Effects of vitamin B12 on the corneal nerve regeneration in rats

Elsevier Editorial System(tm) for Experimental Eye Research Manuscript Draft Manuscript Number: EXER13-368 Title: Effects of vitamin B12 on the corneal nerve regeneration in rats Article Type: Research Article Keywords: Corneal injury Vitamin B12 BetaIII-tubulin Neurofilament 160 Corneal nerve regeneration Corresponding Author: Dr Maria Rosaria Romano, Ph.D. Corresponding Author's Institution: University of Bari First Author: Maria Rosaria Romano, Ph.D. Order of Authors: Maria Rosaria Romano, Ph.D.; Francesca Biagioni, Ph.D.; Albino Carrizzo; Massimo Lorusso, M.D.; Angelo Spadaro, Prof.; Tommaso Micelli Ferrari, M.D.; Carmine Vecchione, Prof.; Monia Zurria; Michele Madonna; Francesco Fornai, Prof.; Ferdinando Nicoletti, Prof.; Marcello Diego Lograno, Prof. Abstract: This study was designed to investigate the effects of a new ophthalmic solution containing vitamin B12 0.05% on corneal nerve regeneration in rats after corneal insult. Eyes of anesthetized male Wistar rats were subjected to corneal injury by removing the corneal epithelium with corneal brush (Algerbrush). After the epithelial injury, the right eye of each animal received the instillation of one drop of the ophthalmic solution containing vitamin B12 0.05% plus taurine 0.5% and sodium hyaluronate 0.5% four time per day for 10 or 30 days. Left eyes were used as control and treated with solution containing taurine 0.5% and sodium hyaluronate 0.5% alone following the same regimen. Re-epithelialization was assessed by fluorescein staining using slit-lamp biomicroscopy. Morphological evaluation was carried out by histological techniques whereas corneal innervation was evaluated by immunohistochemical or immunoblot analysis of neurofilament 160 and β-III tubulin. Slit-lamp and histological analyses showed that re-epithelization of the corneas was accelerated in rats treated with vitamin B12 as compared to rats treated with control solution. A clear-cut difference between the two groups of rats was seen after 10 days of treatment, whereas a near-to-complete re-epithelization was observed in both groups at 30 days. Vitamin B12 treatment had also a remarkable effect on corneal innervation, as shown by substantial increases in the expression of neurofilament 160 and β-III tubulin at both 10 and 30 days. The present study suggests that topic treatment with vitamin B12 could represent a powerful strategy to accelerate re-epithelization and innervation after corneal injury. Suggested Reviewers: Maurizio Rolando Prof. Dipartimento di Scienze Neurologiche Oftalmologia e Genetica, University of Genova [email protected] Filippo Drago Prof. Dip. Biomedica Clinica e Molecolare, University of Catania [email protected]

Terry Kim Department of Ophthalmology, Duke University Medical Center [email protected] Ariel Miller Division of Neuroimmunology [email protected] Ula Jurkunas Department of Ophthalmology, [email protected]

August 06, 2013

Dear Joe Hollyfield, PhD,

we would like to submit our manuscript, entitled: “Effects of vitamin B12 on the corneal nerve

regeneration in rats”.

We feel this work is well suited for Experimental Eye Research since it describes the effects of

vitamin B12 on corneal regeneration in experimental animal model of corneal injury. We think that

this effect is noteworthy since it represents a new pharmacological approach for corneal innervation

alterations.

This manuscript contains original work has not been submitted for consideration elsewhere even if

some preliminary data have been presented as poster presentation at the Association for Research in

Vision and Ophthalmology Annual Meeting, May 1-5, 2012, Fort Lauderdale, Florida.

Looking forward to your reply, we wish you our best regards

Maria Rosaria Romano

Cover Letter

Corneal nerves have an important role in the recovery of mechanical and chemical insult to

cornea.

Topical treatment with vitamin B12 produce a nerve regeneration after corneal injury.

The βIII-tubulin and neurofilament demonstrated a striking vitamin B12 effect on re-innervation.

