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RESEARCH Open Access
Jose A Martinez1, Manami Kasamatsu1, Alma Rosales-Hernandez1,
Leah R Hanson2, William H Frey2,3 andCory C Toth1*
Abstract
Background: Although pregabalin therapy is beneficial for
neuropathic pain (NeP) by targeting the CaVa2δ-1subunit, its site
of action is uncertain. Direct targeting of the central nervous
system may be beneficial for theavoidance of systemic side
effects.
Results: We used intranasal, intrathecal, and near-nerve chamber
forms of delivery of varying concentrations ofpregabalin or saline
delivered over 14 days in rat models of experimental diabetic
peripheral neuropathy andspinal nerve ligation. As well,
radiolabelled pregabalin was administered to determine localization
with differentdeliveries. We evaluated tactile allodynia and
thermal hyperalgesia at multiple time points, and then
analyzedharvested nervous system tissues for molecular and
immunohistochemical changes in CaVa2δ-1 protein expression.Both
intrathecal and intranasal pregabalin administration at high
concentrations relieved NeP behaviors, while near-nerve pregabalin
delivery had no effect. NeP was associated with upregulation of
CACNA2D1 mRNA and CaVa2δ-1protein within peripheral nerve, dorsal
root ganglia (DRG), and dorsal spinal cord, but not brain.
Pregabalin’s effectwas limited to suppression of CaVa2δ-1 protein
(but not CACNA2D1 mRNA) expression at the spinal dorsal horn
inneuropathic pain states. Dorsal root ligation prevented CaVa2δ-1
protein trafficking anterograde from the dorsalroot ganglia to the
dorsal horn after neuropathic pain initiation.
Conclusions: Either intranasal or intrathecal pregabalin
relieves neuropathic pain behaviours, perhaps due topregabalin’s
effect upon anterograde CaVa2δ-1 protein trafficking from the DRG
to the dorsal horn. Intranasaldelivery of agents such as pregabalin
may be an attractive alternative to systemic therapy for management
ofneuropathic pain states.
Keywords: neuropathic pain, pregabalin, diabetic peripheral
neuropathy, spinal nerve ligation
BackgroundNeuropathic pain is a consequence of nerve damage
ordisease in the central and/or peripheral nervous systemsuch as
with diabetes and trauma. The clinical presenta-tion of neuropathic
pain includes hyperalgesia, allodynia,and spontaneous pain [1]. Its
high prevalence in humans[2-4] has led to the development of a
number of animalmodels of neuropathic pain, including diabetic
periph-eral neuropathy and spinal nerve ligation.Changes within the
nervous system associated with
neuropathic pain include critical upregulation of the
calcium channel subunit CaVa2δ-1 [5-7], particularly atthe
dorsal horn [8,9]. This is of importance for the clini-cal utility
and potential mechanism of two differentpharmacotherapies,
gabapentin and pregabalin, both ofwhich are indicated for the
management of neuropathicpain due to multiple etiologies. There is
uncertaintyabout the anatomical location of pregabalin’s
beneficialeffect, as CaVa2δ-1 subunit upregulation is
multifocal[10]. In addition to the known expression within theDRG,
CACNA2D1 mRNA is also localized to brainregions known to be
involved in cortical processing,sensory conduction, and arousal
[11]. Also, there is alsomarked CaVa2δ-1 subunit expression in
spinal cordregions where DRG projections occur. As a result,
thelocalization of pregabalin’s greatest therapeutic effect is
* Correspondence: [email protected] of Clinical
Neurosciences and the University of Calgary, Calgary,AB, CanadaFull
list of author information is available at the end of the
article
Martinez et al. Molecular Pain 2012,
8:3http://www.molecularpain.com/content/8/1/3 MOLECULAR PAIN
© 2012 Martinez et al; licensee BioMed Central Ltd. This is an
Open Access article distributed under the terms of the
CreativeCommons Attribution License
(http://creativecommons.org/licenses/by/2.0), which permits
unrestricted use, distribution, andreproduction in any medium,
provided the original work is properly cited.
Retracted: Comparison of central versus peripheraldelivery of
pregabalin in neuropathic pain states
A retraction article was published for this article. It is
available from the following link; http://www.molecularpain.
RETR
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D, William H Frey
RETR
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D, William H Frey
RETR
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D
RETR
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D
RETR
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DAlthough pregabalin therapy is beneficial for neuropathic pain
(NeP) by targeting the CaV
RETR
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DAlthough pregabalin therapy is beneficial for neuropathic pain
(NeP) by targeting the CaVsubunit, its site of action is uncertain.
Direct targeting of the central nervous system may be beneficial
for the
RETR
ACTE
Dsubunit, its site of action is uncertain. Direct targeting of
the central nervous system may be beneficial for theWe used
intranasal, intrathecal, and near-nerve chamber forms of delivery
of varying concentrations of
RETR
ACTE
DWe used intranasal, intrathecal, and near-nerve chamber forms
of delivery of varying concentrations of
pregabalin or saline delivered over 14 days in rat models of
experimental diabetic peripheral neuropathy and
RETR
ACTE
Dpregabalin or saline delivered over 14 days in rat models of
experimental diabetic peripheral neuropathy andspinal nerve
ligation. As well, radiolabelled pregabalin was administered to
determine localization with different
RETR
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Dspinal nerve ligation. As well, radiolabelled pregabalin was
administered to determine localization with differentdeliveries. We
evaluated tactile allodynia and thermal hyperalgesia at multiple
time points, and then analyzed
RETR
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Ddeliveries. We evaluated tactile allodynia and thermal
hyperalgesia at multiple time points, and then analyzedharvested
nervous system tissues for molecular and immunohistochemical
changes in CaV
RETR
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Dharvested nervous system tissues for molecular and
immunohistochemical changes in CaVBoth intrathecal and intranasal
pregabalin administration at high concentrations relieved NeP
behaviors, while near-
RETR
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DBoth intrathecal and intranasal pregabalin administration at
high concentrations relieved NeP behaviors, while near-nerve
pregabalin delivery had no effect. NeP was associated with
upregulation of CACNA2D1 mRNA and CaV
RETR
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Dnerve pregabalin delivery had no effect. NeP was associated
with upregulation of CACNA2D1 mRNA and CaVprotein within peripheral
nerve, dorsal root ganglia (DRG), and dorsal spinal cord, but not
brain. Pregabalin
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Dprotein within peripheral nerve, dorsal root ganglia (DRG), and
dorsal spinal cord, but not brain. Pregabalin
-1 protein (but not CACNA2D1 mRNA) expression at the spinal
dorsal horn in
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D-1 protein (but not CACNA2D1 mRNA) expression at the spinal
dorsal horn in
neuropathic pain states. Dorsal root ligation prevented CaV
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Dneuropathic pain states. Dorsal root ligation prevented
CaVa
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Daneuropathic pain states. Dorsal root ligation prevented
CaVaneuropathic pain states. Dorsal root ligation prevented CaV
RETR
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Dneuropathic pain states. Dorsal root ligation prevented
CaVaneuropathic pain states. Dorsal root ligation prevented CaV
2
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D2δ
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Dδ-1 protein trafficking anterograde from the dorsal
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D-1 protein trafficking anterograde from the dorsal
root ganglia to the dorsal horn after neuropathic pain
initiation.
