o secundarios.

En ambos casos el inmunomarcaje fue negativo.

Tabla 3. Anticuerpos primarios utilizados en el estudio.

Antígeno (clon) Origen Dilución Fabricante

Axón

Pan-NF (2F11) Ratón Prediluido Biocare Medical1

P200 kDa NF (RT-97) Mouse 1 μg/ml Boehringer-Mannheim2

NSE (BBS/NC/IV-H14) Mouse 1:1000 DAKO3

NSE (E27) Mouse Prediluido Master Diagnostica4

Peripherin Rabbit 1:5000 Chemicon International5

PGP9.5 Conejo 1:1000 Abcam6

PGP9.5 Conejo 1:1000 Biogenesis7

Células de Schwann modificadas

S100P (4C4.9) Ratón 1:1000 Thermo Fisher Scientific8

S100P Conejo Prediluido Novocastra9

S100P Conejo 1:1000 DAKO3

Vimentina (C-20) Conejo 1:200 Santa Cruz Biotechnology10

Vimentina Ratón Prediluido Santa Cruz Biotechnology10

Células de Merkel

Citoqueratina 20 Mouse prediluted DAKO3

Marcadores de origen endoneural/perineural

CD34 (QB-END/10) Ratón Prediluido Master Diagnostica4

Glut1 Conejo Prediluido Cell Marque13

EMA (GP1.4) Ratón Prediluido Novocastra9

Membrana basal

Colágeno IV Conejo 1:100 Abcam6

Perlecano (H300) Conejo 1:1000 Santa Cruz Biotechnology10

Glicosaminoglicanos

CS (CS-56) Ratón 1:100 Sigma Aldrich11

HS (JM403) Ratón 1:100 Amsbio14

SLRP clase I

Biglicano Ratón 1:200 Abcam6

Decorina Ratón 1:100 Santa Cruz Biotechnology10

Asporina Conejo Thermo Fisher Scientific8

SLRP clase II

Fibromodulina Ratón 1:500 Santa Cruz Biotechnology10

Lumicano Conejo 1:2000 Abcam6

PRELP Conejo 1:100 Abcam6

Keratocano Conejo 1:100 Santa Cruz Biotechnology10

Osteoadherina Ratón 1:100 Santa Cruz Biotechnology10

1 Concord, CA, USA. 2 Mannheim, Alemania. 3 Glostrup, Dinamarca. 4 Granada, España. 5 Billerica, MA, USA. 6 Hamburgo, Alemania. 7

Poole, UK. 8 Rockford, IL, USA. 9 Concord, Ontario, Canada. 10 Santa Cruz, CA, USA.

En relación con el estudio de los proteoglicanos de la matriz extracelular, en esta tesis

doctoral se abarca la mayor parte del espectro de proteoglicanos que se expresan en

mamíferos (Figura 16). A pesar de la limitación de no disponer de anticuerpos

específicos para todos los proteoglicanos, se ha podido realizar un estudio lo

suficientemente exhaustivo como para llegar a analizar un gran número de las

moléculas totales: proteoglicanos de heparan sulfato, proteoglicanos de condroitín

sulfato y proteoglicanos de la familia SLRP de las clases I y II.

Figura 16. Proteoglicanos analizados de forma general o específica y proteoglicanos no analizados.

4.2.3. Doble inmunohistoquímica indirecta

Esta técnica fue empleada exclusivamente para la inmunodetección de las proteínas

Glut1/EMA y CD34 correspondiente al primer resultado de la tesis (ver apartado 5.1).

Las secciones fueron procesadas con el sistema de tinción automático Leica Bond™

Max (Leica Biosystems™) ubicado en el Servicio de Anatomía Patológica del Complejo

Hospitalario Universitario de Salamanca. Una vez visualizada la inmunorreacción

correspondiente al primer antígeno con diaminobencidina como cromógeno (color

marrón) (Leica Bond™ Polymer Refine Detection Kit, Leica Biosystems™), se volvió a

realizar un segundo proceso de inmunohistoquímica para el segundo antígeno. En esta

ocasión, se utilizó un método de revelado diferente: el anticuerpo secundario está

conjugado con fosfatasa alcalina y el cromógeno utilizado es Fast Red (Leica Bond™

Polymer Red Refine Detection Kit, Leica Biosystems™), de forma que la segunda

inmunorreacción fue visualizada en color rojo.

4.2.4. Doble inmunofluorescencia indirecta con microscopía confocal

Las muestras de 10 µm se desparafinaron y rehidrataron como se describe en el

apartado de la inmunohistoquímica simple. Seguidamente, se lavaron en PBS-T

durante 20 minutos y se incubaron durante toda la noche a 4oC en cámara húmeda

con una mezcla 1:1 de dos anticuerpos primarios para la detección simultanea de dos

antígenos. Posteriormente las preparaciones se lavaron con PBS-T durante 30 minutos

y se incubaron con los anticuerpos secundarios durante 90 minutos: en primer lugar

IgG de cabra anti-conejo conjugado con Alexa Fluor 488 (1:1000; Serotec™, Oxford,

Reino Unido) y a continuación, con IgG de asno anti-ratón conjugado con Cy3 (1:50;

Jackson-ImmunoResearch™, Baltimore, MD, USA). Ambas incubaciones se realizaron

en cámara húmeda, en oscuridad y a temperatura ambiente. Se realizó un lavado con

PBS-T entre ambas incubaciones y luego se montó la preparación con DAPI (4',6-

diamino-2-fenilindol; 10 ng/ml) diluido en medio de montaje Fluoromount-G

(Southern-Biotech, Alabama, USA) para contrastar los núcleos (color azul).

En el caso de la doble inmunofluorescencia en las que ambos anticuerpos primarios

eran monoclonales de ratón, primero las muestras se incubaron con uno de los dos

anticuerpos contra el primer epítopo. Al día siguiente, se incubaron con IgG de conejo

anti-ratón (Novolink™ Polymer Detection Systems, Leica Biosystems™) durante 90

minutos y, tras un lavado en PBS-T, con anticuerpo secundario IgG de cabra anti-

conejo conjugado con Alexa Fluor 488 durante otros 90 minutos. Posteriormente, las

secciones fueron incubadas con el segundo anticuerpo primario para el otro epítopo

durante toda la noche. El tercer día, las preparaciones se incubaron con anticuerpo

secundario IgG de asno anti-ratón conjugado con Cy3 durante 90 minutos y se

montaron con DAPI diluido en medio de montaje Fluoromount-G.

La inmunofluorescencia fue detectada mediante un microscopio automático de

fluorescencia Leica DMR-XA acoplado a un software de captación de fluorescencia

Leica Confocal Software v2.5 (Leica Microsystems, Heidelberg GmbH, Germany),

ubicado en el Servicio de Procesado de Imágenes de la Universidad de Oviedo. Los

controles específicos de la reacción fueron ejecutados de la misma forma que para la

inmunohistoquímica simple. Además, se llevaron a cabo controles adicionales con el

fin de confirmar la ausencia de procesos de autofluorescencia del tejido o producidos

por el proceso de fijación omitiendo ambos anticuerpos.

4.2.5. Análisis cuantitativo

Se realizaron estudios cuantitativos para los corpúsculos de Meissner y las células de

Merkel en secciones independientes, pero no para los corpúsculos de Pacini debido a

su distribución irregular en la dermis y a su gran tamaño.

Para detectar los corpúsculos de Meissner y las células de Merkel, se realizó

inmunohistoquímica simple para la proteína S100 y CK20 respectivamente, en varios

cortes de 5 µm de espesor, separados entre sí una distancia mínima de 100 µm. Una

vez visualizada la inmunorreacción y montado el cubreobjetos, las secciones fueron

escaneadas mediante el escáner SCN400F (Leica Biosystems™) ubicado en el Instituto

Universitario de Oncología del Principado de Asturias (IUOPA) y procesadas

informáticamente con el programa SlidePath Gateway LAN (Leica Biosystems™).

Para determinar la densidad, el método cuantitativo aplicado fue el utilizado

previamente por Verendeev y Cols. (2015) en un estudio comparativo sobre los

corpúsculos de Meissner de las yemas de los dedos de diferentes primates.

En cada sección, se contabilizó el número de corpúsculos de Meissner o de células de

Merkel y se midió la longitud de la epidermis de la piel tranzando una línea sobre la

misma con una de las herramientas del programa informático anterior (Figura 16).

Para estimar la densidad de forma adecuada, hay que tener en cuenta que en una

sección de tejido podemos estar observando células de Merkel completas pero

también incompletas (el diámetro de una célula de Merkel es de 5-15 µm y el grosor

de la muestra es de 5 µm). Esto se conoce como el efecto del grosor de la sección en

relación al diámetro de la célula (Figura 17). En el caso de los corpúsculos de Meissner,

debido a su mayor tamaño, observamos exclusivamente corpúsculos incompletos

(diámetro de 20-60 µm y grosor de la sección de 5 µm).

Figura 16. Esquema representativo del método empleado para la cuantificación de corpúsculos

sensitivos.

Figura 17. Efecto del grosor de la muestra en relación al diámetro de la célula. Corrección de

Abercrombie (modificado de Técnicas de análisis de imagen: Aplicaciones en Biología).

Por lo que el número obtenido directamente de la cuantificación deber ser corregido

mediante un factor matemático para evitar una sobreestimación de la densidad. Dicho

factor de corrección fue formulado por Abercrombie en 1946 (Verendeev y Cols.,

2015) para estimar poblaciones nucleares a partir de secciones cortadas con

micrótomo y su fórmula es la siguiente:

𝑁 =𝑛 ∗ 𝑇

𝑇 + 𝐻

N = valor medio corregido; n = valor medio observado; T = espesor medio de las

secciones cuantificadas; H = diámetro medio de las células de Merkel o de los

corpúsculos de Meissner cuantificados en las secciones.

El diámetro de las células de Merkel o de los corpúsculos de Meissner fue medido con

la misma herramienta del software SlidePath Gateway LAN, trazando una línea recta

sobre el eje transversal de la célula de Merkel o el corpúsculo de Meissner

respectivamente.

La densidad media (nº de estructuras sensitivas/mm2 de piel) se calculó dividiendo el

valor medio corregido (N) entre el área superficial de piel (mm2), la cual se obtuvo a

partir del grosor medio de las secciones (5 µm = 0,005 mm) y la longitud media de la

piel de las muestras (X mm):

𝐷𝑒𝑛𝑠𝑖𝑑𝑎𝑑 =𝑁ú𝑚𝑒𝑟𝑜 𝑒𝑠𝑡𝑟𝑢𝑐𝑡𝑢𝑟𝑎𝑠 𝑠𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑎𝑠

Á𝑟𝑒𝑎 𝑑𝑒 𝑝𝑖𝑒𝑙

Para el estudio correspondiente al envejecimiento, los individuos fueron clasificados

en tres grupos de edad: a) [20-39 años], b) [40-59 años] y c) [60-90 años]. Se

determinó la densidad media para cada grupo y se extrapolaron los datos

construyendo las gráficas correspondientes mediante el programa Excel.

El desarrollo del proceso de cuantificación, aplicado al presente trabajo, es susceptible

de ser utilizado en la evaluación de los cambios en la inervación cutánea debidos a

neuropatías periféricas y centrales, lo cual dio lugar al capítulo de un libro que se

incluye en esta Tesis doctoral.

4.2.6. Análisis estadístico

Las diferencias en la densidad de corpúsculos de Meissner y células de Merkel entre

los tres grupos de edad pre-establecidos se valoró mediantes el test H de Kruskal-

Wallis H test, y valores de p ≤0.05 se consideraron como estadísticamente

significativos (marcados en las figuras como *p<0.05, **p<0.01).

5. RESULTADOS

Esta parte del capítulo se corresponde con el artículo:

García Piqueras, Jorge et al. Endoneurial-CD34 positive cells define an intermediate layer in

human digital Pacinian corpuscles. En Annals of Anatomy, 211, p. 55-60 (2017);

doi:10.1016/j.aanat.2017.01.006

Debido a la política de autoarchivo de la publicación la versión de la editorial está disponible, únicamente para usuarios con suscripción de pago a la revista, en el siguiente enlace: http://dx.doi.org/10.1016/j.aanat.2017.01.006

Información facilitada por equipo RUO

http://dx.doi.org/10.1016/j.aanat.2017.01.006

Esta parte del capítulo se corresponde con el artículo:

García Piqueras, Jorge et al. Chondroitin Sulfate in Human Cutaneous Meissner and Pacinian

Sensory Corpuscles. En Anatomical Record, 302, p. 325-331 (2018); doi:10.1002/ar.23951

Debido a la política de autoarchivo de la publicación la versión de la editorial está disponible, únicamente para usuarios con suscripción de pago a la revista, en el siguiente enlace: http://dx.doi.org/10.1002/ar.23951

Información facilitada por equipo RUO

http://dx.doi.org/10.1002/ar.23951

For Peer ReviewHEPARAN SULFATE IS ASSOCIATED TO THE BASEMENT MEMBRANE IN HUMAN CUTANEOUS MEISSNER’S AND

PACINIAN CORPUSCLES

Journal: Anatomical Record

Manuscript ID Draft

Wiley - Manuscript type: Full Length Article

Date Submitted by the Author: n/a

Complete List of Authors: García-Piqueras, Jorge; Universidad de Oviedo , Morfología y Biología Celular García-Suarez, Olivia; Universidad de Oviedo, Departamento de Morfología y Biología CelularGarcia-Mesa , Yolanda ; Universidad de Oviedo , Morfología y Biología Celular García-Fernandez, Beatriz; Universidad de OviedoQuiros , Luis; Universidad de Oviedo , Biología Funcional Cobo, Juan ; Universidad de Oviedo , Morfología y Biología Celular Martín-Biedma, Benjamin ; Universidade de Santiago de Compostela, Departamento de Cirugía y Especialidades Médico-QuirúrgicasFeito , Jorge; Complejo Hostipalatio Salamanca , Anatomia Patologica Vega, Jose; Universidad de Oviedo , Morfología y Biología Celular

Keywords: cutaneous sensory corpuscles, basement membrane, extracellular matrix, heparan sulfate proteoglycans, human

John Wiley & Sons, Inc.

The Anatomical Record

For Peer ReviewHEPARAN SULFATE IS ASSOCIATED TO THE BASEMENT

MEMBRANE IN HUMAN CUTANEOUS MEISSNER’S AND

PACINIAN CORPUSCLES

J. García-Piqueras1*, O. García-Suárez1*, Y. García-Mesa1, B. García2, L.

Quirós2,

J.L. Cobo1,3, B. Martín-Biedma4, J. Feito5, J.A. Vega1,6

1Departamento de Morfología y Biología Celular, Grupo SINPOS Universidad

de Oviedo, Spain2Departamento de Biología Funcional, Área de Microbiología, Universidad de

Oviedo, Spain 3Servicio de Cirugía Maxilofacial, Hospital Universitario Central de Asturias,

Oviedo, Spain 4Departamento de Cirugía y Especialidades Médico-Quirúrgicas, Universidad

de Santiago de Compostela, Santiago de Compostela, Spain5Servicio de Anatomía Patológica, Hospital Universitario de Salamanca,

Salamanca, Spain

6Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Chile

*Both authors contributed equally to this paper

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Short title: Extracellular matrix in Pacinian corpuscles

Address for correspondence

José A. Vega, M.D., Ph.D. Departamento de Morfología y Biología Celular Facultad de Medicina y Ciencias de la Salud – Universidad de Oviedo Avd. JualianClaveria, 6, Planta 9ª 33006 OVIEDO – Spain Telf. (0034) 985104097 E-mail: [email protected]

Abstract.- Heparan sulfate proteoglycans are pericellular/cellular-surface

molecules involved in somatosensory axon guidance in the peripheral nervous

system. However, the distribution of heparan sulfate proteoglycans in the

extracellular matrix of human cutaneous sensory corpuscles is unknown.

Immunohistochemistry and immunofluorescence were performed in order to

define the localization of heparan sulfate proteoglycans in Meissner’s and

Pacinian corpuscles using an anti-heparan sulfate antibody together with anti-

type IV collagen, anti-S100 protein, anti-PGP9.5 and anti-CD34 antibodies (to

immunolabel basement membranes, Schwann cells, axon and the endoneurial

intermediate layer of the Pacinian corpuscles, respectively). In Meissner’s

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corpuscles heparan sulfate proteoglycans were found co-localized with type IV

collagen. In Pacinian corpuscles heparan sulfate proteoglycans co-localized

with type IV collagen of the outer core lamellae and capsule, but not in the inner

core or the intermediate layer. Present results strongly suggest that heparan

sulfate proteoglycans are associated to basement membranes of the lamellar

cells in Meissner’s corpuscles, and the complex outer core-capsule in Pacinian

corpuscles. The functional significance of these results, if any, remains to be

elucidated.

Key words.- cutaneous sensory corpuscles, basement membrane, extracellular

matrix, heparan sulfate proteoglycans, human

Introduction

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Heparan Sulfate (HS) proteoglycans (HSPGs) are basic constituents of the

extracellular matrix (ECM). Structurally they are glycoproteins formed by a

variable number of HS chains attached covalently to a core protein localized at

the cell surface (i.e., syndecan or glypican families) or in the pericellular basal

membrane (BM; i.e., agrin, perlecan, type XV collagen and type XVIII) (Sarrazin

et al., 2011; Iozzo and Schaefer, 2015) that act principally as structural scaffold,

but also are involved in other cellular processes such as differentiation,

proliferation or survival (Schwarzbauer, 1999; LeBleu et al., 2007). The BM

architecture is the result of polymeric assemblies constituted by two principal

components that are laminins and type IV collagen, and other molecules as

nidogen glycoproteins, HSPGs, integrins or dystroglycan (Sasaki et al., 2004;

Yurchenco and Patton, 2009).

HSPGs are essential molecules for nervous system development and axonal

regeneration (Bovolenta and Fernaud-Espinosa, 2000; Yamaguchi, 2001;

Poulain and Yost, 2015), and are required for somatosensitive axons guidance

to the skin (Wang et al., 2012). Consistently, HSPGs have been detected in the

peripheral nerves and sensory ganglia (Ma et al., 1994; Halfter et al., 1997,

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1998; Chernousov et al., 1998). Nevertheless, the occurrence and distribution

of HSPGs in human cutaneous mechanoreceptors, the most peripheral

segment of the somatosensory system, have been never investigated.

Meissner’s and Pacinian sensory corpuscles are low-threshold

mechanoreceptors that consist of one axon surrounded by Schwann-related

cells and in the case of the Pacinian corpuscles, by endoneurial- and

perineurial-related cells (Munger and Idé, 1988; Zelená, 1994). Each corpuscle

cell-type is continuous with its counterpart of the nerve fibre supplying the

corpuscle, thus sharing their immunohistochemical features (Vega et al., 1996,

2009; García-Piqueras et al., 2017). Despite the scarce knowledge of the

protein composition of ECM in sensory corpuscles (Vega et al., 1995; Dubový

and Bednárová, 1999; Sames et al., 2001), a BM has been described and

partially characterized in cutaneous sensory corpuscles (Malinovský and Pac,

1982; Malinovský, 1986; Ide et al., 1987; Ide and Hayashi, 1987; Munger and

Ide, 1988; Chouchkov et al., 2003). In particular a BM was found underlying the

surface of the lamellar cells of Meissner’s corpuscles and in the inner core,

intermediate layer, outer core and capsule cells of Pacinian corpuscles

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(Malinovský, 1986; Vega et al., 1995). Nevertheless, other studies have shown

that BM is absent or discontinuous in the inner core and the intermediate layer

(Ide et al., 1987; Ide and Hayashi, 1987; Munger and Ide, 1988). Of particular

interest is the fact that some BM components, and other ECM molecules could

take part in the mechanotransduction process in sensory neurons (Wetzel et al.,

2007; Hu and Chiang, 2010; Chiang et al., 2011).

Since the occurrence and distribution of HSPGs in sensory corpuscles are

known only partially, we have designed the present research on human

cutaneous Meissner’s and Pacinian corpuscles. The study was aimed to

analyze the composition of ECM in the sensory corpuscles, in particular its

presence in BM.

Material and methods

Skin samples were obtained from the palmar aspect of the distal phalanx of the

first and second fingers of both hands (n = 11). Human samples were collected

from autopsies of non-demonstrable neurological disease subjects (age range

20 to 55 years) at the Departments of Pathology of the Hospital Universitario

Central de Asturias (Oviedo, Spain) and Complejo Hospitalario Universitario de

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Salamanca (Salamanca, Spain). These materials were obtained according to

the guidelines of the Helsinki Declaration (World Medical Association, 2013)

and in compliance with Spanish Laws. Tissue samples were fixed in 10%

formaldehyde in 1 M phosphate buffer saline (pH 7.4) for 24 h, dehydrated and

routinely embedded in paraffin. The pieces were cut into 7 μm thick sections

perpendicular to the skin surface and mounted on gelatine-coated microscope

slides. To confirm the presence of Pacinian and Meissner’s corpuscles in the

skin samples, several random sections were staining with hematoxylin and

eosin.