*Highlights (for review)

1

Effects of vitamin B12 on the corneal nerve regeneration in rats

Maria Rosaria Romanoa,*

,≠, Francesca Biagioni

b,*, Albino Carrizzo

b, Massimo Lorusso

c, Angelo

Spadarod, Tommaso Micelli Ferrari

c, Carmine Vecchione

b,e, Monia Zurria

f, Michele Madonna

b,

Francesco Fornaib,g

, Ferdinando Nicolettib,h

, Marcello Diego Lograno

a

aDepartment of Pharmacy-Pharmacological Sciences, University of Bari, Bari, Italy

bIRCSS, I.N.M., Neuromed, Pozzilli (IS), Italia

cEcclesiastical Authority Regional General Hospital Miulli, Acquaviva delle Fonti (BA), Italy

dDepartment of Pharmacological Science, University of Catania, Catania, Italy

eDepartment of Medicine and Surgery, University of Salerno, Salerno, Italy

fR&D Department, Alfa Intes, Casoria (NA), Italy

gDepartment of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy

hDepartment of Phisiology and Pharmacology, Università “Sapienza”, Roma, Italy

#Corresponding author. Tel. and Fax +39 080 5442797

E-mail address: [email protected] (M.R. Romano)

*These authors contributed equally to the work presented here and should therefore be regarded as

equivalent authors.

*ManuscriptClick here to view linked References

2

Abstract

This study was designed to investigate the effects of a new ophthalmic solution containing vitamin

B12 0.05% on corneal nerve regeneration in rats after corneal insult. Eyes of anesthetized male

Wistar rats were subjected to corneal injury by removing the corneal epithelium with corneal brush

(Algerbrush). After the epithelial injury, the right eye of each animal received the instillation of one

drop of the ophthalmic solution containing vitamin B12 0.05% plus taurine 0.5% and sodium

hyaluronate 0.5% four time per day for 10 or 30 days. Left eyes were used as control and treated

with solution containing taurine 0.5% and sodium hyaluronate 0.5% alone following the same

regimen. Re-epithelialization was assessed by fluorescein staining using slit-lamp biomicroscopy.

Morphological evaluation was carried out by histological techniques whereas corneal innervation

was evaluated by immunohistochemical or immunoblot analysis of neurofilament 160 and β-III

tubulin. Slit-lamp and histological analyses showed that re-epithelization of the corneas was

accelerated in rats treated with vitamin B12 as compared to rats treated with control solution. A

clear-cut difference between the two groups of rats was seen after 10 days of treatment, whereas a

near-to-complete re-epithelization was observed in both groups at 30 days. Vitamin B12 treatment

had also a remarkable effect on corneal innervation, as shown by substantial increases in the

expression of neurofilament 160 and β-III tubulin at both 10 and 30 days. The present study

suggests that topic treatment with vitamin B12 could represent a powerful strategy to accelerate re-

epithelization and innervation after corneal injury.

Keywords: Corneal injury, Vitamin B12, BetaIII-tubulin, Neurofilament 160, Corneal nerve

regeneration

3

1. Introduction

The cornea is one of the most innervated and sensitive tissues in the human body. Corneal

nerves and sensation are derived from the nasociliary branch of the ophthalmic division of the

trigeminal nerve (DelMonte and Kim, 2011). The nerves enter the cornea at the limbus in a radial

pattern, loss their myelin sheath thus assuring the transparency of the cornea, and form two stromal

plexuses: the mid-stromal plexus and the sub-epithelial plexus. Hereafter, the nerve trunks divide

into smaller branches to penetrate upwards into the epithelium through the Bowman’s membrane,

and form the sub-basal plexus from which nerve endings with receptors originate (Müller et al.,

2003).

Emerging studies highlight the importance of corneal nerves in protecting corneal tissues

from sensitive, mechanical, chemical and thermal stimuli and producing trophic factors which are

necessary for the maintenance of a healthy ocular surface. This raises the possibility that corneal

nerve insults significantly interfere with corneal healing (Yu and Rosenblatt, 2007). A decreased

corneal nerve function may lead to severe reduction in lacrimal gland secretion producing an

epitheliopathy and poor epithelial healing.

It is known that both surgical (i.e. cataract surgery, refractive surgery, corneal

transplantation) and not surgical (i.e. microbial infections, chemical burns, corneal abrasions and

trauma) conditions can disrupt corneal innervations (Linna et al., 2000; Patel et al., 2002). In

addition, corneal nerve morphology can be altered in diabetes, Sjogren’s syndrome and long-term

contact lenses wearers (Zhang et al., 2005). Complications arising from corneal nerve disruption

may also lead to neurotrophic keratitis which may cause subnormal visual acuity and blindness.

However, to date there are no treatments that efficiently repair the alterations in corneal innervation

although different approaches can relieve the dry eye symptoms and improve the ocular surface.