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Droot ganglia to the dorsal horn after neuropathic pain
initiation.
Either intranasal or intrathecal pregabalin relieves neuropathic
pain behaviours, perhaps due to
RETR
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D
Either intranasal or intrathecal pregabalin relieves neuropathic
pain behaviours, perhaps due tos effect upon anterograde CaV
RETR
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D
s effect upon anterograde CaVa
RETR
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D
as effect upon anterograde CaVas effect upon anterograde CaV
RETR
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D
s effect upon anterograde CaVas effect upon anterograde CaV
2
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D
2
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D
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D
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D
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D
δ
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D
δ-1 protein trafficking from the DRG to the dorsal horn.
Intranasal
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D
-1 protein trafficking from the DRG to the dorsal horn.
Intranasaldelivery of agents such as pregabalin may be an
attractive alternative to systemic therapy for management of
RETR
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D
delivery of agents such as pregabalin may be an attractive
alternative to systemic therapy for management of
neuropathic pain, pregabalin, diabetic peripheral neuropathy,
spinal nerve ligation
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D
neuropathic pain, pregabalin, diabetic peripheral neuropathy,
spinal nerve ligation
Neuropathic pain is a consequence of nerve damage or
RETR
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D
Neuropathic pain is a consequence of nerve damage ordisease in
the central and/or peripheral nervous systemRE
TRAC
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disease in the central and/or peripheral nervous systemsuch as
with diabetes and trauma. The clinical presenta-RE
TRAC
TED
such as with diabetes and trauma. The clinical presenta-tion of
neuropathic pain includes hyperalgesia, allodynia,RE
TRAC
TED
tion of neuropathic pain includes hyperalgesia,
allodynia,RETR
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D
and spontaneous pain [1]. Its high prevalence in humansRETR
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D
and spontaneous pain [1]. Its high prevalence in humans[2-4] has
led to the development of a number of animalRE
TRAC
TED
[2-4] has led to the development of a number of animalmodels of
neuropathic pain, including diabetic periph-RE
TRAC
TED
models of neuropathic pain, including diabetic periph-
A retraction article was published for this article. It is
available from the following link; http://www.molecularpain.
RETR
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DA retraction article was published for this article. It is
available from the following link;
http://www.molecularpain.com/content/10/1/20.
RETR
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D 2
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ril 2
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uncertain. In addition to changes in CaVa2δ-1, otherchanges in
voltage gated channels [12-14] and micro-gliosis with associated
elevation in cytokines may occur[15-18].Pregabalin, as with
gabapentin, interacts specifically
with the CaVa2δ-1 subunit of voltage-gated calciumchannels
[19-21] providing an antihyperalgesic and anti-allodynic effect
specific for its action at the CaVa2δ-1subunit [22]. Recently,
Bauer et al [23] demonstratedthe importance of trafficking of the
CaVa2δ-1 subunitfrom the dorsal root ganglia to the dorsal horn in
thedevelopment of neuropathic pain, with its subsequentalleviation
with pregabalin treatment. In the presentstudy, we attempted to
determine the central and per-ipheral contributions of pregabalin
for relief from neuro-pathic pain in two separate models:
streptozotocin-induced diabetic peripheral neuropathy and
traumaticinjury (spinal nerve ligation). We also examined
thetherapeutic potential for intranasal delivery of pregabalinwith
comparison to more localized delivery systemsusing implantable pump
systems permitting a continu-ous delivery of pregabalin to specific
anatomical loca-tions [10,24]. Intranasal delivery, first developed
tobypass the blood-brain-barrier and directly target thera-peutic
agents to the central nervous system [25-28] ofrodents [29-31] and
humans [30,31], occurs along boththe olfactory and trigeminal
neural pathways usingextracellular pathways rather than axonal
transport [30].Such directed targeting of pregabalin to the brain
canavoid gastrointestinal uptake of oral therapy and maypermit more
potent relief of neuropathic pain behaviorwhile limiting systemic
side effects. For neuroinflamma-tory, neurodegenerative, and
neurovascular disorders[28], intranasal delivery is an attractive
non-invasivemethod to target molecules to the central nervous
sys-tem [32,33] and even the peripheral nervous system[34].Our
primary objective was to study the central deliv-
ery of pregabalin using either intranasal or intrathecaltherapy
and compare to near-nerve delivery for impactupon neuropathic pain
behaviors. Previously, intranasaldelivery targeting the central
nervous system has beenused as a method of delivery for neuropathic
pain states[34-36]. Intranasal delivery is dependent upon the
olfac-tory and trigeminal nerves connecting the nose to thebrain,
the rostral migratory stream, and less so uponvasculature and
lymphatic pathways [28,37]. The benefitof intranasal delivery is
the concentration of effect inthe central nervous system; systemic
delivery to someextent will occur with small, lipophilic molecules
deliv-ered intranasally but is otherwise quite limited [28].
Asmall, lipophilic molecule such as pregabalin would beanticipated
to have greater systemic distribution follow-ing intranasal
delivery than other compounds [38].