Immunohistochemistry.- Deparaffinized and rehydrated 7-μm thick sections

were processed for immunohistochemistry using EnVision antibody complex

detection kit (Dako, Copenhagen, Denmark) following manufacturer’s

instructions. The endogenous peroxidase activity and the non-specific binding

were blocked with 3% H2O2 and 10% foetal calf serum respectively for 30 min

each one. After that, the sections were incubated overnight in a humid chamber

at 4° C with the following primary antibodies: mouse monoclonal anti-HS

antibody (1:100; clone JM-403, AMSBIO; Abingdon, UK); rabbit polyclonal anti-

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type IV collagen (1:100; Abcam; Hamburg, Germany); mouse monoclonal anti-

CD34 (prediluted; clone QB-END/10, Master Diagnóstica™; Granada, Spain).

Moreover, rabbit polyclonal antibodies against S100 protein (1:1000; DAKO;

Glostrup, Denmark) and PGP9.5 (1:500; abcam; Cambridge, UK) were used to

selectively label the lamellar cells and inner-core cells, and the axon,

respectively. After incubation, the sections were rinsed in TBS-T and incubated

with anti-rabbit and anti-mouse EnVision system-labelled polymer

(DakoCytomation) for 60 minutes. After rising again, the immunoreaction was

visualized using 3-3’-diaminobenzidine as a chromogen. Finally, sections were

counterstained with hematoxylin-eosin to ascertain structural details,

dehydrated and mounted with Entellan (Merck, Dramstadt, Germany). Controls

were made by omitting the primary antibody or replacing it with a non-immune

rabbit or mouse serum. No immunostaining was observed (data not shown).

Double immunofluorescence.- Sections were processed as described before

but the primary antibodies were incubated simultaneously overnight at 4oC in a

humid chamber with a 1:1 v/v mixture of anti-HS antibody with anti-S100P

antibody, anti-HS antibody with anti-PGP9.5 antibody (1:300; abcam;

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Cambridge, UK) and anti-HS antibody with anti-type IV collagen antibody. After

rising with TBS, sections were incubated with Alexa fluor 488-conjugated goat

anti-rabbit IgG (1:1000; Serotec, Oxford, UK) for 90 minutes, then rinsed again

and incubated other 90 minutes with Cy3-conjugated donkey anti-mouse

antibody (1:50; Jackson-ImmunoResearch, Baltimore, MD, USA).

In the case of double immunodetection of HS and CD34, sections were

incubated overnight in a humid chamber at 4o C with primary anti-CD34

antibody (to label the intermediate layer; García-Piqueras et al., 2017, 2018).

After a TBS-rinsing, sections were incubated with Post-Primary (Novolink

Polymer Detection System®, Leica-Novocastra Biosystems, Newclastle, UK) in

a humid chamber at room temperature for 90 minutes. Post-Primary is a

prediluted rabbit IgG which recognizes mouse antibodies. After that, sections

were incubated for 90 minutes at room temperature with Alexa fluor 488-

conjugated goat anti-rabbit IgG as described above. Then, sections were

incubated again overnight at 4o C with mouse anti-HS antibody, followed by

incubation with Cy3-conjugated donkey anti-mouse antibody for 90 minutes.

Finally, to label nuclei, sections were counterstained with DAPI (10 ng/ml) and

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mounted with Fluoromount Gold, as previously. Immunofluorescence was

detected using a Leica TCS AOBS SP8 X confocal microscopy (Leica

Microsystem GmbH, Heidelberg, Germany; Servicios Científico-Técnicos de la

Universidad de Oviedo, Oviedo, Spain) and images processed by Leica

Application Suite X software v1.8.1.13759 (Leica Microsystem Gmbh,

Heidelberg, Germany). Representative samples for control purposes were

carried out as described before (data not shown).

Results

Human Meissner’s and Pacinian corpuscles display immunoreactivity for

HSPGs. Both types of sensory corpuscles analysed display immunoreactivity

for HSPGs. In Meissner’s corpuscles, positivity was observed in the surface of

the lamellar cells forming parallel lines which presumably corresponds to a BM

pattern (Figs. 1a-c). In Pacinian corpuscles, HSPGs immunoreactivity occurred

in the outer core and the capsule being especially intense at the innermost

lamellae of the outer core (Figure 2a). No HSPGs immunostaining was

observed in the inner core.

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In order to establish the precise location of HSPGs in Meissner’s and Pacinian

corpuscles, double immunofluorescence using different combinations of anti-HS

with anti-S100P or anti-PGP9.5 antibodies (to label lamellar/inner core cells and

axon, respectively) was performed. No co-localization of HSPGs and S100P

was observed in Meissner’s corpuscles thus supporting the idea that HSPGs

are localized in the pericellular BM of the lamellar cells (Figs. 1d-f).

Nevertheless, in some areas there was partial co-localization HSPGs and

S100P that presumably identified non-BM HSPGs. Moreover, the no co-

localization of HSPGs and PGP9.5 confirmed that HSPGs do not encircle the

axon (Figure 1g-i). Double immunofluorescence also confirmed that HSPGs are

not present in the inner core of Pacinian corpuscles (Figs. 2b-g).

HSPGs in Meissner’s and Pacinian corpuscles are localized in BM. The

immunohistochemical patterns of distribution of HSPGs and type IV collagen

were similar in both Meissner’s and Pacinian corpuscles (Figs. 3a-d; 4a,b

respectively), and also in the BM of skin epidermis (Figs. 3a-d).

To verify this almost identical localization, double immunofluorescence for

HSPGs and type IV collagen was performed. The results demonstrate that they

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were mainly co-localized at BM of Meissner’s (Figs. 3e-g) and Pacinian

corpuscles (Figs. 4c-h). In Pacinian corpuscles, HSPGs immunoreactivity was

detected in the outer part of the capsule, not co-localized with type IV collagen,

which indicates the occurrence of HSPGs independent of BM (Figure 4c-e).

HSPGs are localized in a specific perineural area in the innermost lamellae of

the outer core of Pacinian corpuscles. Since human Pacinian corpuscles show

an intermediate layer between the inner and the outer cores, and the intensity of

immunostaining for HSPGs (as well for type IV collagen) was strong in this

limiting zone, we decided to elucidate where HSPGs are localized. Light

microscopy images shown that HSPGs immunoreactivity coincides with the

endoneural CD34-positive intermediate layer (Figure 5a,b). Nevertheless,

double immunofluorescence showed no co-localization of HSPGs and CD34

(Figure 5c-h). This together with the no co-localization of HSPGs and S100

protein that exclude the presence of HSPGs in the outer lamellae of the inner

core (Figs. 2b-g), strongly suggesting that HSPGs in this corpuscular region are

localized in the innermost lamellae of the outer core.

Discussion

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In the present study we have demonstrate the presence of HSPGs

immunoreactivity in human cutaneous Meissner’s and Pacinian corpuscles,

primarily associated to BM. It must be clarified that the antibody used here

recognizes an epitope that includes N-unsubstituted glucosamine residues of

HS chains. Thus the results apply only for that molecule and not for the different

HSPGs BM or cell surface (Sarrazin et al., 2011; Iozzo and Schaefer, 2015).

Nevertheless, it can be assumed that HS present in basal membrane represent

the HSPGs perlecan, agrin, endostatin and type XV collagen (Yurchenco and

Patton, 2009; Iozzo and Schaefer, 2015).

BMs have been described of Meissner’s and Pacinian corpuscles using

transmission electron microscopy (Malinovsky and Pac, 1982; Malinovsky,

1986; Ide et al., 1987; Ide and Hayashi, 1987; Munger and Ide, 1988) as well as

immunohistochemistry (Vega et al., 1995). Two main components of BM in

human sensory corpuscles are laminin and type IV collagen (Vega et al., 1995).

Here we used as a criterion to determine that HSPGs are present in BM their

co-localization with type IV collagen. Conversely, whereas the absence of co-

localization was interpreted as an evidence of the presence of HSPGs outside

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BM presumably associated to HSPGs like syndecan or glypican families (Iozzo

and Schaefer, 2015).

In Meissner’s corpuscles, HSPGs were mostly found as a part of BM, although

occasionally HSPGs were detected in the cytoplasm of the lamellar cells. This

may be due to cytoplasmic production of HSPGs (Carey et al., 1993; Mehta et

al., 1985; Dziadek et al., 1986). The distribution of type IV collagen matched

and confirms previous studies form our group (Vega et al., 1995). Regarding

Pacinian corpuscles, HSPGs were exclusively co-localized with type IV

collagen, indicating localization in BM. Nevertheless, whether or not BM is

present in all the compartments of Pacinian corpuscle is still matter of

discussion. Based on the present results alone, it seems that the cells of the

inner core and those of the intermediate layer as well lack BM, although it has

been described using transmission electron microscopy (Ide and Hayashi,

1987; Munger and Ide, 1988; Malinovsky, 1986), and the presence of type IV

collagen and laminin was reported in those localizations (Vega et al., 1995).

The present study partially agrees with that by Vega and co-workers since we

have not found immunohistochemical evidences of BM in the inner core or the

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intermediate layer. So, the matter remains still open. In fact, while Ide et al.

(1987), Ide and Hayashi (1987) and Munger and Ide (1988) failed to

demonstrate BM in the inner core cells, Malinovsky and Pac (1982) and

Malinovsky (1986) found it regularly in these places. Further studies are

necessaries to elucidate these discrepancies. On the other hand, and

supporting present finding, there is consensus that the outer core and the

capsule have BM (Munger and Ide, 1988; Malinovsky, 1986; Ide et al., 1987).

The most interesting finding of our study refers to a strong localization of

HSPGs in a corpuscular area that comprises the innermost lamellae of the outer

core. This coincides with the so-called transitional area by Vega et al. (1995)

regarded of perineurial origin (Halata et al., 1990), although recently it was

demonstrated that this zone (also denominated intermediate layer) shares

endoneurial characteristics (García-Piqueras et al., 2017). Here we

demonstrate that the intermediate layer lack BM.

The cells forming Pacinian corpuscles are coupled by different types of

membrane junctions. Whilst the inner core cells communicate among

themselves by gap junctions, the outer core cells are coupled with thigh

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junctions, which are “especially prominent in the first and second (or sometimes

third) innermost lamellae of the outer core” (Ide and Hayashi, 1987). So, the

lamellae with the stronger expression of type IV collagen and HSPGs

correspond to the lamellae with prominent thigh junctions. Special organization

of thigh junction in these first outer core lamellae avoid the leak of fluid and ions

between the inner and outer core, and prevent a perturbation of the electrical

composition in the inner core from changings in the outer core composition (Ide

and Hayashi, 1987; Munger and Ide, 1988). Therefore, BM of the outer core

itself (Bush and Allt, 1990) or HSPGs components (Piña-Oviedo and Ortiz-

Hidalgo, 2008) might act as a barrier that isolates the axon and the inner core,

and presumably also the intermediate layer, from the outer core core-capsule.

ECM molecules of perineural cells are able to regulate the endoneural pressure

(Piña-Oviedo and Ortiz-Hidalgo, 2008).

It is also accepted now that some BM components, as well as ECM molecules,

are implicated in mechanotransduction (Wetzel et al., 2007; Hu and Chiang,

2010; Chiang et al., 2011). Whether or not HSPGs are involved in those

functions remains to be established.

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As a summary, in the present study we have demonstrated that HSPGs are

mainly associated to BM in human cutaneous Meissner’s and Pacinian

corpuscles, and that the inner core and the intermediate layer lack HSPGs thus

suggesting the lack BM.

Acknowledgements.- This study was supported by a grant from Gerencia

Regional de Salud de Castilla y León to JF (GRS 1615/A/17). The authors

thanks Dr. Marta Guervos (Servicios Comunes de Investigación, Microscopia

Confocal, Universidad de Oviedo) and Marta Sánchez-Pitiot (Grupo de

Histopatología molecular, Instituto Universitario de Oncología del Principado de

Asturias) for technical assistance.

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LEGENDS FOR FIGURES

Figure 1. Human Meissner’s corpuscles showing immunoreactivity for heparan

sulfate. Objective 40x (a-c). Double immunofluorescence using anti-heparan

sulfate antibody conjugated with CyTM3 (d, g) and anti-S100P (e) or PGP9.5

(h) antibodies conjugated with Alexa Fluor 488. Predominately no colocalization

or partially colocalization between heparan sulfate and S100P was indicative of

a Schwann cell pericellular expression pattern (f). No colocalization with

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PGP9.5 excluded the presence of heparan sulfate in the axon (i). Objective

63x/1.40 oil; pinhole 1.37; XY resolution 139.4 nm and Z resolution 235.8 nm

(d-i).

Figure 2.- Single immunohistochemistry for heparan sulfate in human

cutaneous Pacinian corpuscles. Heparan sulfate is present in all outer core-

lamellae including the capsule and more intensely in a thin layer around the

inner core. Objective 20x (a). Double immunofluorescence for heparan sulfate

(b, e) and S100P (c, f) confirmed absence of heparan sulfate in the inner core of

Pacinian corpuscles (d, g). Objective 20x/1.25 oil; pinhole 1.30; XY resolution

156 nm and Z resolution 266 nm (b-d); Objective 63x/1.40 oil; pinhole 1.37; XY

resolution 139.4 nm and Z resolution 235.8 (e-g).

Figure 3. Single immunohistochemistry for heparan sulfate (a, b) and type IV

collagen (c, d). Heparan sulfate expression was observed in basement

membranes of Meissner’s corpuscles similar to type IV collagen pattern. Note

equal immunoreactivity in basement membrane of epidermis. Objective 10x (a-

d); Colocalization of heparan sulfate (e) and type IV collagen (f) using double

immunofluorescence (secondary antibodies conjugated with CyTM3 and Alexa

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fluor 488 respectively) confirmed the presence of heparan sulfate in basement

membranes of Meissner’s corpuscles (g). Objective 63x/1.40 oil; pinhole 1.37;

XY resolution 139.4 nm and Z resolution 235.8 nm (e-g).

Figure 4. Single immunohistochemistry for type IV collagen (a) and heparan

sulfate (b) antibodies in Pacinian corpuscles. A similar pattern was observed:

immunoreactivity in all outer core lamellae including the capsule and more

intensive expression in a layer at the periphery of the inner core. Objective 20x

(a, b); Double immunofluorescence confirmed the co-localization of heparan

sulfate (c, f) and type IV collagen (d, g) in Pacinian corpuscles (e, h). Note that

some outer core lamellae were immunopositive just for heparan sulfate and vice

versa. Objective 20x/1.25 oil; pinhole 1.30; XY resolution 156 nm and Z

resolution 266 nm (c-e). Objective 63x/1.40 oil; pinhole 1.37; XY resolution

139.4 nm and Z resolution 235.8 nm (f-h).

Figure 5. Single immunohistochemistry for heparan sulfate (a) and CD34 (b) in

Pacinian corpuscles. HS-immunoreactivity was observed more intensively in a

thin layer around the inner core, similar to the intermediate layer described by

CD34-positive cells. Objective 20x (a, b); Double immunofluorescence using

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anti-HS (c, f) and anti-CD34 (d, g) antibodies (conjugated with CyTM3 and

Alexa fluor 488 respectively) showed no colocalization between them (e, h). HS

is expressed in the first lamellae of the outer core but not in the positive-CD34

intermediate layer of Pacinian corpuscles. Objective 20x/1.25 oil; pinhole 1.30;

XY resolution 156 nm and Z resolution 266 nm µm (c-e). Objective 63x/1.40 oil;

pinhole 1.37; XY resolution 139.4 nm and Z resolution 235.8 nm (f-h).

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254x190mm (96 x 96 DPI)

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254x190mm (96 x 96 DPI)

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254x190mm (96 x 96 DPI)

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254x190mm (96 x 96 DPI)

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254x190mm (96 x 96 DPI)

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Elsevier Editorial System(tm) for Annals of

Anatomy

Manuscript Draft

Manuscript Number:

Title: CLASS I AND CLASS II SMALL LEUCINE-RICH PROTEOGLYCANS IN HUMAN

CUTANEOUS PACINIAN CORPUSCLES

Article Type: Research Article

Keywords: Pacinian corpuscles; extracellular matrix; small leucine-rich

proteoglycans; human

Corresponding Author: Professor Jose A Vega, Ph.D, M.D.

Corresponding Author's Institution: Universidad de Oviedo

First Author: Jorge Garcia-Piqueras

Order of Authors: Jorge Garcia-Piqueras; Yolanda García-Mesa; Jorge

Feito; Beatriz Garcia; Luis M Quiros; Benjamin Martin-Biedma; Juan Cobo;

Jose A Vega, Ph.D, M.D.; Olivia Garcia

Abstract: Pacinian corpuscles are onion bulb-like multilayered

mechanoreceptors that consist of a complicated structure of axon

terminals, Schwann related cells (inner core), endoneural related cells

(intermediate layer) and perineurial related cells (outer core-capsule).

The cells forming those compartments are continuous and share the

properties of that covering the nerve fibers. Small leucine-rich

proteoglycans are major proteoglycans of the extracellular matrix and

regulate collagen fibrillogenesis, cell signalling pathways and

extracellular matrix assembly. Here we used immunohistochemistry to

investigate the distribution of class I (biglycan, decorin, asporin, ECM2

and ECMX) and class II (fibromodulin, lumican, prolargin, keratocan and

osteoadherin) small leucine-rich proteoglycans in human cutaneous

Pacinian corpuscles. The distribution of these compounds was: the inner

core express decorin, biglycan, lumican, fibromodulin, osteoadherin; the

intermediate layer display immunoreactivity for osteoadherin; the outer

core biglycan, decorin, lumican, fibromodulin and osteoadherine; and the

capsule contains biglycane, decorin, fibromoduline, and lumican.

Aspronin, prolargin and keratocan were undetectable. These results

complement our knowledge about the distribution of small leucine-rich

proteoglycans in human Pacinian corpuscles, and help to understand the

composition of the extracellular matrix in these sensory formations.

December 21, 2018

Dear Editor,

We are pleased to submit for publication in Annals of Anatomy a manuscript entitled

“CLASS I AND CLASS II SMALL LEUCINE-RICH PROTEOGLYCANS IN HUMAN CUTANEOUS

PACINIAN CORPUSCLES”, by J. García-Piqueas et al. The manuscript consists of 22

typewritten pages (including 1 table, references and legends for figures), and 8 figures.

The manuscript was prepared following the Authors Guide of Annals of Anatomy. I hope

de manuscript can be of interest for the Journal.

I am looking forward to hearing from you, and I would like to thank you in advance for

your attention and your time. I wish you Merry Christmas and a Happy New Year.

Sincerely yours.

Best regards,

Prof. J.A. Vega

Cover Letter

December 21, 2018

Dear Editor,

Manuscript: “CLASS I AND CLASS II SMALL LEUCINE-RICH PROTEOGLYCANS IN HUMAN

CUTANEOUS PACINIAN CORPUSCLES”, by J. García-Piqueas et al.

Conflict of Interest: The authors declare no conflict of interest

Conflict of Interest

CLASS I AND CLASS II SMALL LEUCINE-RICH PROTEOGLYCANS IN HUMAN

CUTANEOUS PACINIAN CORPUSCLES

J. García-Piqueras1, Y. García-Mesa1, J. Feito1,2, B. García3, L.M. Quiros3,4,

B. Martín-Biedma5, J. Cobo6,7, J.A. Vega1,8, O. García-Suárez1

1Departamento de Morfología y Biología Celular, Grupo SINPOS Universidad de Oviedo,

Spain 2Servicio de Anatomía Patológica, Hospital Universitario de Salamanca, Salamanca, 3 Instituto Universitario Fernández-Vega, Universidad de Oviedo & Fundación de

Investigación Oftalmológica, Oviedo, Spain 4Departmento de Biología Funcional, Universidad de Oviedo, Spain 5Departamento de Cirugía y Especialidades Médico-Quirúrgicas, Universidad de

Santiago de Compostela, Spain 6Departamento de Cirugía y Especialidades Médico-Quirúrgicas, Universidad de Oviedo,

Spain 7Instituto Asturiano de Odontología, Oviedo, Spain 8Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Chile

Short title: Extracellular matrix in Pacinian corpuscles

Address for correspondence José A. Vega, M.D., Ph.D. Departamento de Morfología y Biología Celular Facultad de Medicina y Ciencias de la Salud – Universidad de Oviedo Avd. JualianClaveria, 6, Planta 9ª 33006 OVIEDO – Spain Telf. (0034) 985104097 E-mail: [email protected]

*Manuscript

ABSTRACT

Pacinian corpuscles are onion bulb-like multilayered mechanoreceptors that consist of a

complicated structure of axon terminals, Schwann related cells (inner core), endoneural

related cells (intermediate layer) and perineurial related cells (outer core-capsule). The

cells forming those compartments are continuous and share the properties of that

covering the nerve fibers. Small leucine-rich proteoglycans are major proteoglycans of

the extracellular matrix and regulate collagen fibrillogenesis, cell signalling pathways

and extracellular matrix assembly. Here we used immunohistochemistry to investigate

the distribution of class I (biglycan, decorin, asporin, ECM2 and ECMX) and class II

(fibromodulin, lumican, prolargin, keratocan and osteoadherin) small leucine-rich

proteoglycans in human cutaneous Pacinian corpuscles. The distribution of these

compounds was: the inner core express decorin, biglycan, lumican, fibromodulin,

osteoadherin; the intermediate layer display immunoreactivity for osteoadherin; the

outer core biglycan, decorin, lumican, fibromodulin and osteoadherin; and the capsule

contains biglycan, decorin, fibromodulin, and lumican. Asporin, prolargin and keratocan

were undetectable. These results complement our knowledge about the distribution of

small leucine-rich proteoglycans in human Pacinian corpuscles, and help to understand

the composition of the extracellular matrix in these sensory formations.