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Vitamin B12 (known also as cyanocobalamin) is a dietary essential nutrient and is important

for metabolic functions of the nervous system. Its deficiency is associated with neurological

disorders characterized by demyelination followed by axonal degeneration and irreversible neuronal

damage (Oh and Brown, 2003). The spinal cord, brain, peripheral nerves and optic nerve may all be

affected by vitamin B12 deficiency. Previous studies have shown that vitamin B12 is able to

promote neurite outgrowth and protect cortical neurons and retinal cell cultures against glutamate

cytotoxicity (Okada et al., 2010). To date, no study has examined the therapeutic effect of vitamin

B12 on corneal nerve damage.

The purpose of the present investigation was to evaluate the effects of 0.05% vitamin B12

plus 0.5% taurine eyedrops on corneal nerve regeneration in rats after corneal mechanic injury. This

new ophthalmic solution was designed to vehiculate cyanocobalamin and taurine in an advanced

artificial tear medium based on 0.5% sodium hyaluronate. Hence, a formulation containing

cyanocobalamin in association with taurine and sodium hyaluronate could act not only as a

“traditional” artificial tear but also as an adjuvant to corneal nerve health.

2. Materials and methods

2.1. Chemical

All solvents and chemicals (reagent grade or better) were obtained from Sigma-Aldrich

(Milano, Italy) and used without further preparation. Sodium hyaluronate was obtained from

HTL (Javené, France).

2.2. Formulation

A formulation containing 0.05% vitamin B12 was prepared in the dark by dissolution of

cyanocobalamin in a premixed solution containing 0.5% taurine, 0.5% sodium hyaluronate and

appropriate amount of sodium chloride, potassium chloride, sodium citrate, magnesium chloride,

5

sorbitol and phosphates buffer (vitamin B12 plus THy). In addition, we have also prepared a control

formulation containing the same ingredients of the above mentioned formulation without vitamin

B12 (THy). The resulting formulations were sterilized by filtration on 0.20 µm hydrophilic

polyvinylidene fluoride (PVDF) membrane. The amounts of salts were regulated, in both

formulations, in order to obtain a Na+/K

+ ratio of 5.2. The amount of vitamin B12 was quantified by

the HPLC method described below.

2.3. Characteristic of ophthalmic formulation

2.3.1. pH and osmolarity

A 713 pH Meter (Metrohm, Herisau, Switzerland), equipped with a combined Ag/AgCl

glass electrode was used. The pH measurements were performed in triplicate at 25°C. Osmotic

activities were analyzed by using an automatic cryoscopic osmometer (Osmomat 030-D Gonotech,

GmbH, Berlin, Germany). Before the analyses, the osmometer was calibrated with 300 mOsM

NaCl standard and ultrapure bidistilled water. The measurements were made in triplicate at 25°C.

2.3.2. Rheology

Dynamic rheology analyses of the formulations were performed in triplicate at 25° C by

shear rate experiments in a range D = 5–700 s−1

using a controlled a rotational rheometer Haake

Rheostress 600 rheometer (Haake, Karlsruhe, Germany), equipped with a cone-plate probe (C-

60/1° Ti) system. The flow curves, which represent viscosity as a function of the shear rate, were

used to study the rheologic behavior of the formulations. Each experimental run had a duration of 5

min with shear rate ranging from 0.5 to 700 s-1

.

2.3.3. HPLC analysis

HPLC separations were performed on a HP 1100 chromatographic system (Agilent

technologies, Milan, Italy) equipped with a HP ChemStation software, a binary pump

6

G1312A, a diode array detector G1315A and a thermostated column compartment G1316A. A

range of chromatographic conditions was employed in an attempt to optimise peak resolution and

response (peak area). Optimum conditions were found when the analytical column used was a

Alltech Alltima C18 column (250mm×4.6mm, 5µ particle size, Alltech, Milano, Italy)

maintained a room temperature (21° C). The isocratic mobile phase consisted of methanol and

NaH2PO4 buffer (0.05 M, pH 4.5) solution (33:67, v/v) at a flow rate of 1.0 mL/min with an

injection volume of 20 µL. The wavelength was set at 550 nm and external standardization was

used. 500 µL of the final formulations were diluted to 10 mL using the mobile phase, filtered on

0.20 µm Teflon membrane and injected into the HPLC.

2.4. Animals

Forty male Wistar rats (Harlan, S. Pietro al Natisone (UD), Italy) weighing 200-220 g were

used in this study. The rats were housed for one week in paired cage at light/darkness cycles of 12

hours and with free access to food and beverage. All experiments were performed in agreement

with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in

compliance with the Italian law on Animal Care No. 116/1992 and the Directive 2010/63/EU. The

protocols were approved by the local Animal Care and Use Committee of the University of Bari.

All efforts were made to reduce the number of animals used.