Rarely used in human clinical management at present[35], the
non-invasive nature of intranasal delivery alongwith limited
systemic exposure make it potentiallyattractive for use in the
clinic with human patients.Intrathecal delivery of compounds with
various mechan-isms [10,39,40] has also led to amelioration of
neuro-pathic pain states. Already used in the clinic for deliveryof
opioids, baclofen and conotoxins, intrathecal deliveryis an
invasive technique used in specific situations ofrefractoriness or
intolerance of oral delivery, but its usedepends upon the agent
used, its site of action, and itsability to cross the blood-brain
barrier. Finally, nearnerve delivery has also been used to modulate
sensorynerve regeneration and impact upon pain behaviors[24,41,42].
Our secondary objectives of this study wereto evaluate changes in
expression of the CaVa2δ-1 subu-nit in the dorsal spinal cord, DRG
peripheral nerve andbrain, evaluate potential changes in other
voltage-gatedcalcium and sodium channels in the spinal cord, DRGand
nerve, evaluate for changes in accumulation ofmicroglia, and to
evaluate the safety and tolerability ofpregabalin using the various
delivery routes in rodents,including the use of intranasal
delivery. We also exam-ined the previously demonstrated role of
CaVa2δ-1 sub-unit trafficking [23] in a dynamic and progressive
modelof neuropathic pain, diabetic peripheral neuropathy.
Thedetermination of impact of pregabalin upon theCaVa2δ-1 subunit
within nervous system tissuesdepending upon the mode of delivery
would assist inthe understanding of the pharmacological action
ofpregabalin upon the CaVa2δ-1 subunit and its effectsupon pathways
relevant in the development of neuro-pathic pain.
ResultsRadiolabelled Detection of Pregabalin LocalizationAt 73
hours of intrathecal or intranasal delivery, prega-balin
concentrations were detected throughout the ner-vous system (Figure
1). Intranasal delivery gaveconsistently higher elevated pregabalin
levels throughoutthe central nervous system structures at and above
thecephalad portion of the cervical spinal cord. In
contrast,intrathecal delivery was associated with higher
pregaba-lin penetration into the lumbar spinal cord and muchhigher
levels within lumbar dorsal root ganglia. Systemicpresence of
pregabalin was much higher at 73 hourswith intranasal pregabalin
delivery, but was similar toconcentrations achieved with
intrathecal delivery at 74and 77 hours.After 74 hours of chronic
delivery, concentrations
with intranasally delivered pregabalin began to fall
whileintrathecal levels were remaining stable throughout thenervous
system and systemically (Additional File 1). At77 hours after
intranasal delivery, both systemic and
Martinez et al. Molecular Pain 2012,
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Page 2 of 20
RETR
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Dd in specific situations of
RETR
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Dd in specific situations of
refractoriness or intolerance of oral delivery, but its use
RETR
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Drefractoriness or intolerance of oral delivery, but its
usedepends upon the agent used, its site of action, and its
RETR
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Ddepends upon the agent used, its site of action, and itsability
to cross the blood-brain barrier. Finally, near
RETR
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Dability to cross the blood-brain barrier. Finally, nearnerve
delivery has also been used to modulate sensory
RETR
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Dnerve delivery has also been used to modulate sensorynerve
regeneration and impact upon pain behaviors
RETR
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Dnerve regeneration and impact upon pain behaviors[24,41,42].
Our secondary objectives of this study were
RETR
ACTE
D[24,41,42]. Our secondary objectives of this study wereto
evaluate changes in expression of the CaV
RETR
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Dto evaluate changes in expression of the CaVnit in the dorsal
spinal cord, DRG peripheral nerve and
RETR
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Dnit in the dorsal spinal cord, DRG peripheral nerve andbrain,
evaluate potential changes in other voltage-gated
RETR
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Dbrain, evaluate potential changes in other voltage-gatedcalcium
and sodium channels in the spinal cord, DRG
RETR
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Dcalcium and sodium channels in the spinal cord, DRGand nerve,
evaluate for changes in accumulation of
RETR
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Dand nerve, evaluate for changes in accumulation of
RETR
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Dtions [10,24]. Intranasal delivery, first developed to
RETR
ACTE
Dtions [10,24]. Intranasal delivery, first developed tobypass
the blood-brain-barrier and directly target thera-
RETR
ACTE
Dbypass the blood-brain-barrier and directly target thera-peutic
agents to the central nervous system [25-28] of
RETR
ACTE
Dpeutic agents to the central nervous system [25-28] ofrodents
[29-31] and humans [30,31], occurs along both
RETR
ACTE
Drodents [29-31] and humans [30,31], occurs along boththe
olfactory and trigeminal neural pathways using
RETR
ACTE
Dthe olfactory and trigeminal neural pathways usingextracellular
pathways rather than axonal transport [30].
RETR
ACTE
Dextracellular pathways rather than axonal transport [30].Such
directed targeting of pregabalin to the brain can
RETR
ACTE
D
Such directed targeting of pregabalin to the brain canavoid
gastrointestinal uptake of oral therapy and may
RETR
ACTE
D
avoid gastrointestinal uptake of oral therapy and maypermit more
potent relief of neuropathic pain behavior
RETR
ACTE
D
permit more potent relief of neuropathic pain behaviorwhile
limiting systemic side effects. For neuroinflamma-
RETR
ACTE
D
while limiting systemic side effects. For neuroinflamma-tory,
neurodegenerative, and neurovascular disorders
RETR
ACTE
D
tory, neurodegenerative, and neurovascular disorders[28],
intranasal delivery is an attractive non-invasive
RETR
ACTE
D
[28], intranasal delivery is an attractive non-invasivemethod to
target molecules to the central nervous sys-
RETR
ACTE
D
method to target molecules to the central nervous sys-tem
[32,33] and even the peripheral nervous system
RETR
ACTE
D
tem [32,33] and even the peripheral nervous system
Our primary objective was to study the central deliv-
RETR
ACTE
D
Our primary objective was to study the central deliv-ery of
pregabalin using either intranasal or intrathecalRE
TRAC
TED
ery of pregabalin using either intranasal or intrathecaltherapy
and compare to near-nerve delivery for impactRE
TRAC
TED
therapy and compare to near-nerve delivery for impactupon
neuropathic pain behavioRE
TRAC
TED
upon neuropathic pain behaviodelivery targeting the central
nervous system has beenRE
TRAC
TED
delivery targeting the central nervous system has beenused as a
method of delivery for neuropathic pain statesRE
TRAC
TED
used as a method of delivery for neuropathic pain states
microglia, and to evaluate the safety and tolerability of
RETR
ACTE
Dmicroglia, and to evaluate the safety and tolerability
ofpregabalin using the various delivery routes in rodents,
RETR
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Dpregabalin using the various delivery routes in
rodents,including the use of intranasal delivery. We also exam-
RETR
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Dincluding the use of intranasal delivery. We also exam-ined the
previously demonstrated role of CaV
RETR
ACTE
Dined the previously demonstrated role of CaVunit trafficking
[23] in a dynamic and progressive model
RETR
ACTE
Dunit trafficking [23] in a dynamic and progressive modelof
neuropathic pain, diabetic peripheral neuropathy. The
RETR
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Dof neuropathic pain, diabetic peripheral neuropathy.