Keywords: Pacinian corpuscles; extracellular matrix; small leucine-rich proteoglycans;

human.

INTRODUCTION

The Pacini’s corpuscles are rapidly-adapting low-threshold mechanoreceptors

functionally connected with myelinated A or A nerve fibres that detect gross

pressure changes and vibrations (Johnson, 2001; Roudaut et al., 2012; Fleming and Luo,

2013; Jones and Smith, 2014). They are widely distributed throughout the body (Bell et

al., 1994; Zelena, 1994), and their structure is independent of the anatomical

localization (Pawson et al., 2008). Typically they are oval-shaped multilayered

capsulated formations that consist of one central axon surrounded by non-neuronal

cells arranged in concentric lamellae, that form the inner core, intermediate layer, outer

core, and capsule (see Bell et al., 1994; Zelena, 1994). The inner core is composed of

terminal non-myelinating Schwann-like cells, the intermediate layer is formed by

endoneurial-related cells, and the outer core-capsule cells are perineurial-related cells

(Munger and Ide, 1988; Zelena, 1994, Vega et al., 2009; Feito et al., 2016; García-

Piqueras et al., 2017). Consistently, the different parts of the Pacinian corpuscles share

the immunohistochemical profile of their counterparts in nerves (Vega et al., 1996,

2009). Interposed between the different cell layers forming the Pacinian corpuscles

there is an extracellular matrix (ECM) occasionally organized as a basement membrane

(BM). Thus, it has been described a BM in relation to the inner core, outer core and

capsule of Pacinian corpuscles of different mammalian species (Munger and Ide, 1988;

Bell et al., 1994; Zelena, 1994; Vega et al., 1995; Dubový and Bednarova, 1999).

Recently, the presence of chondroitin sulfate proteoglycan chains was found associated

to the intermediate layer in human cutaneous Pacinian corpuscles (García-Piqueras et

al., 2018).

ECM is an organized complex network primarily formed of fibrillary proteins, such as

collagens and laminins, and proteoglycans (PGs; Iozzo and Schaefer, 2015; Theocharis et

al., 2016). Collagens are localized in the interlamellar spaces of Pacinian corpuscles:

type II collagen predominates in the outer core and capsule, type V collagen in the

intermediate layer, and collagen IV is present in all three extra-neuronal compartments

(Vega et al., 1995; Pawson et al., 2000). Conversely, little information is available about

the occurrence and distribution of other ECM components, especially PGs, in Pacinian

corpuscles.

PGs fall into two main groups: small and large. The group of small PGs (also known as

small leucine-rich proteoglycans; SLRPs), contains 18 members that are subclassified

into classes I to V. In particular class I is formed by biglycan (BGN), decorin (DCN),

asporin (ASPN), extracellular matrix protein 2 (ECM2) and ECMX, whereas class II is

formed by fibromodulin (FMOD), lumican (LUM), prolargin (PRELP), keratocan (KERA)

and osteoadherin (OMD) (McEwan et al., 2006; Schaefer and Schaefer, 2010; Chen and

Birk, 2013). Sames et al. (2001) are the only authors reporting the presence of DCN and

BGN in the cat mesentery Pacinian corpuscles.

SLRPs are expressed mainly during neural development but also in adult neural tissues

(Le Goff and Bishop, 2007; Dellett et al., 2012). In the peripheral nervous system,

including cat Pacinian corpuscles (Sames et al., 2001), some SLRPs have been detected

(Hanemann et al., 1993; Wilda et al., 2000). Nevertheless, as far as we know, SLRPs have

not been systematically explored in the human Pacinian corpuscles. Thus, the present

study was designed to investigate the occurrence and distribution of classes I and II of

SLRPs in human digital Pacinian corpuscles, and thus to contribute to the knowledge of

ECM composition in sensory corpuscles.

MATERIAL AND METHODS

Skin samples were obtained from the palmar side of the distal phalanx of amputated

hand fingers (n = 15) collected within 6-8 h after the accident from otherwise healthy

subjects (age range 24–42 years). Also, samples of finger tip skin (n = 7; age range 8–

56 years) were obtained from necropsies of neurological-disease-free subjects at the

Department of Pathology of the Hospital Universitario Central de Asturias (Oviedo,

Spain) and Complejo Hospitalario Universitario de Salamanca (Salamanca, Spain). The

specimens were fixed in 4% formaldehyde in 0.1 M phosphate buffer saline (pH 7.4) for

24 h, dehydrated, and embedded in paraffin.

All the tissue samples used in this study were obtained in compliance with Spanish Law

and the guidelines of the Helsinki Declaration II, and the study was approved by the

Ethical Committee for Biomedical Research of the Principality of Asturias, Spain (Ref.

266/18). The paraffin-embedded digital skin was cut into 10 μm-thick sections

perpendicular to the skin surface and mounted on gelatine-coated microscope slides.

The presence of Pacinian corpuscles was ensured staining randomly selected sections

with hematoxylin & eosin.

Immunohistochemistry.- Sections were deparaffinized, rehydrated and rinsed in 0.05 M

HCl Tris buffer (pH 7.5) containing 0.1% bovine serum albumin and 0.1% Triton X-100.

Thereafter, the endogenous peroxidase activity (3% H2O2) and non-specific binding

(10% fetal calf serum) were blocked, and the sections were incubated overnight in a

humid chamber at 4° C with the primary antibodies listed in Table 1. The antibodies

against protein gene product 9.5 (PGP9.5), neurofilaments proteins (NFP), peripherin,

S100 protein, CD34 and vimentin, were used to immunolabel the axon, inner core,

intermediate layer and outer core-capsule, respectively, within Pacinian corpuscles

(Vega et al., 2009; García-Piqueras et al., 2017). For immunohistochemical detection of

BGN the sections were heated in Envision FLEX target retrieval solution of pH6 (Dako) at

65°C for 20 min and then incubated for another 20 min at room temperature in the

same solution, previously to incubation of the primary antibody. After incubation, the

sections were rinsed in the same buffer as above and incubated with Dako EnVision

System labeled polymer-HR anti-rabbit IgG or anti-mouse IgG (DakoCytomation,

Denmark) for 30 minutes at room temperature. Finally, sections were washed, and the

immunoreaction visualized using 3-3’-diaminobenzidine as a chromogen. To ascertain

structural details, sections were slightly counterstained with hematoxylin & eosin.

For control purposes, representative sections were processed in the same way as

described above using non-immune rabbit or mouse sera instead of the primary

antibodies or omitting the primary antibodies in the incubation. Under these conditions,

no positive immunostaining was observed (data not shown).

Double immunofluorescence.- Based on the results of positivity for SLRPs in simple

immunohistochemistry, double immunofluorescence was carried out for DCN or BGN or

LUM or FMOD or OMD with axonal markers, S100 protein, vimentin and CD34.

Deparaffinized and rehydrated 10 µm thick sections were processed for reduction of

non-specific bindings (30 minutes with a solution of 5% bovine serum albumin in tris-

buffer saline –TBS- pH 7,4). Them they were incubated overnight, at 4° C in a humid

chamber with a 1:1 v/v mixture of the selected antibodies. After rinsing with TBS, the

sections were incubated for 1 hour with Alexa fluor 488-conjugated goat anti-rabbit IgG

(Invitrogen, Eugene, OR, USA), diluted 1:1000 in TBS containing 5% mouse serum

(Serotec, Oxford, UK), then rinsed again and incubated for another hour with Cy 3-

conjugated donkey anti-mouse antibody (Jackson-ImmunoResearch, Baltimore, MD,

USA) diluted 1:50 in TBS. Both steps were performed at room temperature in a dark

humid chamber. Sections were then washed and mounted with Fluoromount Gold.

Finally, to ascertain structural details sections were counterstained with DAPI (10

ng/ml). In the case of double immunodetection of CD34 with DCN, FMOD or OMD,

sections were incubated overnight in a humid chamber at 4o C with primary mouse anti-

DCN, anti-FMOD or anti-OMD, followed by incubation with Cy3-conjugated donkey anti-

mouse antibody for 90 minutes. Thereafter samples were incubated overnight with

mouse anti-CD34 antibody. After rinsing with TBS, sections were incubated with

prediluted rabbit Post-Primary IgG, which recognizes mouse antibodies (Novolink

Polymer Detection System®, Leica-Novocastra Biosystems, Newclastle, UK) at room

temperature for 90 minutes. After that, sections were incubated for 90 minutes at room

temperature with Alexa fluor 488-conjugated goat anti-rabbit IgG. Finally, to label

nuclei, sections were counterstained with DAPI (10 ng/ml) and mounted with

Fluoromount Gold as described above. For control purposes representative sections

were processed in the same way as described above using non-immune rabbit or mouse

sera instead of the primary antibodies or omitting the primary antibodies in the

incubation. Triple staining was detected using a Leica DMR-XA automatic fluorescence

microscope coupled with a Leica Confocal Software, version 2.5 (Leica Microsystems,

Heidelberg GmbH, Germany) and the images captured were processed using the

software Image J version 1.43 g Master Biophotonics Facility, Mac Master University

Ontario (www.macbiophotonics.ca).

RESULTS

Immunohistochemistry was performed to investigate the occurrence and localization of

some members of the class I and class II SLRPs in human digital Pacinian corpuscles. So,

http://www.macbiophotonics.ca/

we investigated the distribution within them of ASPN, BGN, DCN, FMOD, OMD, LUM,

PRELP and KERA. As a rule ASPN, PRELP and KERA were undetectable (data not shown)

whereas specific immunostaining was detected for the other SLRPs investigated with

different patterns of distribution within the corpuscles.

Class I SLRPs.- Serial sections of Pacinian corpuscles showed different patterns of

distribution for DCN and BGN. In light microscopy DCN and BGN immunoreactivity was

observed in all the corpuscular compartments including the intermediate layer, inner

and the outer core (Figs. 1a-e). Double immunofluorescence for DCN in combination

with specific markers of Pacinian corpuscular constituents (S100 protein, vimentin and

CD34) demonstrate that DCN surround the S100 protein positive cells, thus it is

localized outside the inner core (Fig. 2a-c). In addition, the relation of DCN

immunoreactive structures with the intermediate layer suggests that DCN is also

outside this layer (Fig. 2d-f). Therefore, DCN should be expressed in the interlamellar

spaces of the outer core and the capsule (Fig. 2g-i).

A strong and specific immunoreactivity for BGN was found in the inner core, outer core

and capsule (Fig. 1a-d). However, because the lack of immunoreactivity observed

encircling the inner core, the presence of BGN in the intermediate layer cannot be

ensured (Fig. 1d). To ascertain whether BGN is localized in the inner segment of the

Pacinian corpuscles simultaneous detection of BGN and S100 protein, or BGN and

peripherin, was performed. The results demonstrated that while BGN highly co-localizes

with S100 protein, thus supporting its presence in the inner core (Figs. 3a-c), it was

absent from the central axon (Fig. 3d-f). In addition, co-localization of BGN and

vimentin, but not of BGN and CD34, demonstrates that BGN is present in the outer core

lamellae and capsule (Fig. 3j-l) but not in the intermediate layer (Fig 3g-i).

http://link.springer.com/article/10.1007/s10571-010-9511-2/fulltext.html?view=classic#Fig1

Class II SLRPs.- Comparing serial sections processed for detection of S100P (Fig. 4a),

CD34 (Fig. 4c) and vimentin (Fig. 4e) with those used for detection of class II SLRPs, it is

apparent that the distribution of LUM immunoreactivity overlaps with the inner core,

intermediate layer and the outermost lamellae of the capsule (Fig. 4b); OMD has the

same distribution observed with LUM without the capsule´s outermost enhancement

(Fig. 4f); FMOD was only detected in the inner core and outer core-capsule (Fig. 4d).

In order to determine the precise localization of these molecules we performed double

immunofluorescence with specific antigens of the different corpuscular compartments

(Fig. 5). The results of these experiments demonstrated that LUM is absent from the

axon (Fig. 5a-c) and the intermediate layer (Fig. 5g-i) while it is localized in the inner

core (Fig. 5d-f), and widely in the outer core and capsule (Fig. 5l-k). Regarding FMOD, it

was confirmed that FMOD is absent from the axon (Fig. 6a-c) and the intermediate layer

(Fig. 6g-i). Conversely, a faint but specific reaction was detected in the inner core,

although in many cases do not match completely with S100 protein (Fig. 6d-f); the outer

core-capsule display solid co-localization (Fig. 6j-l). The inner core, intermediate layer

and outer core displayed OMD immunoreactivity (Fig. 7). Although in most cases co-

localization of OMD and S100P was observed, we have occasionally found that a

subpopulation of Schwan cells OMD positive do not express S100 and vice versa (Fig.

7c).

The results, based on the results of double immunofluorescence, are summarized in the

figure 8.

DICUSSION

ECM is composed by network of secreted proteins surrounding cells, either as an

interstitial matrix or organized into basement membranes. In Pacinian corpuscles ECM

fills the spaces between lamellae that form the inner core, intermediate layer and outer

core-capsule (Ide, 1986). In some of these compartments ECM is organized as a BM

(Malinovský et al., 1986; Vega et al., 1995; Dubový and Aldskogius, 1996; García-

Piqueras et al., 2018). The current information about the composition of ECM in

vertebrate Pacinian corpuscles regards to different types of collagen, laminin and two

PGs (Malinovský et al., 1986; Vega et al., 1995, 1996; Sames et al., 2001). Thus, in the

present study we have investigated the occurrence of other constituents of ECM, in

particular class I (DCN, BGN and ASPN) and Class II (LUM, FMOD, OMD, KERA and

PRELP) SLRPs.

A previous study carried out on Pacinian corpuscles from the cat mesentery detected

DCN and BGN (Sames et al., 2001), being found DCN in the outer core and BGN in the

inner core. Present results in human digital Pacinian corpuscles matches those for DCN

in the cat, but differs with respect to BGN distribution since they found it restricted to

the inner core while we observed immunoreactivity in the inner core and also in the

outer core. The role of these two molecules in Pacinian corpuscles is unknown.

Evidence of the influence of DCN PGs in the cellular morphology via the stabilization of

the cytoskeletal vimentin intermediate filaments has been recently reported (Jungmann

et al., 2012) but the importance of this relationship in the Pacinian corpuscles outer

core must be elucidated in additional studies.

As far as we know the occurrence and distribution of class II SLRPs in vertebrate

Pacinian corpuscles was never investigated. Therefore, data provided in the present

study cannot be contrasted with other studies.

Biochemical and cell culture assays have shown that SLRPs modulate ECM organization

by binding to various collagens (I, II, III, IV, V, VI, XII and XIV; Chen and Birk 2013;

Gubbiotti et al., 2016). In particular, LUM binds collagen II, BGN and FMOD interact with

collagens II and VI, and DCN binds collagens II, IV, V and VI (Chen and Birk, 2013). The

interlamellar spaces of Pacinian corpuscles contain different types of collagen (II, IV, V,

VI; Tachibana et al., 1995; Vega et al., 1996; Pawson et al., 2000) and members of the

class I and class II SLRPs proteins participate in the regulation of collagen fibril diameter

and interfibrillar spacings (Chen and Birk, 2013; Tashima et al., 2015). SLRPs modulate

ECM fibrillogenesis, organization and assembly (Kalamajski and Oldberg, 2010; Chen

and Birk, 2013; Gubbiotti et al., 2016).

On the other hand, SLRPs regulate cell activity interacting with growth factor receptors

(Hocking et al., 1998; Svensson et al., 1999; Ezura et al., 2000; Merline et al., 2009;

Dellett et al., 2012; Iozzo and Schaefer 2015; Neill et al., 2015). The relative proportion

of collagen and SLRPs determine the mechanical properties of the tissues (Hansen et al.,

2015). DCN and BGN bind to epidermal growth factor receptor (EGFR) and Erb4

(Schaefer and Iozzo, 2008; Schaefer and Schaefer, 2010; Iacob and Cs-Szabo, 2012) and

the localization of those SLRPs in human Pacinian corpuscles observed here is similar to

that reported for EGFR in human Pacinian corpuscles (Vega et al., 1994) or Erb4 in the

murine Pacinian corpuscles (González-Martínez et al., 2007). Interestingly DCN interacts

with EGFR to regulate collagen fibrillogenesis (Mohan et al., 2011).

SLRPs are expressed during neural development and in adult neural tissues (Le Goff and

Bishop, 2007; Dellett et al., 2012), including Schwann cells (Hanemann et al., 1993),

dorsal root ganglia (Wilda et al., 2000), and cat mesentery Pacinian corpuscles (Sames

et al., 2001). PGs, including the molecules studied here, are involved in regenerative

process of peripheral nervous system, both as neuritogenic molecules and as inhibitors

of axonal regeneration (Braunewell et al 1995; Oohira et al., 2000; Bradbury et al.,

2002; Maeda et al., 2011; Soleman et al., 2013; Silver and Silver, 2014). DCN inhibits

neurite outgrowth (Stichel et al., 1995; Davies et al., 2004), whereas BGN is a

neurotrophic molecule (Junghans et al., 1995; Koops et al., 1996).

In conclusion, this study provides a comprehensive analysis of SLRPs localization in

human cutaneous Pacinian corpuscle. Their complex expression patterns suggest a

compartment-specific regulation of collagen fibrillogenesis and stromal matrix assembly

and hydration. Considering the numerous SLRPs functions, they could be essential for

determining Pacinian corpuscle elasticity, mechanical properties and structural integrity

for the correct transmission of pressure resulting from indentation or stretch. The roles

of all the SLRPs in the human Pacinian corpuscles await elucidation in the future.

Acknowledgements.- This study was partially supported by a grant from Gerencia

Regional de Salud de Castilla y León to JF (GRS 1615/A/17). The authors thank Dr. Marta

Guervos (Servicios Comunes de Investigación, Microscopia Confocal, Universidad de

Oviedo) and Marta Sánchez-Pitiot (Grupo de Histopatología molecular, Instituto

Universitario de Oncología del Principado de Asturias) for technical assistance.

http://www.jneurosci.org/content/25/20/4964.long#ref-40

http://www.jneurosci.org/content/25/20/4964.long#ref-42

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LEGENDS FOR FIGURES

Figure 1.- Serial sections (a-e) of a human digital Pacinian corpuscle showing the

immunohistochemical distribution of BGN (d) and DCN (e). Figures a-c correspond to

immunodetection of VIM, CD34 and S100P respectively. DCN was mainly found in

outer-core cells and capsule, in a thin layer between both inner and outer cores and in

the S100P-positive inner core (e). BGN was expressed in all the compartments of

Pacinian corpuscles except the axon and the intermediate layer (d). Negative control (f).

ic: inner core; oc-c: outer core-capsula; il: intermediate layer. Scale bar: 100 µm, a to f.

Objective: 20X, a to f.

Figure 2.- Double immunofluorescence in Pacinian corpuscles showing the simultaneous

localization of DCN in red (a, d and g) with S100P (b), CD34 (e) and VIM (h) in green.

Although DCN did not colocalize with S100P (a-c), CD34 (d-f) or VIM (g-i), it was found in

both inner and outer cores. In the inner core, DCN was localized around the Schwann-

related cells (a-c), and sometimes its immunostaining was weaker (d). Moreover, a close

association between DCN and CD34 or VIM was observed, especially at the periphery of

the inner core (d-f) and in the outer core (g-i), respectively. Scale bar: 20 µm, a to f; 40

µm, g to i. Objective: 63X/1.40 oil, pinhole 1.37, XY resolution 139.4 nm and Z resolution

235.8 nm, a to f; 40X/1.25 oil, pinhole 1.00, XY resolution 156 nm and Z resolution 334

nm, g to i.

Figure 3.- Double immunofluorescence using anti-BGN antibodies (a, d, g and j) and anti-

S100P (b), Peripherin (e), VIM (h) or CD34 (k) antibodies in Pacinian corpuscles. The

expression of BGN was observed in both the inner core (a-c) and the outer core-capsule

(g-i). Conversely, BGN was not present in the intermediate layer (j-l), but its presence in

the axon can be ruled out (d-f). Scale bar: 20 µm, a to c, g to i, j to l; 10 µm, d to f.

Objective: 63X/1.40 oil, pinhole 1.37, XY resolution 139.4 nm and Z resolution 235.8 nm,

a to f, j to l; 40X, g to i.

Figure 4.- Immunodetection of S100P (a), LUM (b), CD34 (c), FMOD (d), VIM (e) and

OMD (f) in consecutive sections of a Pacinian corpuscle using single

immunohistochemistry. LUM was found in the inner core, the intermediate layer and

the outermost lamellae of the outer core-capsule (b). The expression patterns of FMOD

(d) and OMD (f) were similar, and both proteoglycans were present in the inner core

and outer core. However OMD was detected in the intermediate layer but not in the

capsule, contrary to FMOD. ic: inner core; oc: outer core; c: capsule; il: intermediate

layer. Scale bar: 100 µm, a to f. Objective: 20X, a to f.

Figure 5.- Double immunofluorescence in Pacinian corpuscles for LUM in green (a, d, g

and j) and NFP (b), S100P (e), CD34 (h) or VIM (k) in red. LUM colocalized with S100P in

the inner core (d-f) but not in the axon with NFP (a-c). It was also observed in the outer

core and capsule associated to VIM (j-l), but not in the CD34-positive intermediate layer

(g-i). Scale bar: 20 µm, a to i; 40 µm, j to l. Objective: 63X/1.40 oil, pinhole 1.37, XY

resolution 139.4 nm and Z resolution 235.8 nm, a to i; 40X/1.25 oil, pinhole 1.00, XY

resolution 156 nm and Z resolution 334 nm, j to l.