2.5. Corneal abrasion in rats

Rats were anesthetized with ketamine (40 mg/Kg) and xylazine (8 mg/Kg) by intraperitoneal

injection and a drop of benoxinate-HCl 0.4% were applied to the eye to deliver local corneal

anesthesia before animals were subjected to injury. Animals was divided in two groups, one group

was treated and monitored for ten days whereas the second group was treated and monitored for

thirty days. Animals were examined pre- and post-operatively to exclude ocular surface diseases

and removed from the study if inflammation or infection occurred. The corneal epithelium was

7

removed with 0.5 mm corneal rust ring remover (Algerbrush, EyeBM Vet, Milan, Italy), under a

dissecting microscope. The right eye was treated with 0.05% vitamin B12 plus 0.5% taurine and

0.5% hyaluronic acid eyedrops (vitamin B12 plus THy), topically administered (one drop, four

times per day) starting the same day of corneal epithelial debridement and the left eye was used as

control, received eyedrops containing 0.5% taurine and 0.5% hyaluronic acid (THy) (one drop, four

times per day). The corneal surface healing was monitored daily for seven days after injury using

fluorescein staining and photographing the corneas at the slit lamp with a digital camera and

following weekly up to thirty days. Rats were sacrificed 24 hours after the last treatment, eyes were

enucleated immediately and whole corneas were rapidly removed.

2.6. Histological and immunohistochemical assays

Dissected corneas were placed in 4% paraformaldehyde for 5 hours at 4°C. After two

thorough washing in H2O distilled for 5 minutes each, corneas were placed in 70% ethanol at 4°C

until paraffin inclusion. The corneas were cut into 8 µm medio-lateral sections and used for

histological and immunohistochemical analyses. Sections were deparafinized and processed for

staining with hematoxylin & eosin (H&E).

To evaluate the corneal nerve regeneration the immunohistochemical analysis was

performed. The deparaffinized tissue sections were incubated overnight with monoclonal mouse

antibody anti-neurofilament 160 (1:100; Sigma Aldrich, Milan, Italy) or with monoclonal mouse

antibody anti-tubulin betaIII isoform (TUJ1) (1:100; Millipore, Temecula, CA, USA), and then for

1 h with secondary biotin-coupled anti-mouse (1:200; Vector Laboratories, Burlingame, CA).

Control staining was performed without the primary antibodies.

2.7. Immunoblotting

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Corneal tissues were homogenized at 4°C in extraction buffer (Tris pH 7.4, 100 mM; EDTA

10 mM; PMSF 2 mM; aprotinin 0.01 mg/mL). The homogenates were centrifuged at 12,000 rpm at

4°C for 20 min, and the protein concentration of the supernatant was determined using a protein

assay kit (Bio-Rad, Hercules, CA, USA). 40 μg of total proteins in Laemmli buffer were boiled for

5 min and separated by 6.5% and 8% acrylamide SDS gels at 25 mA and electrophoretically

transferred to nitrocellulose membranes (Amersham, GE Healthcare Life Science, Milan, Italy) at

90 V for 90 min. The membranes were then blocked for 2 h at room temperature with phosphate-

buffered saline (PBS) containing 0.05% Tween-20 (TTBS) and 5% non-fat milk, and incubated

overnight at 4 °C with the primary antibodies anti-neurofilament 160 at a concentration of 1:1000 or

anti beta-III tubulin at a concentration of 1:2000. The membrane was washed three times for 5 min

in TBS, 0.05% Tween-20 before a 2 h incubation in a buffer (TBS 0.05% Tween-20 and 2.5%

nonfat milk) containing horseradish peroxidase-linked anti-mouse IgG (Calbiochem, Merk

Millipore, Italy) at 1:3000 dilution. The membrane was then treated for 40 min at 54 °C with

stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7), then was

incubated with primary antibody anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at a

concentration of 1:3000 (Cell Signaling, Merk Millipore, Milan, Italy) and subsequently with

horseradish peroxidase-linked anti-rabbit IgG to quantify loading control. The probed proteins were

developed by using a chemiluminescent kit (ECL, Amersham).

2.8. Data analysis

Western blot data were analyzed using ImageJ software (developed by Wayne Rasband,

National Institutes of Health, USA) to determine optical density (OD) of the bands. The OD reading

was normalized to GAPDH to account for variations in loading. Data analysis was performed using

statistical software (GraphPad Prism Software, version 5.0). Analysis of variance (one-way

ANOVA) was used to compare all conditions and Bonferroni post-hoc test was used to compare

9

mean values for all groups. P < 0.05 was considered statistically significant. All data are presented

as mean SEM.