Thedetermination of impact of pregabalin upon the
RETR
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Ddetermination of impact of pregabalin upon theCaV
RETR
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DCaVdepending upon the mode of delivery would assist in
RETR
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D
depending upon the mode of delivery would assist in
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Intranasal and Intrathecal Pregabalin Delivery
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nervous system levels of intranasally delivered pregaba-lin
began to fall while intrathecal dosing provided rela-tively stable
pregabalin concentrations.Near nerve delivery of pregabalin only
led to detection
at the proximal sciatic nerve, with zero detection ofmeaningful
radioactive pregabalin at other systemic ornervous system sites.
The concentrations of pregabalinreceived at the proximal sciatic
nerve were several foldhigher with near nerve delivery than could
be achievedwith intrathecal or intranasal delivery (Figure
1).Maintenance of Diabetes in the Diabetic PeripheralNeuropathy
ModelAfter streptozotocin injection, rats developed diabeteswithin
1 week in greater than 80% of animals, and ineach case, diabetes
was maintained over the length ofthe study. Diabetic rats were
smaller than non-diabeticrats at endpoint (201.8 ± 14.7 grams as
compared to234.5 ± 15.2 grams). Diabetic rat glycemia was
increasedas compared to non-diabetic rats at endpoint (26.4 ±
3.1mmol/L as compared to 6.1 ± 2.2 mmol/L).Neuropathic Pain
BehaviorDespite different methods and dosing of pregabalindelivery,
there was no impact upon locomotor function-ing in rat cohorts
studied with Rotarod testing at 72-74hours post-pregabalin delivery
(data not shown). Dia-betic rats and rats subjected to spinal nerve
ligationboth developed tactile allodynia and thermal hyperalge-sia
during experimentation (Figure 2). Both intrathecaland intranasal
pregabalin provision led to improvementin tactile allodynia and
thermal hyperalgesia at highdoses (2.04 mg/kg/d) in both diabetic
peripheral neuro-pathy and spinal nerve ligation models. Medium
(0.51mg/kg/d) doses of pregabalin decreased thermal hyperal-gesia
due to diabetic peripheral neuropathy and spinalnerve ligation
without impact upon tactile allodynia.Lower doses (0.051 mg/kg/d)
of intrathecal or intranasalpregabalin had no impact upon either
tactile allodyniaor thermal hyperalgesia in either model.
Near-nervedelivery of pregabalin had no impact upon tactile
allody-nia or thermal hyperalgesia with any dose (data notshown) in
either model of neuropathic pain.mRNA Expression for CACNA2D1 and
Other Voltage GatedChannels in Diabetic Peripheral NeuropathyBoth
diabetic peripheral neuropathy and spinal nerveligation were
associated with elevation of CACNA2D1mRNA expression within both
the DRG and dorsalspinal cord (Figure 3). However, there was no
observedimpact of pregabalin delivery upon CACNA2D1 mRNAexpression
within either location when compared to sal-ine delivery in either
model.Protein Expression for a2δ-1 and other Voltage
GatedChannelsImmunohistochemical detection of CaVa2δ-1 protein
innerves exposed to diabetes or spinal nerve ligation
demonstrated an increased number of CaVa2δ-1 posi-tive profiles
near the site of spinal nerve ligation, butwithout impact of
pregabalin delivery, irrespective ofsite of delivery (Additional
File 2). The presence of dia-betes or the provision of spinal nerve
ligation was alsoassociated with a shift to a greater number of
CaVa2δ-1positive DRG neurons within the DRG, but again therewas no
impact with delivery of pregabalin regardless ofmethod of delivery
or form of neuropathic pain (Addi-tional File 2). Both diabetes and
spinal nerve ligation ledto greater expression of CaVa2δ-1 protein
at the dorsalhorn; at this location, however, receipt of high
dosepregabalin via intrathecal or intranasal delivery wasassociated
with relatively diminished expression ofCaVa2δ-1 protein (Figure 4)
at the dorsal horn.Dorsal spinal cord protein expression revealed
by Wes-tern blotting for CaVa2δ-1 protein was elevated in
thepresence of diabetes or after spinal nerve ligation (Figure5).