Figure 6.- Double detection of FMOD in red (a, d, g and j) and PGP9.5 (b), S100P (e),

CD34 (h) or VIM (k) in green by using confocal immunofluorescence in Pacinian

corpuscles. Immunolabeling of FMOD was observed in the inner core but colocalizing

irregularly with S100P (d-f). It was also found in the outer core and capsule, adjacent to

VIM (j-l). On the other hand, FMOD was neither present in the axon (a-c), nor in the

intermediate layer (g-i). Scale bar: 10 µm, a to l. Objective: 63X/1.40 oil, pinhole 1.37, XY

resolution 139.4 nm and Z resolution 235.8 nm, a to l.

Figure 7.- Double immunofluorescence for simultaneous detection of OMD in red (a, d,

g and j) with S100P (b), PGP9.5 (e), CD34 (h) and VIM (k) in green, in Pacinian

corpuscles. OMD was expressed in the axon weakly (d-f), the inner core with an

undefined pattern (a-c), the intermediate layer (g-i) and the outer core (j-l). VIM and

OMD do not colocalized in the outer core, but both molecules were adjacent in every

single lamella (j-l). Scale bar: 10 µm, a to f and g to i; 40 µm, j to l. Objective: 63X/1.40

oil, pinhole 1.37, XY resolution 139.4 nm and Z resolution 235.8 nm, a to I; 40X/1.25 oil,

pinhole 1.00, XY resolution 156 nm and Z resolution 334 nm, j to l.

Figure 8.- Schematic representation of the transverse section of a Pacinian corpuscle

showing the distribution of the analyzed SLRPs in the different corpuscular

compartments, based on the results of double immunofluorescence.

Table 1. Primary antibodies used in the study

Antibody (clone) Abbreviation Catalog number Host Dilution Source

Primary antibodies to general Pacinian corpuscles markers

CD34 (QB-END/10)

MAD-001613QD-8 Mouse Prediluted A

Neurofilament proteins (RNF402)

NFP sc-32729 Mouse 1:1000 B

Peripherin AB1530 Rabbit 1:500 C

PGP9.5 ab10404 Rabbit 1:300 D

S100 protein S100P IS504 Rabbit 1:1000 E

S100 protein (4C4.9)

S100P GTX24066 Mouse 1:2000 F

Vimentin VIM sc-7557 Rabbit 1:200 B

Vimentin (V9)

VIM IS630 Mouse Prediluted E

Primary antibodies to SLRPs

Asporin ASPN PA5-13553 Rabbit 1:200 G

Biglycan BGN PA5-13700 Rabbit 1:500 G

Biglycan (4E1-1G7)

BGN ab54855 Mouse 1:100 D

Decorin (9XX)

DCN sc-73896 Mouse 1:100 B

Fibromodulin (H-11)

FMOD sc-166406 Mouse 1:500 B

Keratocan KERA sc-66941 Rabbit 1:100 B

Lumican LUM ab168348 Rabbit 1:2000 D

Osteoadherin (B-10)

OMD sc-271102 Mouse 1:100 B

Prolargin PRELP ab103868 Rabbit 1:100 D

Secondary antibodies

Anti-rabbit IgG K4002 Goat Prediluted E

Anti-mouse IgG K4000 Goat Prediluted E

Anti-mouse IgG RE7111 Rabbit Prediluted H

Alexa Fluor 488-conjugated anti-rabbit IgG

A11034 Goat 1:1000 I

Cy3-conjugated anti-mouse IgG

715-166-150 Donkey 1:50 J

Source: A: Master Diagnóstica (Granada, Spain); B: Santa Cruz Biotechnology (Santa Cruz, CA, USA); C: Chemicon International (Billerica, MA, USA); D: Abcam (Cambridge, UK); E: Dako (Glostrup, Denmark); F: GeneTex (Irvine, CA, USA); G: Thermo Scientific (Freemont, CA, EEUU); H: Leica-Novocastra Byosistems (Newcastle, UK); I: Invitrogen (Eugene, OR, USA); J: Jackson-ImmunoResearch (Baltimore, MD, USA)

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December 21, 2018

Dear Editor,

Manuscript: “CLASS I AND CLASS II SMALL LEUCINE-RICH PROTEOGLYCANS IN HUMAN

CUTANEOUS PACINIAN CORPUSCLES”, by J. García-Piqueas et al.

Ethical statement: this study was performed in compliance with Spanish Law and the

guidelines of the Helsinki Declaration II, and the study was approved by the Ethical

Committee for Biomedical Research of the Principality of Asturias, Spain (Ref. 266/18).

*ethical statement

Chapter

The Cutaneous Biopsy for theDiagnosis of PeripheralNeuropathies: Meissner’sCorpuscles and Merkel’s CellsOlivia García-Suárez, Yolanda García-Mesa,Jorge García-Piqueras, Giuseppina Salvo, Juan L. Cobo,Elda Alba, Ramón Cobo, Jorge Feito and José A. Vega

Abstract

Cutaneous biopsy is a complementary method, alternative to peripheral nervebiopsy, for the analysis of nerve involvement in peripheral neuropathies, systemicdiseases, and several pathologies of the central nervous system. Most of theseneuropathological studies were focused on the intraepithelial nerve fibers (thin-myelinated Aδ fibers and unmyelinated C fibers), and few studies investigated thevariations in dermal innervation, that is, large myelinated fibers, Merkel’s cell-neurite complexes, and Meissner’s corpuscles. Here, we updated and summarizedthe current data about the quantitative and qualitative changes that undergo MCsand MkCs in peripheral neuropathies. Moreover, we provide a comprehensiverationale to include MCs in the study of cutaneous biopsies when analyzing theperipheral neuropathies and aim to provide a protocol to study them.

Keywords: skin biopsy, peripheral neuropathy, Meissner’s corpuscles,Merkel’s cells

1. Introduction

Since the last half of the past century, the analysis in the cutaneous biopsy ofnerves, Merkel’s cells (MkCs), and sensory corpuscles, especially Meissner’s cor-puscles (MCs), become a complementary method to diagnose peripheral neuropa-thies [1] and a reliable alternative to peripheral nerve biopsy. Nevertheless, it hasbeen during the last decade that numerous studies have provided consistent evi-dence to support this technique as a valuable tool to understand the etiologies ofsome neurological diseases and to follow up clinical trials [2–4] (Figures 1 and 2).

Most of the neuropathological studies on cutaneous biopsies were focused onintraepithelial nerve fibers, which are thin-myelinated Aδ fibers or unmyelinated Cfibers [2, 3, 5–9]. Conversely, few studies have investigated the large myelinated fibers(although it can offer notable advantages over the unmyelinated ones [10]). Also, thequantitative and qualitatively changes in MCs and MkCs associated to peripheralneuropathies are poorly known although the study of MCs has gained interest [11–13].

1

The evaluation of the dermal innervation, including large fibers, MCs, andMkCs, is not currently included within the routine analysis of skin biopsies becauseof the lack of a validated protocol. Changes in the density and size of MC and MkCs(i.e., variations in number/unit of surface, atrophy and/or hypertrophy, proteinexpression, etc.), can reflect quantitative or qualitative variations in the number ofsensory neurons or nerve fibers innervating them or in the cells forming MCsthemselves. Even more, they might also reflect pathologies of the central nervoussystem, and in these cases, the cutaneous biopsy becomes a method to study dis-eases difficult to be analyzed without invasive surgery.

This chapter is aimed to update the current data about the quantitative andqualitative changes in MCs and MkCs in peripheral neuropathies, as well as toprovide a comprehensive rationale to include them in the study of cutaneous biop-sies when analyzing the peripheral neuropathies. Furthermore, our purpose is toprovide a technical protocol for analyzing MCs and MkCs in cutaneous biopsies. Wehave excluded from this review the intraepidermic nerve fibers because they havebeen extensively studied in peripheral neuropathies, and standardized method hasbeen proposed and accepted [4, 9].

2. State of the art: a review and update of the literature

2.1 Why do we study Meissner’s corpuscles and Merkel’s cells for clinicalpurposes

The cutaneous MCs are sensory structures placed just beneath the epidermiswithin the dermal papillae in areas especially sensitive to light touch, like the finger-tips, palms, soles, lips, and male and female genital skin [14–16]. They show anellipsoid morphology with the main axis perpendicular to the skin surface and a sizelargely variable (length of 80–150 μm and diameter of 20–40 μm). Structurally, theyconsist of an axon that runs between the stacked nonmyelinating Schwann-like cells(the so-called lamellar cells) and habitually lacks a differentiated capsule [14, 16, 17].

Figure 1.Meissner’s corpuscles (arrows) and Merkel’s cells in the first toe skin of nondiabetic (nd) and diabetic (d)subjects as observed using immunohistochemistry for S100 protein (S100P) and cytokeratin-20 (CK20),specific markers for lamellar cells and Merkel’s cells, respectively.

2

Neuropathies

MCs are particularly abundant in the fingers and palm hand, which are twozones easily accessible for biopsy. Nevertheless, the analysis of MCs from thesezones has many problems. First of all, the normal density (MCs/mm2) at thislocalization should be determined to compare normal and pathological conditions.The most ancient studies established that the density of MCs in the human hand is�10–24 MCs/mm2 [18–20], it is higher in the fingertip (2.7/mm2 � 0.68) than inthe palm (1.33/mm2 � 0.6), and it does not change significantly with age [21].Nolano et al. [22] found 33.02/mm2 � 13.2 in the fingertip of digit III and 45/mm2 inthe digit V; Herrmann et al. [12] determined that the density of MCs on the palmarside of digit V is 12/mm2 � 5.3, whereas in the skin of the thenar eminence, it is5.1/mm2 � 2.2.

The second trouble for the use of MCs in the diagnosis of neuropathies iswhether or not MCs change in density and characteristic with aging. A reduction innumber and size of MCs in elderly is generally assumed [18, 23–25], but detailedstudies are not available. Preliminary data from our laboratory demonstrate thataging is accomplished of a reduction in the number and size of digital MCs, as wellas changes in their architecture and immunohistochemical properties (García-Piqueras et al., unpublished). However, the variations in the corpuscular size andmorphology of MCs are difficult to evaluate because of their large variability withinthe same skin sample. Therefore, in the absence of evident atrophy, hypertrophy, or

Figure 2.Meissner’s corpuscles (arrows) of the palmar aspect of the fingers of patients diagnosed of Alzheimer’s disease,amyotrophic lateral sclerosis, and multiple sclerosis, as observed using immunohistochemistry for S100 protein(S100P). The samples were obtained during necropsy and in compliance with Spanish law.

3

The Cutaneous Biopsy for the Diagnosis of Peripheral Neuropathies: Meissner’s Corpuscles…DOI: http://dx.doi.org/10.5772/intechopen.81687

corpuscular disruption, the evaluation of these parameters must be cautiously con-sidered when evaluating cutaneous biopsies.

The main constituents of MCs, that is, the axon and lamellar cells, contain specificproteins as widely demonstrated using immunohistochemistry [17, 26, 27]. Thesestudies reported a large volume of information, but they are purely descriptive and donot consent to quantify those proteins and their possible variations in neuropathies.The central axon displays immunoreactivity for general neuronal markers (neuron-specific enolase, protein gene product 9.5, neurofilament subunit proteins). They alsoexpress Ca2+-binding proteins such as calbindin D28k, parvalbumin, calretinin, andneurocalcin, which presumably regulate the axonic Ca2+ homeostasis and thereforeparticipate in the mechanoelectric transduction. Recently, our research’s groupdetected axonic TRPC6, TRPV4, ASIC2, and Piezo2 ion channels that work as puta-tive mechanoproteins [28–30]. Regarding lamellar cells, the vimentin is the interme-diate filament filling their cytoplasm, while the glial fibrillary acidic protein is alwaysabsent. They strongly express S100 protein colocated with parvalbumin or calbindinD-28 kDa. The lamellar cells also display immunoreactivity for TrkB, the signalingreceptor for the neurotrophins BDNF/NT-4 [31]. Apart from axon- or lamellar cell-specific proteins, there are some others shared by both corpuscular constituents. Theyinclude p75NTR and TrkA (low-affinity pan-neurotrophin receptor and the high-affinity receptor for nerve growth factor, respectively; [32, 33]), the epidermalgrowth factor receptor [34], or cell death protein Bcl-2 [35]. The presence of some ionchannels in the lamellar cells has been also reported [28–30]. It is possible that someof these proteins undergo changes during peripheral neuropathies, but limited infor-mation is so far available in this topic (see [17]). The proteins present in human MCsare summarized in Table 1.

The cutaneous MkCs are special epidermal cells placed in the basal layer of theepidermis, isolated or forming clusters, in both the glabrous and hairy skin. Theyare innervated by Aβ sensory axons connected through synapse-like contactsforming the so-called MkCs-neurite complexes. MkCs are involved in fine touchworking as a part of slowly adapting type I low-threshold mechanoreceptors andexpress specific mechanoproteins [16, 30, 36–39]. MkCs have an epithelial originand do not originate from the neural crest, as classically accepted [40–42].

Using immunohistochemistry, diverse proteins have been detected in the MkC-neurite complexes. They include low-molecular-weight cytokeratins and a reper-tory of synaptic vesicles-related proteins (chromogranin A, synaptophysin), differ-ent neuropeptides as well as neurotransmitter receptors, neurotrophin receptors,ion channels (ASIC2 and Piezo2), and neuron-specific enolase [28, 43–46]. Theaxon of the MkC-neurite complexes displays immunoreactivity for general neuronalmarkers (Table 1).

The density of MCs varies from an anatomical region to another, and it is directlyrelated to the sensibility of those zones [47]. In terms of density as far as we know, noage-dependent changes have been communicated. Recently, we have found signifi-cant reduction in of digital MkCs with aging (García-Piqueras et al., unpublished).On the other hand, whether or not MkCs, or the nerve fibers innervating them, areinvolved in peripheral neuropathies has been poorly studied, but this possibilityshould be explored because the easily accessibility to MkCs-neurite complexes.

2.2 Variations in MCs and MKCs in peripheral neuropathies

Data reporting changes in MCs in peripheral neuropathies are scarce and arerestricted to diabetes and other rare inheretary neuropathies, HIV infection,mechanical or traumatic nerve entrapment, and a miscellaneous group of systemicdiseases with neurological symptoms.

4

Neuropathies

Meissner’s corpuscles Merkel’s cell-neuritecomplex

Protein Ax LC Ax MC

Axonal proteins

Neuron-specific enolase

Protein gene product 9.5

β-Arrestin 1

GAP-43

Ca2+-binding proteins

S100 protein

Calbindin D28K

Calretinin

Neurocalcin

Cytoskeletal proteins

Neurofilament proteins

Vimentin

Growth factor receptors

p75NTR (pan-neurotrophin receptor)

TrkA (NGF receptor)

TrkB (BDNF/NT4 receptor)

EGF receptor

Putative mechanoproteins (ion channels)

ASIC2

Piezo2

TRPC6

TRPV4

TRPM8

Cell death-live proteins

Bcl-2

Neuropeptides and bioactive amines

Serotonin

Bombesin

Vasoactive intestinal polypeptide

Substance P

CCK8

Calcitonin gene-related peptide

Neuropeptide receptors

NMDA

Synaptic vesicle-associated proteins

Chromogranin A

Synaptophysin

Table 1.Proteins detected in human Meissner’s corpuscles and Merkel’s cell neurite complexes usingimmunohistochemistry. Red: positivity for a protein in the axon of Meissner's corpuscles; Blue: positivty for aprotein in the lamellar cells (LC) of Meissner's corpuscles.

5


2.2.1 Diabetic neuropathy

Distal symmetric peripheral neuropathy is one of the most common complica-tions of diabetes [48] and involves motor, autonomic, and sensory nerve fibers. Thehistopathological studies have provided evidence that both the thin unmyelinated Cfibers and the large myelinated ones are affected in on diabetic neuropathy. Con-sistently, the two most prominent complaints are peripheral pain and changes intouch [13, 49–52]. The intraepidermic nerve fibers as well as the nerve apparatus ofthe dermis are reduced in the diabetic neuropathy, and the reduction of the dermicnerves involves MCs. Importantly, although some authors have argued their inter-est in studying MCs and MkCs to better understand the diabetic neuropathy [53],only few studies have approached this topic.

In cutaneous biopsies, it was shown that the density of MCs is significantlyreduced in diabetic patients with respect to the controls (10.2 � 8.4 vs.16.2� 9.4/mm2, more evidently in type I than in type II diabetes), and this correlatedwith a reduction inmedian and ulnar nerves sensory amplitude; moreover, someMCswere hypertrophic or showed anomalies in their architecture (disorganization of thelamellar cells and increase in the irregularity of the axons) [54]. Similar findings asthose obtained from cutaneous biopsy were observed using in vivo reflectance confo-cal microscopy at the thenar eminence and digit V [55]. We have recently communi-cated that long-term diabetic neuropathy courses with a reduction in the numberand size of MCs and changes in their immunohistochemical profile [56] (Figure 1).

Nevertheless, the number and size of MCs are probably related with the time ofevolution of the neuropathy. In fact, in an animal model of diabetes that developneuropathy, MCs were found more abundant and hypertrophic during the first fewyears of hyperglycemia, whereas after a long time, the hypertrophy declines but thenumber of corpuscles remained higher than in age-matched nondiabetic subjects;furthermore, the MCs from the diabetic animals found had abnormal structure andimmunochemistry properties [57].

On the other hand, as far as we know, the only study reporting a reduction in thenumber of immunohistochemically demonstrable MkCs in diabetic neuropathy wasfrom our laboratory [56].

2.2.2 Charcot-Marie-Tooth disease

Charcot-Marie-Tooth (CMT) disease is a common inherited neuromuscular dis-order characterized by neuropathies without known metabolic alterations. In theskin of patients with common and rare forms of CMT caused by different muta-tions, the density of MCs is reduced compared with normal controls [58–60].Similar findings were reported by Almodovar et al. [61] using in vivo reflectanceconfocal microscopy.

2.2.3 Human immunodeficiency virus (HIV) neuropathy

HIV-sensory neuropathy is a common complication of HIV infection and may beassociated with significant morbidity due to neuropathic pain [62]. Severalapproaches exist for quantitative assessment of human HIV-associated distal sen-sory polyneuropathy, and some of them have analyzed both unmyelinated andmyelinated nerve fibers, as well as MCs. Using in vivo reflectance confocal micros-copy, it was found a marked reduction in MCs [12, 63] in HIV+ subjects with andwithout distal sensory neuropathy [64].

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Neuropathies

2.2.4 Entrapment neuropathies

Surprisingly, little is known about the impact of entrapment neuropathy ontarget innervation. More than 20 years ago, we reported that human digital MCssurvive to entrapment or section of peripheral nerves for more than 10 years, andalthough its number remains relatively stable, denervated MCs lack some antigensor change the pattern of expression of some others [65–67]. These data were con-firmed recently in subjects undergoing carpal tunnel syndrome [68].

2.2.5 Miscellaneous

A reduction in density or loss of MCs has also been reported in the skin ofpatients suffering from Ross syndrome (a rare disorder of sweating associated withareflexia and tonic pupil) [69], POEMS syndrome (polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, and skin changes) [70], systemic scle-rosis [71], pachyonychia congenita (in contrast, MkC densities are higher) [72],chronic inflammatory demyelinating polyradiculoneuropathy [73], and systemiclupus erythematosus [12].

2.3 MCs are also altered in central nervous system disorders

In addition to the abovementioned peripheral neuropathies, changes in MCshave been reported in Parkinson’s disease associated or not with dementia [74–76],spinobulbar muscular atrophy [77], Friedreich’s ataxia [78], amyotrophic lateralsclerosis [79], or Guillain-Barré syndrome [73]. Furthermore, altered cutaneousinnervation also has been observed in some psychiatric disorders [80] and mentaldeficiencies [81] (Figure 2).

3. Proposal of a method to systematically study MCs and MkCs incutaneous biopsies

MCs are only present in glabrous skin, and therefore fingers or toes are appro-priate regions to take cutaneous biopsies focused to evaluate them; in spite of thediscrepancies regarding their density in these places, they are abundant enough.

In our opinion, the palmar aspect of fingertip IV would be an ideal region to bebiopsied, because it is not involved in handling; the lateral borders should beexcluded to avoid damaging the digital nerves and the formation of neuromas. Onthe other hand, toe pad biopsies can be also useful, but they contain a lower densityof MCs than fingers [82].

The Joint Task Force EFNS/PNS [9] recommends to perform a 3 mm punch skinbiopsy (including epidermis and the subpapillary and reticular dermis), using asterile technique and under local anesthesia. A sample of these dimensions does notneed sutures, heal completely within 1 week, and this normally guaranties no sideeffects or complaints. Informed consent is required, and information on the possiblerisks must be always provided. The fixation of the skin samples is recommended in2% PLP (2% paraformaldehyde, 0.075 M lysine, 0.037 M sodium phosphate, 0.01 Mperiodate) or Zamboni’s solution. We have also obtained excellent structural resultsand good antigen preservation using Bouin’s fixative and buffered 10% formalde-hyde. Conversely, 4% paraformaldehyde masked most of the antigens present inMCs. The thickness of the sections is also important. The Joint Task Force EFNS/

7


PNS especially recommends 50-μm thick sections to perform 3D reconstructions ofMCs. Nevertheless, our experience demonstrates that to demonstrate the occur-rence of most antigens present in the axon or in the lamellar cells of Meissner’scorpuscles, 8 or 10 μm sections are appropriate.