3. Results

3.1. Ophthalmic formulation

The amounts of salt ingredients in both formulations (vitamin B12 plus THy and THy) were

regulated in order to obtain a Na/K ratio 5.2 with pH and osmolarity of 7.20 and 250 mOsm/L,

respectively. Viscosity (η, mPa·s) of the vitamin B12/THy formulation as a function of shear rates

(, s-1

) at 25°C is shown in Fig. 1. Viscosity rapidly decreases with an increase of shear rates. This

demonstrates the pseudoplastic behavior of sodium hyaluronate in the vitamin B12/THy

formulation. At low shear rate of approximately 5 s-1

the dynamic viscosity was 51 mPa·s, whereas

at higher shear rate the viscosity decreased exponentially, and at 700 s-1

was 26 mPa·s. A control

formulation (THy) was also examined under the same conditions and gave a perfectly

superimposable rheogram with respect to the B12/THy formulation (data not shown).

3.2. Determination of Vitamin B12 and preliminary stability data

The HPLC method for the assessment of vitamin B12 was validated with respect to linearity,

accuracy, and reliability in the range of concentrations between 5 and 100 g/mL. Identification and

quantification of vitamin B12 was achieved by comparing its retention times and UV absorption

with pure standard. No interfering peaks were observed when the control formulation (THy)

prepared without vitamin B12 was analysed. Under these conditions, the retention time for

paracetamol was 6.20 min. The standard curve was linear over the range 5.0 - 100 µg/mL. The

equation for the standard curve relating the vitamin B12 concentration (x, µg/mL) to peak area

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(A) was A = -544548.480 + (315167.076 · x) with a correlation coefficient of 0.9945. The

sensitivity of the assay was determined analysing progressively lower concentration and was found

to be 25 ng/mL with a signal noise of 3:1 (n = 6). In all cases, non-significant differences (P > 0.05)

were observed between the theoretical concentration of vitamin B12 used and the experimental

values registered for the vitamin B12 plus THy formulation. Preliminary results confirmed the

stability of vitamin B12 in the tested formulation. Less than 3% of degradation occurred after three

years at room temperature (21°C) on the dark.

3.3. Effect of topical vitamin B12 on corneal epithelium after mechanic injury

Gross examination of the corneal surface by slit-lamp showed an apparent repair at day 7

after abrasion regardless of the treatment (exemplified in Fig. 2). However, a striking difference

between treatments with vitamin B12/THy vs. THy alone was revealed by morphological analysis.

Hematoxilin and eosin (H&E) staining showed a complete wound healing of corneal epithelium at

10 days only in the group of rats treated with vitamin B12 (Fig. 3B). At 30 days, a complete re-

epithelization was seen in both groups of rats (Fig. 3C), although only the group of rats treated with

vitamin B12 showed a dense organization of stromal fibres (Fig. 3C, right panel).

3.4. Effect of topical vitamin B12 on regeneration of corneal nerves

Corneal re-innervation was examined by immunohistochemical and immunoblot analysis of

the selective neuronal markers, -III tubulin and neurofilament 160, after mechanical injury. Since

these proteins are also found in corneal epithelial cells (Kubilus and Linsenmayer, 2010), the

combination of these techniques is necessary to prevent any bias generated by immunoblot analysis.

For immunoblotting, -III tubulin and neurofilament 160 levels were normalized to GAPDH levels

because the other potential housekeeping, β-actin, did not remain stable after injury (See

Supplementary Figure S1). The extent of re-innervation was greater at both 10 and 30 days in the

11

group of rats treated with vitamin B12 than in the control group (Fig. 3 and 4). At 30 days, vitamin

B12 treatment fully restored the normal levels of -III tubulin in injured (Fig. 4A-C). In contrast,

treatment with the control solution has no visible effect on -III tubulin expression at both time

points (Fig. 3A-C and 4A-C). Levels of neurofilament 160 showed a near-to-full recovery after 10

and 30 days of treatment with THy alone. However, recovery was greater after treatment with

vitamin B12 (Fig. 5 and 6). Interestingly, expression levels of neurofilament 160 in injured corneas

after 30 days of treatment with vitamin B12 were higher than those detected in uninjured corneas

(the effect of vitamin B12 on uninjured corneas was not tested) (Fig. 6A-C).

4. Discussion

The present study shows that after a mechanical injury the morphological integrity of the

cornea in all its elements can be fully recovered by a daily local treatment with an ophthalmic

solution containing vitamin B12. It is known that injury to the cornea epithelium disrupts the

homeostasis of the tissue and the role of corneal nerves seems to be important in promoting

epithelial wound healing (Wilson et al., 1999; Cortina et al., 2010). Therefore, following epithelial

debridement, it becomes necessary to restore not only the epithelium and the stroma, but also the

nervous component of the cornea.