In either model of neuropathic pain, delivery ofintranasal or
intrathecal high dose pregabalin was asso-ciated with less
significant elevation of CaVa2δ-1 pro-tein at the dorsal horn;
meanwhile, near-nervepregabalin delivery had no impact upon dorsal
hornexpression of CaVa2δ-1 protein. Similarly, diabetes andspinal
nerve ligation led to elevation of CaVa2δ-1 pro-tein at the dorsal
root ganglia, greatest in smaller noci-ceptive neurons. However, no
method of pregabalindelivery impacted upon CaVa2δ-1 cellular or
axonal pat-terns of expression in the dorsal root ganglia or in
per-ipheral nerve (Additional Files 2, 3).CaVa2δ-1 protein in the
thalamus and primary sen-
sory cortex of the brain from rats exposed to diabeticperipheral
neuropathy, as well as in the contralateralthalamus and primary
sensory cortex, was unchanged inthe presence of neuropathic pain
when compared tocontrol rats without diabetes or traumatic nerve
injury(Additional File 4).Development of Increased Microglial
Density and Activationin Diabetic Peripheral NeuropathyMicroglial
quantification was performed in the dorsalthoracic and lumbar
spinal cords in rats subjected todiabetes and spinal nerve
ligation. There was anincreased density of activated microglia in
the dorsalspinal cord regions in rats in either neuropathic
painmodel when compared to control rat spinal cord speci-mens
(Figure 6). There was no difference in microglialquantification in
rats receiving saline or pregabalin withany method of delivery in
either model of neuropathicpain.Verification of Anterograde
Trafficking of CaVa2δ-1 inDorsal Root Ligation and Spinal Nerve
LigationFor rats undergoing spinal nerve ligation +/- dorsal
rootligation, immunohistochemistry identified accumulationof
CaVa2δ-1 at sites proximal to the ligatures placed at
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Dmethod of delivery or form of neuropathic pain (Addi-
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Dmethod of delivery or form of neuropathic pain (Addi-tional
File 2). Both diabetes and spinal nerve ligation led
RETR
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Dtional File 2). Both diabetes and spinal nerve ligation led
2
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D2δ
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Dδ-1 protein at the dorsal
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D-1 protein at the dorsal
horn; at this location, however, receipt of high dose
RETR
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Dhorn; at this location, however, receipt of high dosepregabalin
via intrathecal or intranasal delivery was
RETR
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Dpregabalin via intrathecal or intranasal delivery wasassociated
with relatively diminished expression of
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Dassociated with relatively diminished expression of-1 protein
(Figure 4) at the dorsal horn.
RETR
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D-1 protein (Figure 4) at the dorsal horn.Dorsal spinal cord
protein expression revealed by Wes-
RETR
ACTE
DDorsal spinal cord protein expression revealed by Wes-
RETR
ACTE
Ddelivery, there was no impact upon locomotor function-
RETR
ACTE
Ddelivery, there was no impact upon locomotor function-ing in
rat cohorts studied with Rotarod testing at 72-74
RETR
ACTE
Ding in rat cohorts studied with Rotarod testing at 72-74hours
post-pregabalin delivery (data not shown). Dia-
RETR
ACTE
Dhours post-pregabalin delivery (data not shown). Dia-betic rats
and rats subjected to spinal nerve ligation
RETR
ACTE
Dbetic rats and rats subjected to spinal nerve ligationboth
developed tactile allodynia and thermal hyperalge-
RETR
ACTE
Dboth developed tactile allodynia and thermal hyperalge-
(Figure 2). Both intrathecal
RETR
ACTE
D(Figure 2). Both intrathecal
and intranasal pregabalin provision led to improvement
RETR
ACTE
D
and intranasal pregabalin provision led to improvementin tactile
allodynia and thermal hyperalgesia at high
RETR
ACTE
D
in tactile allodynia and thermal hyperalgesia at highdoses (2.04
mg/kg/d) in both diabetic peripheral neuro-
RETR
ACTE
D
doses (2.04 mg/kg/d) in both diabetic peripheral neuro-pathy and
spinal nerve ligation models. Medium (0.51
RETR
ACTE
D
pathy and spinal nerve ligation models. Medium (0.51mg/kg/d)
doses of pregabalin decreased thermal hyperal-
RETR
ACTE
D
mg/kg/d) doses of pregabalin decreased thermal hyperal-gesia due
to diabetic peripheral neuropathy and spinal
RETR
ACTE
D
gesia due to diabetic peripheral neuropathy and spinalnerve
ligation without impact upon tactile allodynia.
RETR
ACTE
D
nerve ligation without impact upon tactile allodynia.Lower doses
(0.051 mg/kg/d) of intrathecal or intranasal
RETR
ACTE
D
Lower doses (0.051 mg/kg/d) of intrathecal or
intranasalpregabalin had no impact upon either tactile
allodynia
RETR
ACTE
D
pregabalin had no impact upon either tactile allodyniaor thermal
hyperalgesia in either model. Near-nerve
RETR
ACTE
D
or thermal hyperalgesia in either model. Near-nervedelivery of
pregabalin had no impact upon tactile allody-RE
TRAC
TED
delivery of pregabalin had no impact upon tactile allody-nia or
thermal hyperalgesia with any dose (data notRE
TRAC
TED
nia or thermal hyperalgesia with any dose (data notshown) in
either model of neuropathic pain.RE
TRAC
TED
shown) in either model of neuropathic pain.mRNA Expression for
CACNA2D1 and Other Voltage GatedRE
TRAC
TED
mRNA Expression for CACNA2D1 and Other Voltage GatedChannels in
Diabetic Peripheral NeuropathyRE
TRAC
TED
Channels in Diabetic Peripheral Neuropathy
tern blotting for CaV
RETR
ACTE
Dtern blotting for CaVa
RETR
ACTE
Da2
RETR
ACTE
D2δ
RETR
ACTE
Dδ-1 protein was elevated in the
RETR
ACTE
D-1 protein was elevated in the
presence of diabetes or after spinal nerve ligation (Figure
RETR
ACTE
Dpresence of diabetes or after spinal nerve ligation (Figure5).