There are different techniques for identification and assessment of MCs (silverimpregnation techniques, electron microscopy, immunohistochemistry, and immu-nofluorescence), but the ideal one should allow to the quantification and specificimmunostaining, distinguishing the different MCs constituents. In routine studies,at least one marker for the axon and one for the lamellar cells should be used.Indirect immunofluorescence, especially when associated with confocal micros-copy, provides an opportunity to investigate multiple neuronal and nonneuronalproteins within the same MC and also to perform its 3D reconstruction usingappropriate computerized image analysis systems. Ideally, double immunostainingfor both axon and lamellar cells, associated or not with labeling of the nuclei,provides a global image of the morphology and size of the corpuscle, as well as ofthe arrangement of corpuscular constituents (Figure 3).

To quantify MCs, we use the method proposed by Verendeev et al. [83] toestablish the density of MCs in the fingertips of primates. Briefly, 10-μm-thicksections, 200 μm apart, processed for S100 protein immunohistochemistry, areused. The sections are scanned by SCN400F scanner (Leica, Leica Biosystems™)and computerized using SlidePath Gateway LAN software (Leica, LeicaBiosystems™). Then, in each section, MCs are identified and counted by twoindependent observers. The average numerical values were corrected applyingthe Abercrombie’s formula: N = n*T/(T + H), where N is the corrected averagenumber of MCs, n is the counted average number of MCs in all sections of afingertip, T is the average section’s thickness, and H is the average diameter of thecounted MCs. Through a specific tool of the abovementioned software, the aver-age MCs diameter was determinate measuring the horizontal axis by drawing astraight line approximately in the central region of each corpuscle. The longitu-dinal epidermis of each section (mm) is measured with the same tool, and theaverage length was multiplied by the section’s thickness (mm) to give the mea-sured surface area (mm2). Finally, the average number of Meissner corpuscles(N) was divided by the surface area (mm2) that is the density of MCs by squaredmillimeter of skin (number of MCs/mm2) (Figure 4). To establish the density ofdigital Merkel’s cells, we used the same method immunostaining Merkel’s cells forcytokeratin.

Figure 3.3D reconstruction of a Meissner’s corpuscle in a finger of a 25-year-old male. The axon is labeled in red, and thelamellar cells in green. The cell nuclei were labeled with DAPI. Scale bar = 20 μm.

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Neuropathies

4. Final remarks and future prospectives

Peripheral neuropathies are diverse and require a multidimensional approach fordetection and monitoring clinical and research setting. The minimal invasiveness ofskin biopsy makes it a useful tool not only for diagnostics but also for following theprogression or the effects of a treatment in neuropathies.

Pathophysiological studies in patients with large nerve fiber polyneuropathiesare limited because the difficulty in obtaining nerve samples due to the invasivenature of the procedure. For this reason, some authors utilized skin biopsies toobtain morphological and molecular information from large dermal myelinatednerve fibers. The development of new methods to evaluate skin innervation,including MCs, through noninvasive techniques, that is, in vivo reflectance confocalmicroscopy, may contribute to better understand the changes in sensory corpusclesin neuropathies [12, 55, 61, 84–86].

Nevertheless, to use MCs as a complementary method in the diagnosis of neu-rological diseases, more studies are still necessary. Firstly, the density of MCs mustbe mapped in the specific areas where they are abundant and easily accessible tocutaneous biopsy, especially the hand glabrous skin. Secondly, the physiologicalage-related changes in the number and protein composition of MCs of theseselected areas must be established. Quantitative data, apart from qualitative, on

Figure 4.Schematic representation of the technical procedure to quantify Meissner’s corpuscles in sections of humandigital skin immunostained for the detection of S100 protein.

9


changes in protein composition of MCs with aging are necessary as a baseline forpossible pathological changes. In addition to immunohistochemical studies, skinbiopsy is amenable to the extraction of mRNA, RT-PCR, or microarrays for genesinvolved in neuropathies, and these methods should be used and standardized tostudy MCs. Finally, future studies should include not only neuropathies such asneurofibromatosis [85], or other rare metabolic neuropathies such as Gauchertype 1 disease [86], but also central nervous system diseases such as Alzheimer’sdisease.

Author details

Olivia García-Suárez1, Yolanda García-Mesa1, Jorge García-Piqueras1,Giuseppina Salvo1, Juan L. Cobo1,2, Elda Alba3, Ramón Cobo1, Jorge Feito1,4 andJosé A. Vega1,5*

1 Departamento de Morfología y Biología Celular, Grupo SINPOS, Universidad deOviedo, Spain

2 Servicio de Cirugía Máxilofacial, Hospital Universitario Cental de Astrias, Oviedo,Spain

3 Servicio de Neurología, Hopital Universitario “La Paz”, Madrid, Spain

4 Servicio de Anatomía Patológica, Complejo Hospitalario de Salamanca,Salamanca, Spain

5 Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Santiago deChile, Chile

*Address all correspondence to: [email protected]

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the termsof the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.

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The ageing of the somatosensory system at the periphery: Age-related changes in

cutaneous mechanoreceptors

Short running page heading: Aging of sensory corpuscles

J. García-Piqueras1*, Y. García-Mesa1*, L. Cárcaba1, J. Feito1,2, I. Torres-Parejo1, B.

Martín-Biedma3, J. Cobo4,5, O. García-Suárez1, J. A. Vega1,6

1Departamento de Morfología y Biología Celular, Grupo SINPOS Universidad de

Oviedo, Spain

2Servicio de Anatomía Patológica, Hospital Universitario de Salamanca, Spain

3Departamento de Cirugía y Especialidades Médico-Quirúrgicas, Universidad de

Santiago de Compostela, Spain

4Departamento de Cirugía y Especialidades Médico-Quirúrgicas, Área de

Estomatología, Universidad de Oviedo, Spain

5Instituto Asturiano de Odontología, Oviedo, Spain

6Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Chile

*These authors contributed equally to this paper

ABSTRACT

Decline of tactile sensation associated to aging depends on modifications in skin and

both central and peripheral nervous systems. Nowadays, age-related changes at the

periphery of the somatosensory system, especifically in human mechanoreceptors is

poorly known. Here we used immunohistochemistry to analyze the age-dependent

changes in Meissner’s and Pacinian corpuscles as well as in Merkel’s cells-neurite

complexes. Moreover, the variations in the neurotrophic TrkB-BDNF system and the

mechanoprotein Piezo2 (involved in maintenance of cutaneous mechanoreceptors and

light touch respectively) were evaluated. The number of Meissner’s corpuscles and

Merkel´s cells decreased progressively with aging. Meissner’s corpuscles were smaller,

rounded in morphology and located deeper in the dermis and evidences of corpuscular

denervation were found in the oldest subjects. Pacinian corpuscles showed generally no

relevant age-related alterations. Ageing was accomplished of reduced expression of

Piezo2 expression in the axon of Meissner’s corpuscles and Merkel’s cells, as well as of

a decline in the BDNF-TrkB neurotrophic system. This study demonstrates that

cutaneous Meissner’s corpuscles and Merkel’s cell-neurite complexes (and less

evidently Pacinian corpuscles) undergo morphological and size changes during the

aging process, as well as a reduction in terms of density. Furthermore, the

mechanoprotein Piezo2 and the neurotrophic TrkB-BDNF system are reduced in aged

corpuscles. All together, these alterations might explain part of the impairment of the

somatosensory system associated to aging.

KEYWORDS

ageing, glabrous skin innervation, sensory corpucles, Piezo2, BDNF-TrkB system,

human

INTRODUCTION

The sensory decline in the main sensory modalities is a consequence of the

ageing process. Nevertheless, sensory ageing is not homogeneous across sensory

systems, and large differences exist among these systems (Andersen, 2012; Jayakody et

al., 2018). The experimental and clinical findings regarding the ageing of the

somatosensory system (involving mechanoreception, thermoreception and nociception)

are not conclusive, probably because of the involvement of the central nervous system,

the peripheral nervous system and skin (Shaffer & Harrison, 2007; Goble et al., 2009;

Tseng et al., 2013; Decorps et al., 2014; Heft & Robinson, 2017). In any case, elderly

people have reduced fine touch discrimination ability (Skedung et al., 2018).

Age-dependent changes in the structure and physiology of the somatosensory

cortex are now rather well known (Blatow et al., 2007; Brodoehl et al., 2013; Cheng &

Lin, 2013; Gröschel et al., 2013; Hagiwara et al., 2014). Additionally, the occurrence of

morphological and histological changes in aged skin due to genetic and environmental

factors is widely acknowledged (Khavkin & Ellis, 2011; Rittie & Fisher, 2015; Kanaki

et al., 2016; Lephart, 2016; Krutmann et al., 2017). Conversely, the effects of ageing on

the somatosensory peripheral nervous system (dorsal root ganglia, large nerve fibres,

and sensory corpuscles; Vega et al., 1993; Ulfhake et al., 2002; see Rittie & Fisher,

2015), especially at the periphery, are largely unknown. Skin ageing is associated with

an overall reduction in nerve fibre endings in the epidermis and dermis (Besné et al.,

2002; Panoutsopoulou et al., 2009; Fromy et al., 2010; Namer, 2010), although

photoaging is characterized by increased sensory nerves in skin (Toyoda et al., 2005).

Tactile sensation is one of the most important components of mechanosensation

and is sensed by specific sensory formations localized in the skin and known

collectively as cutaneous sensory corpuscles or receptors (Zimmerman et al., 2014).

Functionally, these receptors fall into two categories: rapidly adapting

mechanoreceptors and slowly adapting mechanoreceptors, which each have two

variants, type I and type II, which differ in terms of their electrophysiological and

morphological characteristics (Jones & Smith, 2014). Slowly adapting type I

mechanoreceptors (SAI) are associated with epidermal Merkel cell-neurite complexes,

and slowly adapting type II mechanoreceptors (SAII) are thought to be located in

dermal Ruffini’s corpuscles, although the function of SAII can be carried out by other

sensory corpuscles in addition to or different from Ruffini’s corpuscles (Wu, 1998; Paré

et al., 2003; Olson et al., 2016). Merkel cells are specialized epidermal cells (Van

Keymeulen et al., 2009) functionally connected to Aβ nerve fibres that act as SAI and

express mechanoproteins (Ikeda et al., 2014; Maksimovic et al., 2014; García-Mesa et

al., 2017). In mammals, rapidly adapting mechanoreceptors are found in Meissner’s and

Pacinian sensory corpuscles (Zimmerman et al., 2014). Meissner’s corpuscles (MCs)

consist of an axon that spirals between stacked Schwann-related cells, are located in the

superficial papillary dermis and represent rapidly adapting type I mechanoreceptors

(RAI; Vega et al., 2012). Cutaneous Pacinian corpuscles are located in the superficial

hypodermis or deep reticular dermis and represent rapidly adapting type II

mechanoreceptors (RAII); their morphology resembles an onion bulb, with a central

axon surrounded by thickly packed lamellar Schwann-related cells that form the inner

core and are surrounded by many loosely packed endoneurial-perineurium-related cells

that constitute the intermediate layer, the outer core and the capsule outer core (Vega et

al., 2009; García-Piqueras et al., 2017).

Because age-dependent impairments in mechanosensory function have been

reported and because limited information is available about age-dependent changes in

mechanosensors, we conducted research on human digital skin to evaluate the

quantitative and qualitative changes that occur in Merkel’s cells, Meissner’s corpuscles

and Pacinian corpuscles with ageing. We have recently studied the development of

these structures until maturity (Feito et al., 2018), demonstrating that human cutaneous

sensory corpuscles are not stable but are dynamic structures that undergo permanent

age-related changes. On the other hand, because the ion channel Piezo2 provides at the

basis of mechanotransduction (see for a review Anderson et al., 2017) and is located in

both Meissner’s corpuscles and Merkel’s cells of human digital skin (García-Mesa et

al., 2017), we investigated whether the pattern of expression of this protein changes

with ageing. Furthermore, it is well known that cutaneous mechanoreceptors depend on

the neurotrophin system of the brain-derived neurotrophic factor (BDNF) and its high-

affinity receptor TrkB for development, growth and maintenance (Botchkarev et al.,

1999; LeMaster et al., 1999; González-Martinez et al., 2004; Perez-Pinera et al., 2008;

Reed-Geaghan et al., 2016). Additionally, both BDNF and TrkB have been detected in

developing and adult sensory corpuscles in different species, including humans (Stark et

al., 2001; Sedy et al., 2004; Calavia et al., 2010; Cabo et al., 2015a). Therefore, we

analysed whether the localization of BDNF and TrkB in mechanoreceptors varies with

age. Reduced expression of BDNF and TrkB has been observed in the central nervous

system of old subjects compared with young and adult subjects (Erickson et al., 2010;

Forlenza et al., 2015), suggesting that these molecules have a role in normal ageing.

The present study aimed to add information about the mechanisms that underly

sensory deficits in elderly subjects and is focused on the peripheral touch components of

somatosensory perception, with analysis of the effects of ageing on the structural and

neurotrophic features of cutaneous sensory corpuscles.

METHODS

Skin samples were obtained from the palmar aspect of the distal phalanx of the

first and second hand fingers during autopsy at the Service of Pathology of the Hospital

Universitario Central de Asturias, Oviedo, Spain (n = 6) and the Service of Pathology of

the Complejo Hospitalario Universitaro de Salamanca (n = 9) and from incidental

amputation at the Service of Plastic Surgery of the Hospital Universitario Central de

Asturias (n = 6). The samples were collected within 12 h after demise from subjects free

of neurological diseases and without a clinical history of fibromatosis or labour-induced

repeated trauma. The age range was 23 to 90 years, and at least one case per decade of

life was analysed.

The specimens were fixed in 4% formaldehyde in 0.1 M phosphate-buffered

saline (pH 7.4) for 24 h, dehydrated and routinely embedded in paraffin. The pieces

were cut to 7 µm thickness in sections perpendicular to the skin surface and mounted on

gelatin-coated microscope slides. The presence of Pacinian and Meissner’s corpuscles

in the skin samples was evaluated by staining with haematoxylin and eosin. The

occurrence of Merkel’s cells was assessed with immunohistochemistry. These materials

were all obtained in compliance with Spanish Law and the guidelines of the Declaration

of Helsinki.

The automated diagnostic platform Leica Bond III was used for

immunohistochemistry with the Leica Bond™ Polymer Refine Detection Kit (Leica

Biosystems™, New Clastle upon Tyne, UK ) according to the manufacturer’s

instructions. The primary antibodies were directed against the main corpuscular

constituents: axons (neurofilament, PGP 9.5) and Schwann cell-derived lamellar and

inner core cells (S100 protein). Moreover, to identify Merkel cells, an antibody against

cytokeratin 20 (CK20) was used. The BDNF-TrkB neurotrophin system was detected

using specific antibodies against specific amino acid sequences within these molecules.

Indirect immunohistochemistry included several negative and positive controls, as well

as the internal positive and negative controls. Additionally, representative sections were

processed in the same way as described above using non-immune rabbit or mouse sera

instead of the primary antibodies or by omitting the primary antibodies during the

incubation.

Data on case material and the characteristics of the primary antibodies used are

summarized in Tables 1 and 2, respectively.

Double immunofluorescence was performed on deparaffinized and rehydrated

sections. Endogenous peroxidase activity and non-specific binding were reduced by

incubating sections with 3% H2O2 and a solution of 10% bovine serum albumin in Tris

buffer solution (TBS), respectively. The sections were then incubated overnight at 4°C

in a humid chamber with a 1:1 mixture of a polyclonal antibody against S100-protein

(diluted 1:1000), PGP 9.5 (diluted 1:1000), BDNF (diluted 1:200) or TrkB (diluted

1:200) and a monoclonal antibody against NFP (diluted 1:1000) or CK20 (prediluted

and 1:200 in the blocking solution). After rinsing with TBS, the sections were incubated

for 1 h with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Serotec, Oxford, UK),

diluted 1:1000 in TBS containing 5% mouse serum (Serotec), rinsed again and

incubated for another hour with Cy3-conjugated donkey anti-mouse antibody (Jackson-

ImmunoResearch, Baltimore, MD, USA) diluted 1:50 in TBS. Both steps were

performed at room temperature in a dark humid chamber. Finally, to ascertain structural

details, sections were counterstained and mounted with DAPI diluted in glycerol

medium (10 ng/ml). Triple fluorescence was detected using a Leica DMR-XA

automatic fluorescence microscope (Photonic Microscopy Service, University of

Oviedo) with Leica Confocal Software, version 2.5 (Leica Microsystems, Heidelberg

GmbH, Germany), and the captured images were processed using the software ImageJ,

version 1.43 g Master Biophotonics Facility, Mac Master University Ontario

(www.macbiophotonics.ca). For control purposes, representative sections were

processed in the same way as described above using non-immune rabbit or mouse sera

instead of the primary antibodies or by omitting the primary antibodies during the

incubation. Furthermore, when available, additional controls were carried out using

specifically preabsorbed antisera. Under these conditions, no positive immunostaining

was observed (data not shown).

Quantitative analyses

Quantitative analyses were performed to determine the density of cutaneous

digital Meissner’s corpuscles and Merkel cells at different ages but not the density of

Pacinian corpuscles because of their irregular distribution in the skin dermis. To

quantify Meissner’s corpuscles, we used the method proposed by Verendeev et al.

(2015) to determine the density of Meissner’s corpuscles in the fingertips of primates.

The samples included in the study were divided into three age groups: 20 to 39 years, 40

to 59 years, and 60 years or older (see Table 1). Briefly, six 7 µm thick sections of each

skin sample that were 200 µm apart and processed for S100 protein

immunohistochemistry were evaluated. The sections were scanned by an SCN400F

scanner (Leica, Leica Biosystems™), and the scans were computerized using SlidePath

Gateway LAN software (Leica, Leica Biosystems™). Then, in each section, Meissner’s

corpuscles were identified and counted by two independent observers (JG-P and JAV).

The average numerical values were corrected by applying Abercrombie’s formula: N =

n*T/(T+H), where N is the corrected average number of Meissner’s corpuscles, n is the

counted average number of Meissner´s corpuscles in all sections of a fingertip, T is the

average section thickness, and H is the average diameter of the counted Meissner

corpuscles. Through a specific tool of the previously mentioned software, the average

Meissner corpuscle’s diameter was determined by measuring its horizontal axis by

drawing a straight line approximately in the central region of each Meissner’s corpuscle.

The epidermal length of each section (mm) was measured with the same tool, and the

average length was multiplied by the section’s thickness (mm) to give the measured

surface area (mm2). Finally, the average number of Meissner corpuscles (N) was

divided by the surface area (mm2) to calculate the density of Meissner corpuscles per

squared millimetre of skin (number of Meissner’s corpuscles/mm2). Afterward, the

average density for each pre-established age group was calculated from the individual

densities. To determine the density of digital Merkel cells, we used the same Merkel

cell immunostaining method for CK20. Significant differences among the three pre-

established age groups were assessed with the Kruskal-Wallis H test, and p-values

<0.05 were considered statistically significant (marked in the figures as *p<0.05,

**p<0.01).

RESULTS

Ageing of digital Meissner’s corpuscles

Meissner’s corpuscles were identified at all ages investigated (Figs. 1-3), but

differences in the morphology, placement within the dermis, intensity of

immunostaining for the evaluated markers, size and number were noted between the

three pre-established age groups. In the age group of 20-39 years, Meissner’s corpuscles

were elongated and always localized in the apex of the dermal papillae. The lamellar

cells were packed, arranged in parallel and displayed strong S100 protein

immunoreactivity (Figs. 1a, 2b and d); within the corpuscle, the axon showed a tortuous

trajectory (Fig. 2a and c). By 40-59 years, the predominant localization of Meissner’s

corpuscles was also within the dermal papillae, but some were also highly displaced

(Fig. 1b); the corpuscles showed no reduction in size, and the architecture and

immunohistochemical profile of the axon and lamellar cells did not vary (Fig. 2e-h).

The scenario changed dramatically in the older subjects (Fig. 1c). In this group, the size

of the Meissner’s corpuscles was reduced, the morphology was rounded (only a few

showed a typical elongated morphology), and most were localized behind rete pegs

instead of inside the dermal papillae. In those corpuscles, the lamellar cells showed

reduced immunostaining for S100 protein, and the axon was sometimes

undistinguishable (Figs. 2i-l and 3). This picture applied to approximately 70% of the

corpuscles, whereas the 30% remaining had a size, morphology and localization similar

to that of the younger subjects.

All these findings were confirmed by confocal microscopy of the Meissner’s

corpuscles of the three age groups, which showed that ageing induced changes in the

size, morphology and immunohistochemical profile of these structures (Fig. 4). Of

particular interest was the absence of axon profiles in a large percentage of Meissner’s

corpuscles from older subjects (Fig. 4i).

In addition to the above-described qualitative changes, ageing is accompanied

by a progressive reduction in the number of Meissner’s corpuscles. Significant

differences were observed between age groups, especially between young subjects vs.

adult and old subjects (Fig. 1).