The new ophthalmic solution tested in the present study was designed to promote the

corneal nerve regeneration by using an active ingredients such as vitamin B12 formulated in an

advanced artificial tear medium based on sodium hyaluronate, sodium chloride, potassium chloride,

magnesium chloride and other important constituents, such as taurine, sorbitol and citrate. The

ingredients were chosen appropriately to perform different functions that can be summarized as

follows: (i) to increase the stability and bioavailability of vitamin B12; (ii) to confer osmoprotection

to the ocular surface; and (iii) to stabilize the lachrymal film. It has been shown that

cyanocobalamine in aqueous solution can be stabilized by taurine through an unknown mechanism

12

(Kazumi and Takumi, 1981). In addition, it is also reported that citrate (Sumi and Matsuki, 1974)

and sorbitol (Barr et al., 1957) can stabilize aqueous solution of vitamin B12. This was confirmed

by the minimal degradation of vitamin B12 observed after three years of incubation at room

temperature (see Results section).

Vitamin B12 is known to be the antipernicious anaemia factor, required for human and

animal metabolism (Ina and Kamei, 2006). Although vitamin B12 has multiple biological activities,

this is the first study which demonstrates its activity in promoting corneal wound healing and nerve

regeneration. This is consistent with the evidence that vitamin B12 promotes the synthesis of

neurotrophic factors, which in turn support neurite outgrowth and survival (Scalabrino and

Peracchi, 2006; Okada et al., 2010). In addition, vitamin B12 deficiency may cause optic

neuropathy, eye movement disorders and corneal epitheliopathy with decreased vision and

photophobia (Chavala et al., 2005; Akdal et al., 2007; Jurkunas et al., 2011).

In our experiments, acute corneal abrasion caused damage in the corneal epithelium and in

the organization of stromal fibres as indicated by histological analysis with H&E staining.

Treatment with vitamin B12 allowed a substantial recovery of all components of the cornea already

at 10 days. The use of two neuronal markers (β-III tubulin and neurofilament 160) demonstrated a

striking effect of vitamin B12 treatment on re-innervation, although data with the two markers were

not homogeneous. Unexpectedly, while β-III tubulin expression recovered only in rats treated with

vitamin B12, neurofilament 160 levels recovered also in response to the THy control solution,

although recovery was greater with vitamin B12. The substantial increase in neurofilament 160

expression seen after 30 days of treatment with vitamin B12, suggests a direct effect of the vitamin

on neurofilament synthesis. Studies on intact corneas are needed to test this hypothesis. The

appearance of neurofilaments in the distal segment of damaged neurites represents the initial phase

of neural regeneration, and re-innervation is a key factor in predicting the prognosis of corneal

health. Many infective diseases, such as herpetic viral infections, and ophthalmic surgical

procedures can corneal innervation, leading to severe conditions such as neurotrophic keratitis with

13

corneal melting and loss of vision (Bonini et al., 2003; Dixit et al., 2008). Dry eye after refractive

surgery is a complication resulting from damage to the nerves because the surgery itself causes the

cut of some nerve branches (Sitompoul et al., 2008) and this damage produces loss of corneal

sensitivity and reduced tear secretion by the lacrimal gland (Stern et al., 1998). Hence, corneal

nerve regeneration is an important target in the treatment of corneal disorders.

In conclusion, we have shown for the first time that local treatment with a solution

containing stabilized vitamin B12 leads to a rapid and complete repair following corneal damage,

and that vitamin B12 is particularly effective in promoting mechanisms that facilitate re-

innervation. Further studies are needed for a detailed molecular characterization of these

mechanisms.

References

Akdal, G., Yener, G.G., Ada, E., Halmagyi, G.M., 2007. Eye movement disorders in vitamin B12

deficiency: two new cases and a review of the literature. Eur. J. Neurol. 10, 1170-1172.

Barr, M., Kohn, S.R., Tice, L.F., 1957. The effect of various sugars and polyols on the stability of

vitamin B12. J. Am. Pharm. Assoc. 46, 650-652.

Bonini, S., Rama, P., Olzi, D., Lambiase, A., 2003. Neurotrophic keratitis. Eye. 17, 989-995.

Chavala, S.H., Kosmorsky, G.S., Lee, M.K., Lee, M.S., 2005. Optic neuropathy in vitamin B12

deficiency. Eur. J. Intern. Med. 16, 447-448.

Cortina, M.S., He J., Li, N., Bazan, N.G., Bazan, H.E.P., 2010. Neuroprotectin D1 synthesis and

corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA.