In either model of neuropathic pain, delivery of
RETR
ACTE
D5). In either model of neuropathic pain, delivery ofintranasal
or intrathecal high dose pregabalin was asso-
RETR
ACTE
Dintranasal or intrathecal high dose pregabalin was asso-ciated
with less significant elevation of CaV
RETR
ACTE
Dciated with less significant elevation of CaVtein at the dorsal
horn; meanwhile, near-nerve
RETR
ACTE
Dtein at the dorsal horn; meanwhile, near-nervepregabalin
delivery had no impact upon dorsal horn
RETR
ACTE
Dpregabalin delivery had no impact upon dorsal hornexpression of
CaV
RETR
ACTE
Dexpression of CaVspinal nerve ligation led to elevation of
CaV
RETR
ACTE
Dspinal nerve ligation led to elevation of CaVtein at the dorsal
root ganglia
RETR
ACTE
Dtein at the dorsal root gangliaceptive neurons. However, no
method of pregabalin
RETR
ACTE
Dceptive neurons. However, no method of pregabalindelivery
impacted upon CaV
RETR
ACTE
Ddelivery impacted upon CaVterns of expression in the dorsal
root ganglia or in per-
RETR
ACTE
D
terns of expression in the dorsal root ganglia or in per-
RETR
ACTE
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42
34
26
Forc
e (g
ram
s)
Day1 4 7 10 13 18
Tactile Allodynia Measurements in DPN with Intranasal
Pregabalin
Intranasal Pregabalin (2.04 mg/kg)Intranasal Saline
Control Rat
Tim
e (s
econ
ds)
Day1 4 7 10 13 18
Thermal Hyperalgesia Measurements in DPN with Intranasal
Pregabalin
Intranasal Pregabalin (2.04 mg/kg)Intranasal Pregabalin (0.51
mg/kg)Intranasal Saline
Control Rat
Forc
e (g
ram
s)
26
34
42
Day1 4 7 10 13 18
Tactile Allodynia Measurements in DPN with Intrathecal
Pregabalin
Intrathecal Pregabalin (2.04 mg/kg)Intrathecal Saline
Control Rat
12
28
44
Tim
e (s
econ
ds)
Day1 4 7 10 13 18
Intrathecal Pregabalin (2.04 mg/kg)Intrathecal Pregabalin (0.51
mg/kg)Intrathecal SalineControl Rat
Forc
e (g
ram
s)
30
10
Day1 4 7 10 13 18
Tactile Allodynia Measurements in SNL with Intranasal
Pregabalin50
Intranasal Pregabalin (2.04 mg/kg)
Intranasal Saline
Control Rat
Tim
e (s
econ
ds)
12
24
36
Day1 4 7 10 13 18
Thermal Hyperalgesia Measurements in SNL with Intranasal
Pregabalin
Intranasal Pregabalin (2.04 mg/kg)
Intranasal Saline
Control Rat
Forc
e (g
ram
s)
10
50
30
Day1 4 7 10 13 18
Tactile Allodynia Measurements in SNL with Intrathecal
Pregabalin
Intrathecal Pregabalin (2.04 mg/kg)
Intrathecal Saline
Control Rat
Tim
e (s
econ
ds)
12
24
36
Day1 4 7 10 13 18
Thermal Hyperalgesia Measurements in SNL with Intrathecal
Pregabalin
Intrathecal Pregabalin (2.04 mg/kg)Intrathecal Pregabalin (0.51
mg/kg)Intrathecal Saline
Control Rat
* **
*
*
* * * *
* *
* *
*
*
*
* * * *
*
* **
*
*
** *
* **
*
*
***
*
θ
θ θθ θ
θ
θθθ
θ
θθ θ
θ
28
44
Thermal Hyperalgesia Measurements in DPN with Intrathecal
Pregabalin
A
B12
C
D
E
F
G
H
Figure 2 Behavioral testing results for both models of
neuropathic pain. Tactile (A) and thermal (B) sensory testing data
for rats with orwithout (control) diabetes and diabetic peripheral
neuropathy (DPN) receiving intranasal pregabalin are presented for
multiple doses ofpregabalin or saline. Intrathecal pregabalin
delivery was also followed by measurements of tactile allodynia (C)
and thermal hyperalgesia indiabetic rats (D). High doses of either
intranasal or intrathecal pregabalin impacted upon these
neuropathic pain behaviours in DPN. For ratswith or without spinal
nerve ligation receiving intranasal pregabalin, tactile (E) and
thermal (F) sensory testing data are presented;
intranasalpregabalin at high dose impacted upon both measures of
neuropathic pain behaviour. Finally, intrathecal pregabalin also
impacted upon bothtactile allodynia (G) and thermal hyperalgesia
(H) due to spinal nerve ligation when compared to saline delivery.
Significant differences weredetected between the diabetic rats
receiving high dose (2.04 mg/kg/d) (*) and medium dose (0.51
mg/kg/d) (θ) pregabalin as compared todiabetic rats receiving
saline delivery (non-matched ANOVA tests, F-values range between
1.98-22.86 for indicated groups and time points, n ≥ 5,p <
0.0125 after Bonferroni correction). Significant differences were
detected between rats with spinal nerve ligation receiving high
dose (2.04mg/kg/d) (*) and medium dose (0.51 mg/kg/d) (θ)
pregabalin as compared to rats with spinal nerve ligation receiving
saline delivery (non-matched ANOVA tests, F-values range between
2.25-8.11 for indicated groups and time points, n ≥ 5, p <
0.0125 after Bonferroni correction) [n= 5-6 rats in each cohort for
each time point]. Data for rats receiving low dose pregabalin were
similar to data for rats receiving saline and arenot shown.