Ageing of digital Pacinian corpuscles

No evident noticeable variations in the Pacinian corpuscles were found between

the three established age groups (Fig. 5). The arrangement of all corpuscular

components was almost identical, and no age-related changes were found in the number

of concentric lamellae forming the outer core and the capsule. The axon displayed NFP

immunoreactivity (Figs. 5a, c, e, g), whereas the lamellae of the inner core were positive

for S100 protein (Figs. 5b, d, f, h, j). Nevertheless, in some Pacinian corpuscles of older

subjects (approximately 15-25%), no immunoreactivity for NFP was detected, and S100

protein immunoreactivity was restricted to the outer lamellae of the inner core (Figs. 5i-

l). Additionally, in some other corpuscles, the neural compartment of the corpuscles

(i.e., the axon and the inner core) was disarranged compared with the typical

arrangement of these structures (Fig. 6).

Ageing of cutaneous Merkel’s cells

Digital Merkel cells are epidermal cells primarily localized in the basal strata

that selectively express the intermediate filament CK20. Using the expression of this

protein as a marker, Merkel’s cells were found in the epidermal rete pegs of all subjects,

and the number of cells progressively decreased with age (Fig. 7). The quantitative

analysis showed significant differences between the young subjects and the adult and

old subjects (graph in Fig. 7).

A graphic summary of the age-related changes in Meissner’s corpuscles and

Merkel’s cells described above is presented in Fig. 8.

Ageing of the BDNF-TrkB system in cutaneous mechanoreceptors

The development and maintenance of Meissner’s corpuscles and, to a lesser

extent, Merkel’s cells and Merkel’s cell-axon complexes are under the control of the

BDNF-TrkB neurotrophin system. Thus, we investigated whether the components of

that system are present in those sensory structures and whether these components

undergo age-dependent variations. In human digital Meissner’s corpuscles from adult

subjects, immunoreactivities for BDNF and TrkB were detected in the lamellar cells

(Supplementary Fig. 1a-c) and the axon (Supplementary Fig. 1d-f), respectively. In the

Pacinian corpuscles, BDNF was found in the inner core cells (Supplementary Fig. 2),

and TrkB was found in the axons (data not shown). Regarding Merkel’s cells, BDNF

was undetectable in these cells (Supplementary Fig. 3a-c), while TrkB

immunoreactivity regularly occurred in their cytoplasms (Supplementary Fig. 3d-f).

The pattern of localization of both BDNF and TrkB with age was previously

described. However, in younger subjects, BDNF immunoreactivity also showed

localization in axons (Fig. 9a). With ageing, the intensity of immunostaining for BDNF

(Figs. 9b-d) and TrkB (Figs. 9e-h) progressively decreased, and TrkB immunoreactivity

was undetectable in older subjects (Fig. 9h). These results applied to approximately

85% of Meissner’s corpuscles, and no apparent age-dependent changes were observed

in the remaining corpuscles (15%). The percentage of TrkB-positive Merkel cells also

declined with age, with a reduction of approximately 60% in the old age group

compared with the young and adult age groups (data not shown).

Piezo2 levels in Meissner’s corpuscles and Merkel’s cells decrease with age

The occurrence of Piezo2 immunoreactivity in the axons of human cutaneous

Meissner’s corpuscles and the cytoplasm of Merkel’s cells has been demonstrated

recently by our research group (García-Mesa et al., 2017). Here, we observed that the

axon of most of the Meissner’s corpuscles of the younger subjects displayed Piezo2

positivity (Figs. 10a and b), which remained unchanged in the middle-age group and

was markedly reduced in the older subject age group (Fig. 10d and e). No Piezo2

immunostaining was noted in the lamellar cells. In agreement with the above data

regarding the age-dependent decrease in the density of Merkel’s cells, there was an age-

dependent decrease in the density of Piezo2-positive Merkel’s cells (Fig. 10c and f). On

the other hand, although no direct contacts between Merkel’s cells and axons were

observed, the density of axons in the vicinity or apparently contacting Merkel’s cell also

decreased with age (Fig. 11).

DISCUSSION

In human skin, a series of sensory formations, collectively known as sensory

corpuscles (Zimmerman et al., 2014), are able to detect different qualities of

somatosensation. In particular, Merkel’s cell-neurite complexes, Meissner’s corpuscles

and Pacinian corpuscles discriminate unique aspects of touch with SAI and SAII

mechanoreceptors (Jones & Smith, 2014; Zimmerman et al., 2014). Ageing is

accompanied by a progressive decrease in the somatosensory system that affects the

quality of life of elderly subjects (Skedung et al., 2018). This deterioration presumably

involves all levels of the somatosensory pathways from the skin to the cerebral cortex,

including the cutaneous receptors in which the mechanical input is transduced into

electrical energy. The present study was designed to investigate the age-dependent

changes in some types of cutaneous mechanoreceptors and in one mechanoprotein

directly involved in mechanotransduction (Piezo2), as well as the variations in a

neurotrophic system (BDNF-TrkB) that is essential for their development and

maintenance.

The effects of ageing on touch have been reviewed in detail by Wickremaratchi

and Llewelyn (2006), Decorps et al. (2014), and Heft and Robinson (2017). Ageing is

also associated with a progressive decline in cutaneous thermic (Guergova & Dufour,

2011) and mechanical (Wickremaratchi & Llewelyn, 2006; Wu et al., 2011) perception,

as well as a marked degradation of tactile spatial acuity (Kalisch et al., 2009). As a

whole, these authors affirm that tactile perceptual ability as well as spatial acucity

decrease with age, and this impairment could be due at least in part to a reduction in the

density and distribution of Meissner’s and Pacinian corpuscles (Wickremaratchi &

Llewelyn, 2006), as well as Merkel’s disc (Lumpkin et al., 2003).

Age-dependent changes in cutaneous Meissner’s and Pacinian corpuscles

Classic studies have demonstrated that Pacinian and Meissner’s corpuscles

decrease in number and undergo structural deterioration upon ageing in humans (Bolton

et al., 1966; Kennedy et al., 2011), monkey (Paré et al., 2007) and mice (Mathewson &

Nava, 1985). Furthermore, deterioration of the discs of Merkel was also observed with

ageing in rats (Fundin et al., 1997), but deterioration was not observed in Merkel’s cell-

neurite complexes (Bolton et al., 1966; Mathewson & Nava, 1985; Paré et al., 2007).

Studies in humans are limited and do not use specific immunohistochemistry

assays for each corpuscular component. Here, we observed that the size, morphology

and structure of Meissner’s corpuscles remained essentially unaltered until 60 years or

age, and these corpuscles then progressively shrank, changed morphology and

topographical localization, and lost their immunohistochemical profile. Interestingly,

the absence of immunoreactivity for axonal markers in older subjects suggests

denervation of those corpuscles, which is supported by the decrease in the expression of

S100 protein by the lamellar cells. In fact, it is well known that lamellar cells of rat pad

Meissner-like corpuscles lack S100 protein (del Valle et al., 1993), with a similar result

in humans (Marquez et al., 1997; Albuerne et al., 1998). Globally, our results are in

good agreement with the data reported by Nava and Mathewson (1996) in Meissner-like

corpuscles of murine forepaw digital pads. These authors observed that in mice aged to

their maximum life expectancy, Meissner-like corpuscles decrease and become

disorganized and lobulated compared with those in middle-age animals, and these

changes are attributed to distal axonopathy and atrophy of the sensory neurons. On the

other hand, these changes in Meissner’s corpuscles affect most but not all the

corpuscles, suggesting that tactile texture discrimination remains partially intact with

age, irrespective of cutaneous condition. The findings observed here are difficult to

apply to the glabrous skin of the entire body. Presumably, differences in ageing exist

between different anatomical zones depending on the environment. In addition,

decreased epidermal innervation of facial skin was found, epidermal innervation of

abdominal skin did not change with age, and an age-associated increase in innervation

was observed in mammary skin (Besné et al., 2002). A recent study by Skedung et al.

(2018) evaluated the density of Meissner’s corpuscles in two groups of aged subjects

with high and low performance, showing that the number was reduced approximately

50% in the subjects with lower performance. Thus, the lower performance of this group

can be confidently linked to a neural decline, although we stress that we do not link the

reduced acuity directly to the lower Meissner’s corpuscle density because there are no

data on the other tactile receptors. Thus, the age-related decline in neural properties is

the primary explanation for the reduction in active touch acuity. This reduction results

in an age-related change in of hand movement perception (Landelle et al., 2018)

Regarding Pacinian corpuscles, the effects of ageing were not evident either in

the number or in the structure. However, a small population of Pacinian corpuscles from

the older subjects seemed to be denervated (no labelling of the axon and absence of

S100 protein in the whole inner core) or the neural compartment was disarranged.

Pacinian corpuscles have been related to the detection of vibration (Bell et al., 1994;

Zelena, 1994; Zimmerman et al., 2014), and these variations might be correlated with

the decreased vibration sensitivity reported in elderly people (see Shaffer & Harrison,

2007; Landelle et al., 2018).

The exhaustive review on the Pacinian corpuscles carried out by Zelena (1994)

in the book “Nerves and Mechanoreceptors” does not mention the age-dependent

changes in Pacinian corpuscles in humans or other mammalian species.

Age-related changes in the expression of basic corpuscular antigens

We observed that in some Meissner’s corpuscles from the aged subjects, there

was a reduction in the immunoreactivity for S100 protein along with an absence of

immunohistochemically detectable axons. These two findings suggest denervation of

these corpuscles because the expression of S100 protein by lamellar cells depends on

the functional integrity of the axon (Marquez et al., 1997; Albuerne et al., 1998).

Age-dependent changes in Merkel’s cells and Merkel’s cell-neurite complexes

In aged mice, we found a reduction in the number of palatal Merkel cells

number at key time-points that correlate with impaired oral abilities, such as swallowing

and mastication. Collectively, this work identified the mechanosensory architecture of

oral tissues involved in feeding (Moayedi et al., 2018). A study carried out in the

Meissner-like corpuscles of the murine palatine mucosa showed that only rudiments of

corpuscles were encountered due to an atrophy of the axon and lamellae (Iida &

Tachibana, 1996). Recently, Moayedi et al. (2018) reported that Merkel cells undergo a

dramatic reduction in density with ageing in murine palatine mucosa, whereas the

authors did not examine variations in Meissner’s corpuscles.

Piezo2 expression is reduced in aged cutaneous mechanoreceptors

Activation of mechanically gated ion channels is at the origin of the detection of

low- or high-threshold mechanical stimuli. Different candidates have been proposed to

be the mechanotransducers in human sensory corpuscles and Merkel’s cells (Calavia et

al., 2010b; Cabo et al., 2015b; Alonso-González et al., 2017). However, only Piezo2 has

been shown to be an essential component of distinct stretch-activated ion channels

involved in the mechanotransduction (see Roudaut et al., 2012; Delmas & Coste, 2013;

Wu et al., 2017. Piezo2 is present in mechanosensory neurons (Coste et al., 2010;

Ranade et al., 2014) and different low-threshold cutaneous mechanoreceptors, including

human Meissner’s corpuscles and Merkel’s cells (García-Mesa et al., 2017). Although

the ageing effects on the structure and/or the function of these mechanosensitive ion

channels have not been described, one can speculate that these channels could

contribute to age-related tactile defects.

The BDNF-TrkB neurotrophin complex is altered in the skin of aged subjects

The development, survival and maintenance of cutaneous sensory corpuscles are

under the control of different neurotrophins and their Trk signalling receptors (see

Montano et al., 2010). The BDNF-TrkB system is involved in different modalities of

touch because it controls Meissner’s corpuscles and, to a lesser extent, Pacinian

corpuscles and Merkel’s cell-neurite complexes (González-Martınez et al., 2004; Sedy

et al., 2004; de Carlos et al., 2006; Perez-Pinera et al., 2008). The BDNF-TrkB system

over sensory corpuscles is controlled by retrograde axonal transport in the sensory

neurons supplying these components or directly by non-neuronal cells surrounding the

peripheral tip of the sensory axon.

BDNF-TrkB continues to be expressed in some neurons of adult sensory ganglia

and in some types of sensory corpuscles throughout the whole lifespan (see Montano et

al., 2010). The present and previous studies (Calavia et al., 2010a) demonstrate that

human cutaneous sensory corpuscles contain immunohistochemically detectable BDNF

and TrkB. Our results strongly suggest that in Meissner’s and Pacinian corpuscles,

retrograde trophic signalling occurs to provide trophic support to the sensory neurons

supplying these components: the lamellar cells and the inner core cells seem to be the

source of BDNF, and the axons that express TrkB are the recipients.

As far as we know, the effects of ageing in this neurotrophic system have never

been investigated. Our results demonstrate that the ageing of sensory corpuscles is

accompanied by a decrease in the immunohistochemically detectable BDNF and TrkB,

which were absent in some Meissner’s corpuscles. Whether the absence of BDNF-TrkB

is responsible, at least in part, for the size and morphological changes observed in

Meissner’s corpuscles and in the density of Merkel cells remains to be investigated. In

any case, our results could explain the reduction in sensory neurons of the dorsal root

ganglia that occurs with ageing (Vega et al., 1993). In the human central nervous

system, low levels of BDNF and reduced expression of TrkB are related to ageing and

the related diseases (Erickson et al., 2010; Forlenza et al., 2015; Nunes et al., 2018).

Overall, the present study demonstrates that the cutaneous Meissner’s corpuscles

and Merkel cell-neurite complexes and, less evidently, Pacinian corpuscles, which

represent the most peripheral part of the sensory nervous system, undergo age-

dependent morphological and size changes as well as a decrease in their density. The

mechanoprotein Piezo2 involved in fine touch is also reduced in aged corpuscles, as is

the trophic system responsible for their maintenance. These changes partially explain

the impairment of the somatosensory with ageing.

ACKNOWLEDGEMENTS

This study was supported in part by a grant from Gerencia Regional de Salud de Castilla

y León (GRS 1615/A/17). The authors thank Dr. Marta Guervos (Servicios Comunes de

Investigación, Microscopia Confocal, Universidad de Oviedo) and Marta Sánchez-Pitiot

(Grupo de Histopatología Molecular, Instituto Universitario de Oncología del

Principado de Asturias) for technical assistance.

AUTHOR CONTRIBUTIONS

J.G-P, Y.G-M and I.TP performed the experiments

O.G-S and JAV designed the stdy abd analysed the data

JF, JC and BM-B collected the material under legal conditions and performed part of

the experiments

J.G-P and JAV wrote the manuscript

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SUPPLEMENTARY MATERIAL

Supplementary Fig. 1. Double immunofluorescence for BDNF (a) and TrkB (d) (green

fluorescence) and S100 protein (red fluorescence) in human digital Meissner’s

corpuscles of one 30-year-old subject. BDNF and S100 protein were colocalized,

suggesting that BDNF was present in the lamellar cells. Conversely, no colocalization

of TrkB and S100 protein was detected, indicating that TrkB is mainly located in axons.

Objective 63×/1.40 oil; pinhole 1.37; XY resolution 139.4 nm and Z resolution 235.8

nm. Scale bar: 30 µm.

Supplementary Fig. 2. Double immunofluorescence for BDNF (green fluorescence)

and NFP (b) and S100 protein (e) (red fluorescence) in human digital Meissner’s

corpuscles of one 30-year-old subject. BDNF and S100 protein were colocalized,

suggesting that BDNF is present in inner core cells. Conversely, no colocalization of

BDNF and NFP was detected, indicating that BDNF is not present in the axon.

Objective 63×/1.40 oil; pinhole 1.37; XY resolution 139.4 nm and Z resolution 235.8

nm. ic: inner core; arrow indicates the axon. Scale bar: 50 µm.

Supplementary Fig. 3. Double immunofluorescence for BDNF (a) and TrkB (d) (green

fluorescence) and CK20 (red fluorescence) in human digital Merkel cells (arrows).

BDNF and CK20 never colocalized, whereas TrkB and CK20 colocalized in the

cytoplasm of Merkel cells. Objective 63×/1.40 oil; pinhole 1.37; XY resolution 139.4

nm and Z resolution 235.8 nm. ic: inner core; arrow indicates the axon. Scale bar: 30

µm.

TABLES

Table 1. Data about the subjects and materials.

Case Age in years Gender M/F Hand/finger

1 23 M L/1/D

2 30 M L/1/D

3 35 F L/1/D

4 37 M L/2/D

5 38 F R/1/D

6 40 M L/1/D

7 42 M L/1/D

8 46 M R/2/D

9 47 M L/1/D

10 55 F R/1/D

11 58 F L/1/D

12 58 M L/2/D

13 63 F R/2/D

14 66 M L/1/D

16 69 M L/1/D

16 72 M R/1/D

17 74 M L/1/D

18 81 F L/1/D

19 85 M L/2/D

20 88 M L/1/D

21 90 M L/1/D

F: female, M: male, L: left, R: right, 1 or 2: first or second fingers, respectively, D:

distal phalanx

Table 2. Primary antibodies used in the study.

Antigen (clone) Origin Dilution Supplier Catalogue number

S100P Rabbit 1:1000 DAKO 1 Z 0311

P200 kDa NF (RT-97) Mouse 1:1000 Boehringer-Mannheim2 1178709

PGP 9.5 Rabbit 1:300 Abcam3 ab10404

Cytokeratin 20 Mouse prediluted Leica 4 PA0022

Piezo2 Rabbit 1:500 Sigma Aldrich5 HPA040616

BDNF Rabbit 1:200 Chemicon Int6 AB1534SP

TrkB Rabbit 1:200 Santa Cruz Biotechnology7 SC12

1Glostrup, Denmark; 2Mannheim, Germany; 3Hamburg, Germany; 4Newcastke, UK;

5Saint Louis, MO, USA; 6Temecula, CA, USA; 7Santa Cruz, CA, USA.

BDNF: brain-derived neurotrophic factor; NF: neurofilament; NSE: neuron-specific

enolase.

The antibody against BDNF is directed against the sequence H2N-HSDPARRGEL-

COOH (manufacturer’s notice); the antibody anti-TrkB was directed against the

residues 794-808 of the intracytoplasmic domain of human TrkB (manufacturer’s

notice); the amino acid sequence recognized by the antibody against Piezo2 is

FEDENKAAVRIMAGDNVEICMNLDAASFSQHNP (manufacturer’s information).

FIGURE LEGENDS

Fig. 1. Sections of glabrous digital skin from 3 subjects with ages of 23 (a), 55 (b) and

88 (c) years; immunostained was performed to evaluate S100 protein (S100P). Nerves

and Meissner’s corpuscles were immunolabelled (arrows). e: epidermis. Scale bar: 100

µm. The density of Meissner corpuscles in the three pre-established age ranges is shown

in the graph. A significant reduction in the density of Meissner’s corpuscles was

observed with ageing. *p<0.05, **p<0.01.

Fig. 2. Immunohistochemical localization of neurofilament protein (NFP) and S100

protein (S100P) in the axon and lamellar cells, respectively, of human digital

Meissner’s corpuscles of subjects of different ages: 23 (a, b), 30 (c, d), 40 (e, f), 55 (g,

h), 85 (I, j) and 90 (k, l) years. The localization, size, morphology and arrangement of

corpuscular constituents varied with ageing. Moreover, the Meissner’s corpuscles from

old subjects showed a marked decrease in the intensity of the immunoreaction for

S100P, and in some of these corpuscles, it was impossible to identify the axon. e:

epidermis. Scale bar: 60 µm.

Fig. 3. Digital Meissner’s corpuscles from three subjects with ages of 85 (a-d), 88 (e-h)

and 90 (i-l) years. Meissner’s corpuscles were localized in the dermal papillae, but most

were found in the reticular dermis. Those found at this location were smaller in the old

subjects than in the adults, showed a rounded morphology and lacked the typical

immunohistochemical profile. e: epidermis. Scale bar: 60 µm.

Fig. 4. Double immunofluorescence for S100 protein (green fluorescence) and

neurofilament protein (red fluorescence) in human digital Meisner’s corpuscles of

subjects with ages of 23 (a-c), 58 (d-f) and 90 (g-i) years. Sections were counterstained

with DAPI to ascertain structural details. Objective 63×/1.40 oil; pinhole 1.37; XY

resolution 139.4 nm and Z resolution 235.8 nm. Scale bar: 20 µm.

Fig. 5. Immunohistochemical localization of neurofilament protein (NFP) and S100

protein (S100P) in the axon and the inner core cells, respectively, of human digital

Pacinian corpuscles of subjects with ages of 23 (a-d), 55 (e-h), and 88 (i-l) years. The

axon supplying the corpuscles was identifiable in all corpuscles of 23- and 55-year-old

subjects and was not identified in the 88-year-old subject. The inner core cells displayed

S100P immunoreactivity in all lamellae in the 23- and 55-year-old subjects, whereas in

the older subjects, S100P immunoreactivity was restricted to the outer core lamellae.

Scale bar: 50 µm.

Fig. 6. Double immunofluorescence for S100 protein (green fluorescence) and

neurofilament protein (red fluorescence, arrows in b and e) in human digital Pacinian

corpuscles of subjects with ages of 23 (a-c) and 88 (d-f) years. Sections were

counterstained with DAPI to ascertain structural details. The axon and the inner core of

the 88-year-old subject are disarranged, although the corpuscle retained the general

organization. Objective 63×/1.40 oil; pinhole 1.37; XY resolution 139.4 nm and Z

resolution 235.8 nm. Scale bar: 50 µm.

Fig. 7. Immunohistochemical characterization of CK20 in human digital cells, identified

based on their localization as Merkel’s cells, in subjects of different ages: 23 (a), 30 (b),

55 (c), 72 (d), 88 (e) and 90 (f) years. Scale bar: 50 µm. The density of Merkel’s cells in

the three pre-established age ranges is shown in the graph. A significant reduction in the

density of Meissner’s corpuscles was observed with ageing. *p<0.05, **p<0.01.