Invest. Ophthalmol. Vis. Sci. 51, 804-810.

DelMonte, D.W., Kim, T., 2011. Anatomy and physiology of the cornea. J. Cataract Refract. Surg.

37, 588-598.

Dixit, R., Tiwari, V., Shukla, D., 2008. Herpes simplex virus type 1 induces filopodia in

14

differentiated P19 neural cells to facilitate viral spread. Neurosci. Lett. 440, 113-118.

Ina, A., Kamei, Y., 2006. Vitamin B(12), a chlorophyll-related analog to pheophytin a from marine

brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells.

Cytotechnology. 52, 181-187.

Jurkunas, U.V., Jakobiec, F.A., Shin, J., Zakka, F.R., Michaud, N., Jethva, R., 2011. Reversible

corneal epitheliopathy caused by vitamin B12 and folate deficiency in a vegan with a genetic

mutation: a new disease. Eye. 25, 1512-1514.

Kazumi, O., Takumi, H., 1981. Method for Stabilizing Aqueous Solution of Cyanocobalamin. From

Jpn. Kokai Tokkyo Koho. Application JP56075438 A.

Kubilus, J.K., Linsenmayer, T.F., 2010. Developmental corneal innervation: interaction between

nerves and specialized apical corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 51,782-789.

Linna, T.U., Vesaluoma, M.H., Perez-Santonja, J.J., 2000. Effects of LASIK on corneal sensitivity

and morphology of subbasal nerves. Invest. Ophthalmol. Vis. Sci. 41, 393-397.

Müller, L.J., Marfurt, C.F., Kruse, F., Tervo, T.M., 2003. Corneal nerves: structure, contents and

function. Exp. Eye Res.76, 521-542.

Oh, R., Brown, D.L., 2003. Vitamin B12 deficiency. Am. Fam. Physician. 27, 979-986.

Okada, K., Tanaka, H., Temporin, K., et al., 2010. Methylcobalamin increases Erk1/2 and Akt

activities through the methylation cycle and promotes nerve regeneration in a rat sciatic nerve

injury model. Exp. Neurol. 222, 191-203.

Patel, S.V., McLaren, J.W., Hodge, D.O., Bourne, W.M., 2002. Confocal microscopy in vivo in

corneas of long-term contact lens wearers. Invest. Ophthalmol. Vis. Sci. 43, 995-1003.

Scalabrino, G., Peracchi, M., 2006. New insights into the pathophysiology of cobalamin deficiency.

Trends Mol. Med. 12, 247-254.

Sitompoul, R., Sancoyo, G.S., Hutauruk, J.A., Gondhowiardjo, T.D., 2008. Sensitivity change in

cornea and tear layer due to incision difference on cataract surgery with either manual small

incision cataract surgery or phacoemulsification. Cornea. 27 (Suppl. 1), S13-S18.

15

Stern, M.E., Beuerman, R.W., Fox, R.I., et al., 1998. The pathology of dry eye: the interaction

between the ocular surface and lacrimal glands. Cornea. 17, 584-589.

Sumi, Y., Matsuki, T., 1974. Stabilization of coenzyme B12 in water. From Jpn Kokai Tokkyo

Koho. Application JP49000418 A.

Wilson, S.E., Chen, L., Mohan, R.R., Liang, Q., Liu, J., 1999. Expression of HGF, KGF, EGF and

receptor messenger RNAs following corneal epithelial wounding. Exp. Eye Res. 68, 377-397.

Yu, C.Q., Rosenblatt, M.I., 2007. Transgenic corneal neurofluorescence in mice: a new model for in

vivo investigation of nerve structure and regeneration. Invest. Ophthalmol. Vis. Sci. 48, 1535-

1542.

Zhang, M., Cheng, J., Luo, L., Xiao, Q., Sun, M., Liu, Z., 2005. Altered corneal nerves in aqueous

tear deficiency viewed by in vivo confocal microscopy. Cornea.24, 818-824.

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Figure captions

Figure 1. Rheogram of vitamin B12 plus THy formulation showing changes in viscosity (, mPa•s)

as a function of shear rate (, s−1

) at 25°C.

Figure 2. Corneal insult induced by mechanic abrasion in rats.

(A) Examination of corneal surface healing with slit-lamp using fluorescein staining. (B) Slit-lamp

photograph of corneal surface after injury using a fluorescein staining. (C) The wound of corneal

surface was monitored using fluorescein staining and photographing the cornea at the slit-lamp with

a digital camera. The recovery of corneal epithelium was observed to 7th

days after injury.