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Page 5 of 20
RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
D
Day
RETR
ACTE
D
DayDay
RETR
ACTE
D
Day1 4 7 10 13 18
RETR
ACTE
D
1 4 7 10 13 18
RETR
ACTE
D1 4 7 10 13 18
RETR
ACTE
D1 4 7 10 13 181 4 7 10 13 18
RETR
ACTE
D1 4 7 10 13 18
RETR
ACTE
DDay
RETR
ACTE
DDay
1 4 7 10 13 18
RETR
ACTE
D1 4 7 10 13 18
RETR
ACTE
DThermal Hyperalgesia Measurements in SNL with Intranasal
Pregabalin
RETR
ACTE
DThermal Hyperalgesia Measurements in SNL with Intranasal
Pregabalin
RETR
ACTE
DIntranasal Pregabalin (2.04 mg
RETR
ACTE
DIntranasal Pregabalin (2.04 mg/
RETR
ACTE
D/kg)
RETR
ACTE
Dkg)kg)
RETR
ACTE
Dkg)Intranasal Saline
RETR
ACTE
DIntranasal SalineControl Rat
RETR
ACTE
DControl Rat
RETR
ACTE
DFo
rce
(gra
ms)
RETR
ACTE
DFo
rce
(gra
ms)
Forc
e (g
ram
s)
RETR
ACTE
DFo
rce
(gra
ms)
10
RETR
ACTE
D
10
50
RETR
ACTE
D5050
RETR
ACTE
D50
30
RETR
ACTE
D30
RETR
ACTE
DTactile Allodynia Measurements in SNL with Intrathecal
Pregabalin
RETR
ACTE
DTactile Allodynia Measurements in SNL with Intrathecal
PregabalinTactile Allodynia Measurements in SNL with Intrathecal
Pregabalin
RETR
ACTE
DTactile Allodynia Measurements in SNL with Intrathecal
Pregabalin
RETR
ACTE
D
*
RETR
ACTE
D
*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D*
RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
D
12 RETR
ACTE
D
1212 RETR
ACTE
D
12
28 RETR
ACTE
D
2828 RETR
ACTE
D
28
Tim
e (s
econ
ds)
RETR
ACTE
D
Tim
e (s
econ
ds)
RETR
ACTE
D
1 4 7 10 13 18RETR
ACTE
D
1 4 7 10 13 18RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
D
Intrathecal Pregabalin (2.04 mg
RETR
ACTE
D
Intrathecal Pregabalin (2.04 mgIntrathecal Pregabalin (0.51
mg
RETR
ACTE
D
Intrathecal Pregabalin (0.51 mg
Intrathecal Saline
RETR
ACTE
D
Intrathecal SalineControl Rat
RETR
ACTE
D
Control RatControl Rat
RETR
ACTE
D
Control Rat
RETR
ACTE
D
*RETR
ACTE
D
*RETR
ACTE
D
*RETR
ACTE
D
*RETR
ACTE
D
θRETR
ACTE
D
θθRETR
ACTE
D
θ
Thermal Hyperalgesia Measurements in DPN with Intrathecal
Pregabalin
RETR
ACTE
D
Thermal Hyperalgesia Measurements in DPN with Intrathecal
PregabalinThermal Hyperalgesia Measurements in DPN with Intrathecal
Pregabalin
RETR
ACTE
D
Thermal Hyperalgesia Measurements in DPN with Intrathecal
Pregabalin
RETR
ACTE
DF
RETR
ACTE
DF
RETR
ACTE
D
G
RETR
ACTE
D
G
RETR
ACTE
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the dorsal root but not spinal nerve at 7 days after pro-cedures
were performed (Figure 7). Dorsal horn expres-sion of CaVa2δ-1 was
elevated withimmunohistochemistry or with Western blotting withthe
absence of dorsal root ligation regardless of deliveryof
intrathecal pregabalin or saline; however, CaVa2δ-1protein levels
at the dorsal horn were suppressed in thepresence of a dorsal root
ligature. When dorsal rootligature was present, CaVa2δ-1 protein
accumulationoccurred at the portion of dorsal root distal (but
notproximal) to dorsal root ligature, with partial suppres-sion due
to intrathecal pregabalin delivery. At the spinalroot, the presence
or absence of spinal nerve ligationdid not influence expression of
CaVa2δ-1 protein. These
findings demonstrate the anterograde transport ofCaVa2δ-1
protein away from the dorsal root gangliaafter traumatic nerve
injury.
DiscussionSufficient doses of pregabalin provided through
intrana-sal or intrathecal methods ameliorated tactile allodyniaand
thermal hypersensitivity due to spinal nerve ligationor diabetic
peripheral neuropathy, but there was no ben-efit of providing
pregabalin at the level of the peripheralnerve in either condition,
suggesting that pregabalin’sbenefit is localized to the dorsal root
ganglia or its cen-tral projections. In particular, pregabalin
provision intra-nasally or intrathecally led to partial reversal
ofupregulation of CaVa2δ-1 at the pre-synaptic nerveterminals in
the dorsal horn of the spinal cord [7,8].This may relate to
postulated central trafficking of theCaVa2δ-1 subunit from the
dorsal root ganglia [23], andits potential block with pregabalin
[23]. Pregabalin’seffect appeared to be limited to impact upon
traffickingof CaVa2δ-1 protein away from the dorsal root
gangliacentrally.Voltage-gated calcium channels are heterogenous
mul-
timeric complexes composed of several different subu-nits
including a1, b, a2δ, and g [43,44]. The CaVa2δ-1subunit is an
auxiliary subunit which facilitates targetingand assembly of
channels at the cell surface [45]. CAC-NA2D1 is transcribed as a
single mRNA from a singlegene, with the translated CaVa2δ-1 subunit
proteincleaved to yield a2 and δ proteins; these are subse-quently
disulfide-linked and glycosylated, leading tofunctional CaVa2δ-1
subunits [46]. There are four dif-ferent CaVa2δ-1 subunits: the
CaVa2δ-1 subunit hasbeen investigated most, due to its ability to
serve as aligand for the gabapentinoids [19]. Expression ofCaVa2δ-1
in normal situations is essentially confined toneurons withn the
brain, spinal cord, and DRG [11]. Inthe brain, CACNA2D1 mRNA is
localized to regionsimportant for cortical processing, primary
sensory trans-mission, and arousal; however, we discovered no
mea-surable changes in CaVa2δ-1 protein expression in
theneuropathic pain states studied, regardless of mode ofpregabalin
delivery. Instead, the observed changes inCaVa2δ-1 protein
expression at the dorsal horn appearto be of greater
importance.Upregulation of CaVa2δ-1 in the spinal cord is
essen-
tial for both initiation and maintenance of neuropathicpain in
many conditions [47]. Although upregulation ofCaVa2δ-1 occurred in
the DRG and at the dorsal rootas well, there was no impact of
pregabalin uponCaVa2δ-1 protein levels at these locations. In
addition,elevation of CACNA2D1 mRNA in neuropathic painstates at
the dorsal horn and dorsal root ganglia was notimpacted by forms of
pregabalin delivery. The isolated
Figure 3 Expression of CACNA2D1 mRNA. Expression ofCACNA2D1 mRNA
was upregulated in both dorsal spinal cord (A)and lumbar DRGs (B)
in the presence of either diabetic peripheralneuropathy (DPN) or
spinal nerve ligation (SNL). Dorsal horn mRNAexpression was
unchanged by presence of high dose pregabalin(2.04 mg/kg/d)
provided either through intrathecal or intranasaldelivery when
compared to saline delivery (A). There was no impactof pregabalin
delivered in any manner upon mRNA expression inlumbar DRGs when
compared to saline delivery, although thepresence of spinal nerve
ligation or diabetic peripheral neuropathywas associated with
elevated CACNA2D1 mRNA expression whencompared to control rat
specimens (B), indicated by the * abovethe Control rat data
(non-matched ANOVA tests, F-values rangebetween 0.42-1.00 for
indicated groups and time points, n ≥ 3, * p< 0.0125) [n = 6-8
specimens for each cohort].