Fig. 8. Schematic representation of Meissner’s corpuscles (MC) and Merkel’s cell-

neurite complexes (McNc) in the human digital glabrous skin of young-adult (top) and

old (bottom) subjects. Ageing results in a reduction in the density of both types of

mechanoreceptors as well as changes in the placement, size, morphology and

immunohistochemical profile of Meissner’s corpuscles. e: epidermis.

Fig. 9. Immunohistochemical detection of brain-derived neurotrophic factor (BDNF)

and its signalling receptor TrkB in Meissner’s corpuscles of subjects with ages of 23 (a,

e), 55 (b, f), 88 (c, g) and 90 (d, h) years. Scale bar: 60 µm.

Fig. 10. Sections of glabrous digital skin from 2 subjects with ages of 30 (a) and 88 (d)

years immunostained to detect Piezo2. The axon of most Meissner’s corpuscles

displayed Piezo2 immunoreactivity in young individuals (arrows in a, b), whereas this

immunoreactivity was restricted to segments of the corpuscle, or even absent in the

older subjects (e). The density of Piezo2-positive Merkel cells in young subjects (c) was

higher than that in the old subjects (f, arrow). e: epidermis. Scale bar: 100 µm a and d;

20 µm b-c and e-f.

Fig. 11. Double immunofluorescence for neuron-specific enolase (green fluorescence a

and b), neurofilament proteins (green fluorescence in c-f; red fluorescence in g),

cytokeratin 20 (red fluorescence in a-f), and Piezo2 (green fluorescence in g) in human

Merkel’s cell-neurite complexes of subjects with ages of 30 (a-c), and 88 (d-f) years.

Axonal profiles are indicated by arrows. e: epidermis. Objective 63×/1.40 oil; pinhole

1.37; XY resolution 139.4 nm and Z resolution 235.8 nm. Scale bar: 30 µm a,c,d,e and

f; 20 µm b and g.

6. DISCUSIÓN

6.1. Consideraciones generales

El presente trabajo de tesis doctoral se enmarca en una de las líneas de investigación

que, durante los últimos 35 años, desarrolla el grupo SINPOS (Cluster de Biomedicina,

Campus de Excelencia internacional de la Universidad de Oviedo) en el Área de

Anatomía y Embriología Humana del Departamento de Morfología y Biología Celular

de la Universidad de Oviedo, y que se dedica al estudio de los corpúsculos sensitivos

los cutáneos. La mayor parte de las contribuciones de esta línea de investigación se

recogen en tres revisiones publicadas por J.A. Vega y colaboradores en 1996, 2009 y

2012.

La supervivencia de un organismo depende en gran medida de su capacidad de

respuesta y adaptación al medio ambiente que lo rodea y una de las formas de

interactuar con él es a través del tacto. Los mamíferos han desarrollado estructuras

sensitivas especializadas que les permiten detectar tacto fino, vibraciones y presión

(Zelena, 1994; Munger e Idé, 1998; Johnson, 2001). En conjunto, estas formaciones

nerviosas se denominan corpúsculos sensitivos y funcionalmente son

mecanorreceptores.

Los corpúsculos de Meissner y de Pacini son dos tipos de formaciones nerviosas

sensitivas que, aunque son diferentes en estructura y organización celular, se

comportan fisiológicamente de forma casi idéntica, ya que ambos son receptores de

bajo umbral y adaptación rápida y dependen de fibras nerviosas sensitivas Aα o Aβ

(Johnson, 2001). En cambio, los complejos célula de Merkel-neurita son estructuras

mecanorreceptoras formadas por una asociación sinapsis-like entre axones de

neuronas sensitivas Aβ y células epiteliales especializadas (Fleming y Lou, 2013). A

pesar del conocimiento sobre las propiedades y características neurofisiológicas, no ha

sido hasta estos últimos años cuando se han empezado a identificar los mecanismos

moleculares y celulares mediante los cuales un estímulo mecánico se convierte en una

señal eléctrica. Detrás de esta transformación mecanoeléctrica se encuentran canales

iónicos que se expresan en las células de los mecanorreceptores y que son capaces de

activarse mediante factores mecánicos (Del Valle y Cols., 2012; Kuipers y Cols., 2012;

Zimmerman y Cols., 2014).

La estructura celular y la composición proteica de los mecanorreceptores sensoriales

están bien definidas y caracterizadas (Vega y Cols., 1996; 2009; Pawson y Cols., 2000).

No obstante, el conocimiento sobre algunos aspectos como el origen celular, la matriz

extracelular y los cambios edad-dependientes de los corpúsculos sensitivos resultan

incompletos o son totalmente desconocidos a día de hoy.

Con el presente trabajo de investigación, se aportan nuevos datos sobre la naturaleza

de las células que componen el núcleo externo de los corpúsculos de Pacini, sobre los

proteoglicanos y glicosaminoglicanos de la matriz extracelular de los corpúsculos

sensitivos y sobre las variaciones cuantitativas, cualitativas y morfológicas que cursan

los mecanorreceptores sensoriales en función de la edad.

6.2. Las células CD34-positivas definen una lámina intermedia en los

corpúsculos de Pacini digitales humanos

En el primer trabajo de esta tesis, se ha analizado el origen celular del núcleo externo

de los corpúsculos de Pacini de la piel digital glabra humana. Para ello se han tenido en

cuenta las siguientes premisas: 1) las vainas endoneurales o perineurales de los nervios

periféricos son continuas con las capas periaxónicas de los corpúsculos de Pacini

(Malinovsky, 1986); 2) el endoneuro de los troncos de los nervios periféricos expresa el

antígeno CD34 (Weiss et al., 1993; Khalifa et al., 2000; Hirose et al., 2003; Richard et

al., 2014); 3) los antígenos Glut1 y EMA se expresan en el núcleo externo y en la

cápsula de los corpúsculos de Pacini (Vega y Cols., 2009; Feito y Cols., 2016).

Diversos estudios realizados en corpúsculos de Pacini de diferentes especies animales

han constatado mediante inmunohistoquímica y, sobre todo, mediante microscopía

electrónica de transmisión, la existencia de una lámina intermedia en los corpúsculos

de Pacini. González-Martínez y Cols. (2007) describieron una lámina Erb4-positiva en el

exterior del núcleo interno de corpúsculos de Pacini de ratón; una lámina intermedia o

de crecimiento ha sido caracterizada al detalle con microscopía electrónica en varias

ocasiones en corpúsculos de Pacini del mesenterio del gato (Pease y Quilliam, 1957;

Munger y Cols., 1988) y de la pata delantera de perro (Rico y Cols., 1996); Chouchkov

(1978) describió un espacio subcapsular estrecho con fibroblastos y macrófagos

dispersos y fibrillas de colágeno alrededor del núcleo interno de corpúsculos de Pacini

del mesenterio de gato; un patrón especial compuesto por lectinas, proteoglicanos y

glicosaminoglicanos fue también descrito en la zona equivalente a la lámina

intermedia en corpúsculos de Pacini del peritoneo de gato (Pawson y Cols., 2000;

Sames y Cols., 2001); una zona inmunorreactiva para proteína quinasa C fue

identificada con la lámina intermedia en corpúsculos de Pacini de las patas traseras de

la rata (Kawakita y Cols., 1992).

Sin embargo, en corpúsculos de Pacini humanos, esta lámina intermedia nunca había

sido identificada como tal. Tan sólo fue sugerida en un primer momento en base a la

presencia de algunos antígenos específicos (como la vimentina) entre el núcleo interno

y el núcleo externo (García-Suárez y Cols., 2010).

La lámina intermedia CD34-positiva identificada y descrita en este trabajo se asemeja a

la lámina intermedia o de crecimiento del corpúsculo de Pacini del mesenterio de gato

y con las células del espacio subcapsular estrecho (Chouchkov, 1978).

Estructuralmente divide el corpúsculo de Pacini en dos compartimentos: el

compartimento interno neural y avascular (axón y células de Schwann-modificadas) y

el compartimento externo no neural y vascular (células perineurales y epineurales,

vasos sanguíneos y macrófagos). Aunque su función queda por concretar, podría estar

relacionada con el potencial crecimiento del corpúsculo de Pacini (Zelena, 1994).

Además, las células que expresan CD34 tienen capacidades potenciales de

diferenciación y proliferación (Sidney y Cols., 2014). CD34 es una glicoproteína de

membrana que actúa principalmente como molécula de adhesión (Silva y Cols., 2010),

conocida principalmente por expresarse en las células madre hematopoyéticas, pero

también en células mesenquimales potencialmente diferenciables (Sidney et al., 2014).

Por otro lado, se puede hipotetizar que CD34 interacciona con la membrana basal de

lamela más externa del núcleo interno o con otros componentes de la matriz

extracelular (Dubový y Brednarova, 1999), aunque se necesitan más estudios sobre la

composición de la matriz extracelular de los corpúsculos de Pacini, ya que se

desconoce casi por completo.

Por otro lado, en base al diámetro de las fibras de colágeno del espacio extracelular,

algunos autores han sugerido que la lámina intermedia de los corpúsculos de Pacini de

felinos es de origen endoneural (Osawa e Ide, 1986), así como en los de rata, según la

expresión de la proteína quinasa C beta (Kawakita et al., 1992). Los resultados

obtenidos en este trabajo demuestran el origen endoneural de la lámina intermedia ya

que se corresponde con la expresión de CD34 en las células endoneurales de los

troncos nerviosos. Además, siguiendo la continuidad anatómica entre los nervios

periféricos y los corpúsculos sensitivos (Malinovsky, 1986) y sus perfiles

inmunohistoquímicos comunes (Vega y Cols., 1996, 2009), se establece una relación

entre el perineuro y el núcleo externo de los corpúsculos de Pacini en base a la

expresión de los antígenos EMA y Glut1 (Vega y Cols., 2009; Feito y Cols., 2016).

6.3. Condroitín sulfato en los corpúsculos sensitivos de Meissner y de

Pacini humanos

En el segundo trabajo que conforma el cuerpo de la tesis, se ha estudiado la expresión

de condroitín sulfato en los corpúsculos sensitivos de piel glabra digital humana.

Las moléculas que conforman la matriz extracelular se clasifican en dos categorías:

proteinas fibrosas (colágenos, elastina, laminina, fibronectina, etc.) y proteoglicanos

(entre ellos, los formados por condroitín sulfato). Dubovy y Bednárová (1999)

demostraron que el núcleo interno y la capsula de los corpúsculos de Pacini de rata

tienen diferentes patrones estructurales de proteoglicanos y que, probablemente,

estén implicados en diferentes funciones.

En esta investigación se ha determinado que los corpúsculos de Pacini humanos

expresan una lámina de condroitín sulfato dispuesta entre el núcleo interno y el núcleo

externo, la cual es similar a la descrita en los corpúsculos de Pacini del mesenterio de

gato (Sames y Cols., 2001).

Además, se ha demostrado que esta lámina de condroitín sulfato está asociada a la

lámina CD34 positiva descrita en el apartado anterior (García-Piqueras y Cols., 2017).

Ocurre lo mismo en el caso de los nervios periféricos de los vertebrados, en los que los

proteoglicanos de condroitín sulfato se asocian a las células endoneurales (CD34-

positivas; García-Piqueras y Cols., 2017) pero no a las epineurales (Graham y Muir,

2016). Este resultado es una evidencia más que avala la continuidad de los corpúsculos

de Pacini con los nervios periféricos y, al mismo tiempo, que están formados por los

mismos elementos (axón, células de Schwann, endoneuro, perineuro y

epineuro/cápsula).

Otro resultado relevante es la no expresión de condroitín sulfato y CD34 en los

corpúsculos de Meissner. La ausencia de elementos perineurales (cápsula) en los

corpúsculos de Meissner puede estar relacionada con la ausencia de elementos

endoneurales (objeto de investigación). El endoneuro actúa probablemente como zona

de crecimiento a partir de la cual se genera el perineuro, lo cual puede ser importante

para los corpúsculos de Pacini pero no para los de Meissner.

A pesar de que la función de los proteoglicanos de condroitín sulfato en los nervios

periféricos ha sido determinada y es bastante bien conocida (Graham y Muir, 2016), su

papel en los corpúsculos sensitivos es todavía dudoso. Los proteoglicanos de

condroitín sulfato pueden estar relacionados con la separación del segmento neural

(axón y núcleo interno) del no neural (núcleo externo y cápsula) del corpúsculo de

Pacini, pero también con la mecanotransducción. Aunque los canales iónicos son los

actores principales de la mecanotransducción, se ha demostrado que en el proceso

también participan algunas moléculas de la matriz extracelular (Silver y Siperko, 2003;

Lumpkin y Caterina, 2007; Arnadottir y Chalfie, 2010; DuFort y Cols., 2011). En este

sentido, Matthews y Cols. (2006) afirman que el potencial de acción depende de

específicas deformaciones mecánicas de la membrana axonal y de interconexiones con

la matriz extracelular en terminales nerviosos. Se desconoce todavía qué moléculas de

dicha matriz pueden estar implicadas directamente en la mecanotransducción.

Respecto a la función de control del crecimiento axónico, los proteoglicanos de

condroitín sulfato son inhibidores del mismo, ya que su degradación enzimática

elimina la actividad inhibitoria (Muir, 2010; Graham y Muir, 2016; Man y Cols., 2014).

Se ha demostrado que aplicando condroitinasa in vivo, se puede mejorar el resultado

de la reparación nerviosa (Graham y Muir, 2016). La asociación de los proteoglicanos

de condroitín sulfato a las células endoneurales CD34-positivas en los corpúsculos de

Pacini probablemente regule el correcto crecimiento axónico dentro del

compartimento neural del corpúsculo, aunque no hay datos sobre ello. Sin embargo,

esto es respaldado parcialmente por el hecho de que los corpúsculos de Pacini

denervados cursan con reinervación poliaxonal (Zelená, 1984; Zelená y Zacharova,

1997; Zelená y Jirmanova, 1988, 1995). La reinervación poliaxonal se debe

probablemente a la lámina intermedia, en concreto a los proteoglicanos de condroitin

sulfato, que resultan alterados gravemente e incapaces de inhibir correctamente el

crecimiento axonal (sprouting) durante la reinervación. También, algunos de estos

axones reinervan el núcleo interno orginal (Zelená, 1994).

6.4. El heparán sulfato está asociado a la membrana basal de los

corpúsculos de Meissner y Pacini cutáneos humanos

El tercer artículo continúa con el estudio de otro de los glicosaminoglicanos, el heparán

sulfato, en los corpúsculos sensitivos cutáneos humanos.

En este estudio, se ha demostrado la expresión de heparán sulfato en los corpúsculos

de Meissner y de Pacini, asociada casi exclusivamente a proteoglicanos que forman

parte de la membrana basal con localización pericelular y que corresponden a

perlecano, agrina, colágeno XV y colágeno XVIII (Yurchenco y Patton, 2009; Iozzo y

Schaefer, 2015).

La membrana basal de los corpúsculos de Meissner y de Pacini ya ha sido estudiada

previamente mediante microscopía electrónica (Malinovsky y Pac, 1982; Malinovsky,

1986; Ide y Cols., 1987; Ide y Hayashi, 1987; Munger y Ide, 1988) y por métodos

inmunohistoquímicos (Vega y Cols., 1995). En el presente trabajo, se ha utilizado la

microscopía confocal, utilizando como base la presencia de colágeno IV (uno de los

componentes principales de las membranas basales junto a las lamininas; Vega y Cols.,

1995) para asociar la expresión de heparán sulfato a la membrana basal. En

consecuencia, la colocalización de heparán sulfato con colágeno IV se interpreta como

proteoglicanos de heparán sulfato pertenecientes a la membrana basal, mientras que

la no colocalización indica la existencia de otros proteoglicanos de heparan sulfato de

superficie celular (no pertenecientes a la membrana basal), como son las familias de

sindecanos o glipicanos (Iozzo y Schaefer, 2015).

En los corpúsculos de Meissner, los proteoglicanos de heparán sulfato se encuentran

asociados de la membrana basal, lo cual confirma estudios previos de nuestro

laboratorio realizados mediante la inmunodetección de colágeno IV y laminina (Vega y

Cols., 1995). Sin embargo, también se encontraron otros proteoglicanos de heparán

sulfato no asociados a la membrana basal.

En los corpúsculos de Pacini, los proteoglicanos de heparan sulfato se encuentran

asociados exclusivamente a la membrana basal. No obstante, la presencia de

membrana basal en todos los compartimentos del corpúsculo sigue siendo objeto de

debate. Mientras que algunos autores han descrito la membrana basal tanto en las

células del núcleo interno como en la lámina intermedia, bien con microscopía

electrónica (Malinovsky y Pac, 1982; Malinovsky, 1986), bien con inmunohistoquímica

para colágeno IV y laminina (Vega y Cols., 1995), otros afirman que dichos

compartimentos celulares carecen de membrana basal (Ide y Hayashi, 1987; Munger y

Ide, 1988). Los resultados de este trabajo apoyan esta segunda teoría, ya que no se ha

detectado ni heparan sulfato ni colágeno IV en estas dos localizaciones. A pesar de

ello, sí hay acuerdo sobre la existencia de membrana basal alrededor de las células del

núcleo externo y de la cápsula (Malinovsky, 1986; Ide y Cols., 1987; Munger y Ide,

1988). Esta investigación también confirma este hecho, a partir de la colocalización del

heparán sulfato con el colágeno IV en todas las lamelas del núcleo externo y la cápsula.

Pero el resultado más relevante es la detección una intensa inmunorreacción de

heparán sulfato en las lamelas más internas del núcleo externo. Este área coincide con

la denominada “zona de transición” (Vega y Cols., 1995), la cual se pensaba

previamente que era de origen perineural (Halata y Cols., 1990), pero que

recientemente se ha caracterizado como endoneural (García-Piqueras y Cols., 2017).

Por lo que a partir de la no colocalización de CD34 y HS, se puede concluir que dicha

lámina intermedia carece de membrana basal.

Por otra parte, es conocido que las células que forman los compartimentos del

corpúsculo de Pacini se comunican entre ellas por diferentes tipos de unión celular en

sus membranas: entre las células del núcleo interno existen uniones tipo “gap”,

mientras que en las células del núcleo externo hay uniones tipo “thigh”. Hide y Hayashi

(1987) describieron que estas uniones tipo “thigh” eran “especialmente prominentes

en la primera y segunda lamelas más internas del núcleo externo (a veces también en

la tercera)”. Estas primeras lamelas del núcleo externo con mayor número de uniones

tipo “thigh” coinciden con la intensa expresión de heparan sulfato y colágeno IV

observada en este trabajo. La especial organización de uniones tipo “thigh” presentes

en las primeras lamelas del núcleo externo guarda relación con el aislamiento del

núcleo interno respecto al externo, en modo tal de evitar pérdidas de iones y fluidos

entre ambos, y prevenir que una posible perturbación en el núcleo externo pueda

cambiar la composición eléctrica del núcleo interno (Ide y Hayashi, 1987; Munger e

Ide, 1988). Es por ello que, la membrana basal por sí misma y/o los proteoglicanos de

heparan sulfato inmunodetectados más intensamente en esta zona podrían participar

en dicho aislamiento del núcleo interno-axón (e incluso también de la lámina

intermedia) respecto al núcleo externo-cápsula. Además, se sabe que las moléculas de

la matriz extracelular de las células perineurales son capaces de regular la presión

endoneural (Piña-Oviedo y Ortiz-Hidalgo, 2008).

Diferentes investigaciones han determinado que algunos componentes de la

membrana basal, así como de la matriz extracelular en general, están implicados en el

proceso de la mecanotransducción (Wetzel y Cols., 2007; Hu y Chiang, 2010; Chiang y

Cols., 2011). Queda por establecer ahora si los proteoglicanos de heparan sulfato

también están implicados en esta función.

En resumen, se ha demostrado que los proteoglicanos de heparán sulfato están

vinculados fundamentalmente a la membrana basal en ambos tipos de corpúsculos y

que la ausencia de los mismos en el núcleo interno y la lámina intermedia de los

corpúsculos de Pacini sugiere que estos compartimentos carecen de membrana basal.

6.5. SLRPs

Otro los de los aspectos abordados en el presente trabajo de tesis doctoral en relación

con la matriz extracelular de los corpúsculos sensitivos ha sido la distribución en ellos

de los componentes de las familias I y II de los proteoglicanos denominados pequeños

y ricos en leucina (small leucine-rich proteoglycans, SLRPs). Al igual que el resto de los

componentes de la matriz extracelular su localización en los corpúsculos es,

necesariamente, en los espacios dispuestos entre las láminas que los forman.

La composición molecular de la matriz extracelular en los corpúsculos sensitivos es

relativamente poco conocida, y no existe casi ninguna información en humanos salvo

el trabajo de Vega y Cols. (1995). En la actualidad se conoce bastante bien la

distribución de los distintos tipos de colágeno en los espacios intercelulares de los

corpúsculos de Pacini: el colágeno de tipo II predomina en el núcleo externo y la

cápsula, el de tipo V en la capa intermedia, y el de tipo IV está presente en todos los

compartimentos (Vega y Cols., 1995; Pawson y Cols., 2000). También se conoce que en

algunas zonas de los corpúsculos la matriz extracelular se organiza para formar una

membrana basal (Malinosky y Cols., 1986; Vega y Cols., 1995; Dubovy y Aldskogius,

1996; García-Piqueras y Cols., 2018). Sin embargo, la distribución en los corpúsculos de

los otros componentes principales de la matriz extracelular, es decir, los

proteoglicanos (PGs; Iozzo y Schaefer, 2015; Theocharis y Cols., 2016), es poco

conocida. Sólo el trabajo de Sames y Cols. (2001) aporta datos muy parciales en los

corpúsculos de Pacini del mesenterio del gato.