Figure 3. Treatment with vitamin B12 accelerates re-hepitalization of the injured cornea.

(A) Comparison of histological structure of the normal rat cornea and after corneal mechanic

abrasion with rust ring remover. (B) Recovery of the corneal wound after treatment for 10 days with

vitamin B12 (Vit B12 + THy) (panel right) as compared to treatment with the control solution

(THy) (panel left). (C) Complete recovery of the corneal wound after treatment for 30 days with

either vitamin B12 or control solution. Scale bar 100 µm.

Figure 4. Treatment with vitamin B12 supports re-innervation of the injured cornea: measurement

of β-III tubulin expression at 10 days.

(A) Immunohistochemical analysis of β-III tubulin in normal and scraped cornea and after the

treatment with solution containing vitamin B12 or control solution. Note stromal nerve bundles

(arrows) entering the peripheral cornea after treatment with vitamin B12. Scale bar 100 µm.

17

(B, C) Immunoblot analysis of β-III tubulin in normal corneas and in scraped corneas treated with a

solution containing vitamin B12 or with control solution. A representative immunoblot is shown in

(B). Densitometric analysis is shown in (C) where data are means S.E.M. of 5-6 determinations.

P< 0.05 (One-way Anova + Bonferroni’s test) vs. the untreated scraped cornea (*) or vs. treatment

with the THy control solution (#).

Figure 5. Treatment with vitamin B12 supports re-innervation of the injured cornea: measurement

of β-III tubulin expression at 30 days.

(A) Immunohistochemical analysis of β-III tubulin in normal and scraped cornea and after the

treatment with solution containing vitamin B12 or control solution. Note stromal nerve bundles

(arrows) entering the peripheral cornea after treatment with vitamin B12. Scale bar 100 µm.

(B, C) Immunoblot analysis of β-III tubulin in normal corneas and in scraped corneas treated with a

solution containing vitamin B12 or with control solution. A representative immunoblot is shown in

(B). Densitometric analysis is shown in (C) where data are means S.E.M. of 5-6 determinations.

p< 0.05 (One-way Anova + Bonferroni’s test) vs. the untreated scraped cornea (*) or vs. treatment

with the THy control solution (#).

Figure 6. Treatment with vitamin B12 supports re-innervation of the injured cornea: measurement

of neurofilament 160 expression at 10 days.

(A) Immunohistochemical analysis of neurofilament 160 in normal and scraped cornea and after the

treatment with solution containing vitamin B12 or control solution. Note stromal nerve bundles

(arrows) entering the peripheral cornea after treatment with vitamin B12. Scale bar 100 µm.

(B, C) Immunoblot analysis of β-III tubulin in normal corneas and in scraped corneas treated with a

solution containing vitamin B12 or with control solution. A representative immunoblot is shown in

(B). Densitometric analysis is shown in (C) where data are means S.E.M. of 5-6 determinations.

18

P< 0.05 (One-way Anova + Bonferroni’s test) vs. the untreated scraped cornea (*) or vs. treatment

with the THy control solution (#).

Figure 7. Treatment with vitamin B12 supports re-innervation of the injured cornea: measurement

of neurofilament 160 expression at 30 days.

(A) Immunohistochemical analysis of neurofilament 160 in normal and scraped cornea and after the

treatment with solution containing vitamin B12 or control solution. Note stromal nerve bundles

(arrows) entering the peripheral cornea after treatment with vitamin B12. Scale bar 100 µm.

(B, C) Immunoblot analysis of β-III tubulin in normal corneas and in scraped corneas treated with a

solution containing vitamin B12 or with control solution. A representative immunoblot is shown in

(B). Densitometric analysis is shown in (C) where data are means S.E.M. of 5-6 determinations.

P< 0.05 (One-way Anova + Bonferroni’s test) vs. the untreated scraped cornea (*, §) or vs.

treatment with the THy control solution (#).

Supplementary data

Figure S1. Western blot analysis with β-actin in cornea rats.

(A) Representative western blot analysis of anti-β-actin shows a decrease in cornea tissues after a

repeated injection both with solution containing vitamin B12 or with control solution (Vit B12 +

THy) after 10 days of treatments compared with normal cornea tissue. The bottom panel shows the

β-actin/GAPDH ratio (optical densities).

(B) Representative western blot analysis of anti-β-actin shows a decrease in cornea tissues treated

for 30 days with THy alone, while after 30 day of Vit B12 + THy there is a restoration of β-actin

levels. The bottom panel shows β-actin/GAPDH ratio (optical density). *P < 0.005 vs scraped

cornea; #P < 0.005 vs THy 30 days.

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