El grupo de los SLRPs está formado por, al menos, 18 miembros subclasificados en las

clases de la I a la V. En concreto, las clases I y II que son las analizadas en este trabajo

incluyen al biglicano (BGN), decorina (DCN), asporina (ASPN), proteína 2 de la matriz

extracelular (ECM2), la proteína X de la matriz extracelular (ECMX), la fibromodulina

(FMOD), el lumicano (LUM), la prolargina (PRELP), el keratocano (KERA) y la

osteoadherina (OMD) (McEwan et al., 2006; Schaefer and Schaefer, 2010; Chen and

Birk, 2013).

Se ha detectado DCN en el núcleo externo y BGN en el núcleo interno; lo primero está

en consonancia con lo observado por Sames y Cols. (2001) en los corpúsculos de Pacini

del mesenterio del gato. La distribución del BGN difiere de la de estos autores ya que

ellos lo encuentran restringido al núcleo interno y en nuestro trabajo la hemos

detectado tanto en el interno como en el externo. La función de estas dos moléculas

en los corpúsculos de Pacini es desconocida en la actualidad. Es posible que DCN

participe en la estabilización de la vimentina del citoesqueleto (Jungmann y Cols.,

2012), que es el filamento intermedio de las células de ambos núcleos corpusculares

(ver Vega y Cols., 2009), pero ello tiene que ser confirmado en estudios posteriores.

Los datos que se aportan sobre la distribución del resto de los SLRPs estudiados son

completamente nuevos y, por tanto, no permite la comparación ni discusión con

estudios previos.

En cuanto al papel de los SLRPs en los corpúsculos de Pacini, todo lo que se puede

hacer en la actual situación, es hipotetizar. Algunos ensayos bioquímicos han

demostrado que los SLRPs modulan la organización de la matriz extracelular uniéndose

a los diferentes tipos de colágeno (Kalamajski and Oldberg, 2010; Chen and Birk 2013;

Gubbiotti et al., 2016). En concreto LUM se une al colágeno II, BGN y FMOD a los

colágenos II y VI, y DCN a los colágenos II, IV, V y VI (Chen and Birk, 2013). La

distribución de SLRPs observada en este trabajo y la previamente aportada por otros

estudios acerca de la localización corpuscular de los diferentes colágenos (Tachibana y

Cols., 1995; Vega y Cols., 1995; Pawson y Cols., 2000) apoya los datos bioquímicos.

Por otro lado, SLRPs regulan la actividad celular al interactuar con receptores para

diferentes factores de crecimiento (Merline y Cols., 2009; Dellet y Cols., 2012; Iozzo y

Schaefer 2015; Neill y Cols., 2015). DCN y BGN se unen al receptor del factor de

crecimiento epidérmico (EGFR) y a Erb4 (Schaefer y Iozzo, 2008; Schaefer y Schaefer,

2010; Jacob y Cs-Szabo, 2012) y la localización de estos SLRPs observada en los

corpúsculos de Pacini es similar a la del ERGR tanto en humanos (Vega et al., 1994)

como en ratón (Gonzalez-Martinez et al., 2007). Y se ha demostrado que la interacción

de DCN con EGFR regula la formación de colágeno (Mohan et al., 2011). Además, la

proporción relativa de colágenos y SLRPs determina las propiedades mecánicas de los

tejidos (Hansen et al., 2015).

En la conclusión, esta parte de nuestro estudio proporciona por primera vez un mapa

de la localización de los SLRPs en los corpúsculos de Pacini humanos. Sus complejos

patrones de expresión sugieren funciones muy diversas desde determinar la

elasticidad de los corpúsculos de Pacini, hasta condicionar sus características

mecánicas para la transmisión correcta de la presión.

6.6. El envejecimiento en la periferia del sistema somatosensorial:

cambios edad-dependientes en los mecanorreceptores cutáneos

En el quinto trabajo de esta tesis se ha realizado un estudio de los cambios edad-

dependientes que aparecen en los mecanorreceptores cutáneos (en concreto, células

de Merkel, corpúsculos de Pacini y corpúsculos de Meissner) como consecuencia del

envejecimiento. Además, se han analizado a diferentes edades, por un lado, el sistema

neurotrófico TrkB-BDNF, del cual depende el desarrollo y mantenimiento de los

corpúsculos sensitivos y, por otro lado, la expresión de la proteína Piezo2, principal

mecanotransductor de los corpúsculos sensitivos para el tacto fino.

Los corpúsculos sensitivos de la piel son capaces de detectar diferentes cualidades de

la somatosensibilidad. Los complejos célula de Merkel-neurita, los corpúsculos de

Meissner y los corpúsculos de Pacini actúan como mecanorreceptores SAI y SAII para

discriminar diferentes aspectos del tacto (Zimmerman et al., 2014). Con la edad, se

experimenta un progresivo deterioro del sistema somatosensorial a todos los niveles,

desde la piel hasta la corteza cerebral, incluyendo los recetores cutáneos en los que

tiene lugar la transducción de los estímulos mecánicos en impulsos eléctricos.

Evidentemente, dicha perdida de sensibilidad táctil afecta a la calidad de vida de las

personas (Skedung et al., 2018).

Diferentes autores afirman que la capacidad de percepción del tacto y por

consiguiente, la agudeza espacial táctil, se deterioran con la edad (Decorps y Cols.,

2014; Heft y Robinson, 2017). Esto puede deberse al menos en parte a una reducción

de la densidad de los corpúsculos de Meissner y de Pacini (Wickremaratchi y Llewelyn,

2006), así como de las células de Merkel (Lumpkin y Cols., 2003).

Mediante estudios inmunohistoquímicos se ha observado que los corpúsculos de

Meissner permanecen invariables en morfología y estructura hasta aproximadamente

los 60 años. A partir de entonces, tienen menor tamaño, adquieren forma redondeada,

aparecen topográficamente más profundos en la dermis (fuera de la papila dérmica) y

pierden parte de su perfil inmunohistoquímico característico. Así sucede en muchos

casos correspondientes a sujetos de edad más avanzada, en los que no se ha

detectado inmunorreacción para los marcadores axonales, lo que sugiere que se

produce una denervación del corpúsculo que, además se refleja en la reducción de

expresión de proteína S100 en las células lamelares. Es conocido que la expresión de la

proteína S100 por las células lamelares depende de la integridad funcional del axón

(Márques y Cols., 1997; Albuerne y Cols., 1998). Estos resultados concuerdan con los

cambios morfológicos descritos por Nava y Mathewson (1996) en los corpúsculos de

Meissner de las almohadillas digitales de ratones de avanzada edad, como

consecuencia de axonopatía distal y atrofia de las neuronas sensitivas. Sin embargo,

hay que mencionar que estos cambios afectan a un gran número de los corpúsculos de

Meissner analizados, pero no a todos, y que seguramente, la denervación observada

no se produzca por igual a lo largo de toda la piel en sus diferentes áreas anatómicas

del cuerpo (Besné y Cols., 2002). Recientemente, un estudio en el que se ensaya un

método para testar la agudeza táctil ha demostrado que los individuos con menor

rendimiento (los de mayor edad) poseen una densidad de corpúsculos de Meissner un

50% menor que los individuos con mayor rendimiento (los más jóvenes) (Skedung y

Cols., 2018). La primera explicación para esta pérdida de agudeza táctil podría ser el

deterioro asociado a la edad de las propiedades neurales, aunque habría que

considerar también el análisis de otros corpúsculos sensitivos.

Respecto a los corpúsculos de Pacini, no se han observado diferencias relevantes ni en

número ni en estructura en función de la edad, aunque en algunos casos

correspondientes a los sujetos de edad más avanzada, no ha sido posible detectar el

axón o presentaban un núcleo interno desestructurado (con pérdida parcial de

inmunorreacción para la proteína S100 en el núcleo interno). Esto podría explicar la

reducida capacidad para detectar las vibraciones que aparece en personas ancianas

(Shaffer y Harrison, 2007; Landelle y Cols., 2018).

Las células de Merkel sufren una reducción significativa asociada a la edad en los

individuos estudiados. Este hecho coincide con estudios realizados en ratones viejos,

en los que se observó una reducción de células de Merkel en la mucosa del paladar y

que precisamente, se correlaciona con pérdidas de las capacidades orales (Moayedi y

Cols., 2018).

La activación de los canales iónicos controlados mecánicamente supone el origen de la

detección de los estímulos mecánicos. Entre todos los candidatos propuestos como

posibles mecanotransductores principales que actúan en los mecanorreceptores

humanos (ASIC2, TRPV4, TRPC6; Calavia y Cols., 2010; Cabo y Cols., 2015; Alonso-

González y Cols., 2017), la mecanoproteína Piezo2 parece ser la única esencial para

que el proceso de mecanotransducción tenga lugar (Roudeaut y Cols., 2012; Delmas y

Coste, 2013; Wu y Cols., 2017). Piezo2 se expresa tanto en las neuronas

mecanosensitivas (Coste y Cols., 2010; Ranade y Cols., 2014) como en los

mecanorreceptores de bajo umbral (células de Merkel y corpúsculos de Meissner;

García-Mesa y Cols., 2017). Los resultados obtenidos demuestran que la expresión de

Piezo2 disminuye con el envejecimiento en ambos tipos de mecanorreceptores

cutáneos y que presumiblemente, dicho déficit esté detrás de la pérdida de

sensibilidad táctil asociada a la edad.

El desarrollo y mantenimiento de los corpúsculos sensitivos cutáneos está controlado

por el sistema de neurotrofinas TrkB-BDNF (González-Martínez y Cols., 2004; Sedý y

Cols., 2004; De Carlos y Cols., 2006; Pérez-Piñera y Cols., 2008; Montaño y Cols., 2010).

En adultos, TrkB y BDNF se expresan en algunas neuronas de ganglios sensitivos

(Montaño y Cols., 2010), así como en los corpúsculos sensitivos cutáneos (Calavia y

Cols., 2010). Los resultados de este trabajo sugieren que en los corpúsculos de

Meissner y de Pacini, tiene lugar la señalización retrograda de TrkB-BDNF para

proporcionar soporte trófico a las neuronas sensitivas de las cuales que derivan. De

esta forma, las células lamelares y el núcleo interno serían la fuente de BDNF, mientras

que el axón sería el receptor al expresar TrkB. El efecto de la edad en este sistema de

señalización TrkB-BDNF nunca ha sido estudiado. A partir de esta investigación se

puede notar un evidente paralelismo entre los cambios que aparecen en los

corpúsculos sensitivos asociados al envejecimiento y la decadencia del sistema TrkB-

BDNF; en algunos corpúsculos de Meissner, su inmunodetección fue completamente

negativa. Queda por determinar si el deterioro del sistema TrkB-BDNF es el

responsable principal de los cambios morfológicos observados en los corpúsculos de

Meissner y la perdida de densidad de los mismos y de las células de Merkel a edades

avanzadas. En el sistema nervioso central, la reducida expresión de TrkB y BDNF está

asociada al envejecimiento y enfermedades relacionadas (Erickson y Cols., 2010;

Forlenza y Cols., 2015; Nunes y Cols., 2018).

En resumen, todos estos resultados demuestran que los corpúsculos de Meissner y las

células de Merkel, y en menor medida los corpúsculos de Pacini, experimentan

cambios morfológicos edad-dependientes, así como una reducción en su densidad.

7. CONCLUSIONES

En base a los resultados obtenidos en este trabajo de tesis doctoral y tras la discusión

de los mismos, se han realizado las siguientes conclusiones:

1. Una lámina intermedia de origen endoneural, definida por células CD34-positivas,

se dispone entre el núcleo interno y el núcleo externo de los corpúsculos de Pacini

cutáneos humanos.

2. Los proteoglicanos de condroitín sulfato se localizan en la lámina intermedia de

origen endoneural CD34-positiva de los corpúsculos de Pacini cutáneos humanos,

pero no se expresan en los de Meissner.

3. El heparán sulfato está asociado a proteoglicanos de membrana basal en los

corpúsculos de Meissner y de Pacini humanos.

4. Los proteoglicanos de heparán sulfato y el colágeno IV de la membrana basal

denotan una fuerte expresión en las primeras lamelas del núcleo externo de los

corpúsculos de Pacini, hecho que vincula la membrana basal al aislamiento del

núcleo interno.

5. Los corpúsculos de Pacini de la piel digital humana expresan inmunorreacción para

los proteoglicanos pequeños ricos en leucina de las familias I y II, con un patrón de

distribución complejo: decorina, biglicano, lumicano, fibromodulina y

osteoadherina se detectan en el núcleo interno; la capa intermedia contiene

osteoadherina; el núcleo externo posee biglicano, decorina, lumicano,

fibromodulina y osteoadherina; y la cápsula contiene biglicano, decorina,

fibromodulina y lumicano. No se ha detectado la presencia de aspronina,

prolargina y keratocano.

6. La densidad de corpúsculos de Meissner y de células de Merkel en piel glabra

digital humana desciende drásticamente con el envejecimiento.

7. Los corpúsculos de Meissner cutáneos presentan una morfología alterada en

individuos de edad avanzada: se reducen en tamaño, se redondean en forma y se

localizan más profundos en la dermis. Los corpúsculos de Pacini no presentan

generalmente cambios morfológicos relevantes.

8. Los corpúsculos sensitivos de Meissner y de Pacini humanos experimentan

procesos de denervación y desorganización lamelar asociado a la edad. Además,

los complejos célula de Merkel-axón presentan menor número de axones en las

proximidades de las células de Merkel.

9. La expresión de la mecanoproteína Piezo2 disminuye en los corpúsculos de

Meissner y células de Merkel de la piel glabra digital de sujetos de edad avanzada.

10. El sistema de neurotrofinas TrkB-BDNF, del que depende el desarrollo y

mantenimiento de los corpúsculos sensitivos, decae con la edad mostrando

patrones de expresión reducidos.

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Curriculum Vitae Jorge García-Piqueras

Personal information

First and last names: Jorge García Piqueras

Nationality: Spanish

Date and place of birth: 15/07/1992, Calatayud (Spain)

Telephone number: 637048404

Current residence: Catedrático Doctor Flemng 6 1B

Calatayud (Spain), 50300

Work experience

October 2017 – February 2018:

Research Laboratory Technician. Department of Surgery and Medical Surgical Specialities of the University of Oviedo (Spain) and Fernández Vega Ophthalmological Institute (Oviedo, Spain). National Research Project: MINECO (Innovative solutions for the treatment of dry eye combined with ocular pain). Immunohistochemical tasks consisted in comparative studies on the innervation of elderly/young mouse eyes.

University laboratory experience

September 2016 – Currently:

PhD thesis. Department of Cellular Biology and Morphology of the University of Oviedo

(Spain). “The extracellular matrix in vertebrate mechanoreceptors”.

Immunohistochemical study of vertebrate sensory corpuscles: quantitative/qualitative

changes in relation to aging and the proteoglycan/glycosaminoglycan composition of

the extracellular matrix.

February 2016 – July 2016:

Master’s thesis. Department of Experimental, Diagnostic and Specialty Medicine of the

University of Bologna (Italy) and S.Orsola-Malpighi Hospital (Bologna, Italy).

“Identification of microRNA expression profile in the adenocarcinoma of the

esophagus and cardia”. Characterization of deregulated microRNA in association to

different histotypes of esophageal-cardia adenocarcinoma, which were determined

previously by immunohistochemical and clinical parameters.

February 2015 – July 2015:

Practicum. Department of Cellular Biology and Morphology of the University of Oviedo

(Spain). Several immunohistochemical/immunocytochemical studies on human tissues

and Zebrafish model: peripheral nervous system, cancer research, virus infection, etc.

March 2014 – June 2014:

Bachelor's thesis. Department of Biosciences of the University of Milano (Italy).

“Cloning of a protozoan protein in E.coli and genotyping of transgenic plants”.


Education and degrees

September 2017 – Currently:

PhD in Health Sciences: University of Oviedo (Spain).

September 2014 – July 2016:

International Double Master’s Degree in (120 ECTS)*:

- Biotechnology of Environment and Health: University of Oviedo (Spain).

- Medical Biotechnology: University of Bologna (Italy).

* 1st academic year at Oviedo University. 2nd academic year at Bologna University.

September 2010 – July 2014:

Bachelor's Degree in Biotechnology (240 ECTS): Universidad of Zaragoza (Spain).

*4th academic year at Milano University (Erasmus Programme).

Supplementary courses

December 2016:

Formation in Laboratory Animal Science (by Animalaria SL according to EU legislation):

- Function B: Euthanasia.

- Function C: Experimental procedures.

- Function D: Procedure and project design.

October 2015:

Diploma of University Expert in Assisted Reproduction (15 ECTS): UNED (Spain).

Diploma of University Expert in Biomedical Research and Law (15 ECTS): UNED (Spain).

Personal skills

Mother tongue: Spanish

Other languages:

- English: B2 level. First Certificate in English: Cambridge University (England).

- Italian: B2 level. CALCIF Certificate: Milano University (Italy).

Grants/Scholarships

1. Erasmus grant (September 2015 - July 2016): Bologna University (Italy).

For the second academic year of the Master’s Degree.

2. Erasmus grant (September 2013 - July 2014): Milano University (Italy).

For the fourth academic year of the Bachelor’s Degree.


Scientific publications

1. García-Piqueras J, Carcaba L, García-Mesa Y, Feito J, García B, Viña E, Suárez-Quintanilla J, Cobo J, Vega JA, García-Suárez O. Chondroitin Sulfate in Human Cutaneous Meissner and Pacinian Sensory Corpuscles. Anat Rec (Hoboken). 2018 Oct 9. doi: 10.1002/ar.23951. [Epub ahead of print].

2. Olivia García-Suárez, Yolanda García-Mesa, Jorge García-Piqueras, Giuseppina Salvo, Juan L. Cobo, Elda Alba, Ramón Cobo, Jorge Feito and José A. Vega. The Cutaneous Biopsy for the Diagnosis of Peripheral Neuropathies: Meissner’s Corpuscles and Merkel’s Cells. DOI: 10.5772/intechopen.81687

3. Feito J, García-Suárez O, García-Piqueras J, García-Mesa Y, Pérez-Sánchez A, Suazo I, Cabo R, Suárez-Quintanilla J, Cobo J, Vega JA. The development of human digital Meissner's and Pacinian corpuscles. Ann Anat. 2018 May 26;219:8-24.

4. García-Mesa Y, García-Piqueras J, García B, Feito J, Cabo R, Cobo J, Vega JA, García-Suárez O. Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity. J Anat. 2017 Dec;231(6):978-989.

5. García-Piqueras J, García-Suárez O, Rodríguez-González MC, Cobo JL, Cabo R, Vega

JA, Feito J. Endoneurial-CD34 positive cells define an intermediate layer in human digital Pacinian corpuscles. Ann Anat. 2017 May;211:55-60.

6. Mohamedi Y, Fontanil T, Solares L, Garcia-Suárez O, García-Piqueras J, Vega JA, Cal

S, Obaya AJ. Fibulin-5 downregulates Ki-67 and inhibits proliferation and invasion

of breast cancer cells. Int J Oncol. 2016 Apr;48(4):1447-56.

Congresses

1. García-Piqueras J, García B, García-Mesa Y, Cabo R, Feito J, Cárcaba-García L, Vega JA, García-Suárez O. Keratan sulfate proteoglycans in the extracellular matrix of human Meissner and Pacinian corpuscles. 28th Congress of the Spanish Anatomical Society. 1-3 February, 2018, Badajoz (Spain).

2. García-Piqueras J, García-Mesa Y, Muriel-Rodríguez JD, Torres-Parejo I, Feito J,

Cabo R, Vega JA, García-Suárez O. Quantitative and qualitative age-dependent changes in Meissner’s corpuscles and Merkel cells in human digital glabrous skin. 28th Congress of the Spanish Anatomical Society. 1-3 February, 2018, Badajoz (Spain).

3. García-Mesa Y, García-Piqueras J, Feito J, Cabo P, García-Suárez O, Vega JA. The

Meissner’s corpuscles and Merkel’s cells in multiple sclerosis and amyotrophic lateral sclerosis. 28th Congress of the Spanish Anatomical Society. 1-3 February, 2018, Badajoz (Spain).

4. García-Mesa Y, García-Piqueras J, Feito J, Torres-Parejo Isidro, Cobo R, Cabo P,

García-Suárez O, Vega JA. The validity of Meissner’s corpuscles and Merkel’s cells


analysis in skin biopsies for the diagnosis of peripheral neuropathies. 28th Congress of the Spanish Anatomical Society. 1-3 February, 2018, Badajoz (Spain).

5. García-Suárez O, García-Piqueras J, Cárcaba-García L, Viña E, Cobo J, Pérez-Moltó

FJ, Vega JA. The extracellular matrix composition of the Pacinian and Meissner’s corpuscles in digital skin. 27th Congress of the Spanish Anatomical Society. 2-4 September 2016, Oviedo (Spain).

La Matriz Extracelular en Mecanorreceptores de Vertebrados

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