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Page 6 of 20
RETR
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Dor diabetic peripheral neuropathy, but there was no ben-
RETR
ACTE
Dor diabetic peripheral neuropathy, but there was no ben-efit of
providing pregabalin at the level of the peripheral
RETR
ACTE
Defit of providing pregabalin at the level of the
peripheralnerve in either condition, suggesting that pregabalin
RETR
ACTE
Dnerve in either condition, suggesting that pregabalinbenefit is
localized to the dorsal root ganglia or its cen-
RETR
ACTE
Dbenefit is localized to the dorsal root ganglia or its cen-tral
projections. In particular, pregabalin provision intra-
RETR
ACTE
Dtral projections. In particular, pregabalin provision
intra-nasally or intrathecally led to partial reversal of
RETR
ACTE
Dnasally or intrathecally led to partial reversal ofupregulation
of CaV
RETR
ACTE
Dupregulation of CaVa
RETR
ACTE
Da2
RETR
ACTE
D2δ
RETR
ACTE
Dδ-1 at the pre-synaptic nerve
RETR
ACTE
D-1 at the pre-synaptic nerveterminals in the dorsal horn of the
spinal cord [7,8].
RETR
ACTE
Dterminals in the dorsal horn of the spinal cord [7,8].This may
relate to postulated central trafficking of the
RETR
ACTE
DThis may relate to postulated central trafficking of the
-1 subunit from the dorsal root ganglia [23], and
RETR
ACTE
D-1 subunit from the dorsal root ganglia [23], and
its potential block with pregabalin [23]. Pregabalin
RETR
ACTE
Dits potential block with pregabalin [23]. Pregabalineffect
appeared to be limited to impact upon trafficking
RETR
ACTE
Deffect appeared to be limited to impact upon traffickingof
CaV
RETR
ACTE
Dof CaVa
RETR
ACTE
Daof CaVaof CaV
RETR
ACTE
Dof CaVaof CaV 2
RETR
ACTE
D2δ
RETR
ACTE
Dδ-1 protein away from the dorsal root ganglia
RETR
ACTE
D-1 protein away from the dorsal root ganglia
centrally.
RETR
ACTE
Dcentrally.Voltage-gated calcium channels are heterogenous
mul-
RETR
ACTE
DVoltage-gated calcium channels are heterogenous mul-
timeric complexes composed of several different subu-
RETR
ACTE
Dtimeric complexes composed of several different subu-nits
including
RETR
ACTE
Dnits includingsubunit is an auxiliary subunit which facilitates
targeting
RETR
ACTE
Dsubunit is an auxiliary subunit which facilitates targetingand
assembly of channels at the cell surface [45]. CAC-
RETR
ACTE
Dand assembly of channels at the cell surface [45]. CAC-NA2D1 is
transcribed as a single mRNA from a single
RETR
ACTE
DNA2D1 is transcribed as a single mRNA from a singlegene, with
the translated CaV
RETR
ACTE
D
gene, with the translated CaV
RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
D. Expression of
RETR
ACTE
D. Expression of
CACNA2D1 mRNA was upregulated in both dorsal spinal cord (
RETR
ACTE
DCACNA2D1 mRNA was upregulated in both dorsal spinal cord (A
RETR
ACTE
DA)
RETR
ACTE
D)
) in the presence of either diabetic peripheral
RETR
ACTE
D) in the presence of either diabetic peripheral
neuropathy (DPN) or spinal nerve ligation (SNL). Dorsal horn
mRNA
RETR
ACTE
D
neuropathy (DPN) or spinal nerve ligation (SNL). Dorsal horn
mRNAexpression was unchanged by presence of high dose
pregabalin
RETR
ACTE
D
expression was unchanged by presence of high dose
pregabalin(2.04 mg/kg/d) provided either through intrathecal or
intranasal
RETR
ACTE
D
(2.04 mg/kg/d) provided either through intrathecal or
intranasaldelivery when compared to saline delivery (
RETR
ACTE
D
delivery when compared to saline delivery (A
RETR
ACTE
D
A). There was no impact
RETR
ACTE
D
). There was no impactof pregabalin delivered in any manner upon
mRNA expression in
RETR
ACTE
D
of pregabalin delivered in any manner upon mRNA expression
in
RETR
ACTE
D
lumbar DRGs when compared to saline delivery, although the
RETR
ACTE
D
lumbar DRGs when compared to saline delivery, although
thepresence of spinal nerve ligation or diabetic peripheral
neuropathy
RETR
ACTE
D
presence of spinal nerve ligation or diabetic peripheral
neuropathywas associated with elevated CACNA2D1 mRNA expression
when
RETR
ACTE
D
was associated with elevated CACNA2D1 mRNA expression
whencompared to control rat specimens (
RETR
ACTE
D
compared to control rat specimens (B
RETR
ACTE
D
B), indicated by the * above
RETR
ACTE
D
), indicated by the * abovethe Control rat data (non-matched
ANOVA tests,
RETR
ACTE
D
the Control rat data (non-matched ANOVA tests,
RETR
ACTE
D
the dorsal root but not spinal nerve at 7 days after
pro-RETR
ACTE
D
the dorsal root but not spinal nerve at 7 days after pro-cedures
were performed (Figure 7). Dorsal horn expres-RE
TRAC
TED
cedures were performed (Figure 7). Dorsal horn expres-sion of
CaVRE
TRAC
TED
sion of CaVaRETR
ACTE
D
aRETR
ACTE
D
RETR
ACTE
D
between 0.42-1.00 for indicated groups and time points,
RETR
ACTE
D
between 0.42-1.00 for indicated groups and time points,<
0.0125) [n = 6-8 specimens for each cohort].
RETR
ACTE
D
< 0.0125) [n = 6-8 specimens for each cohort].
RETR
ACTE
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Figure 4 CaVa2δ-1 protein expression. CaVa2δ-1 protein
expression was detected by immunohistochemistry at the dorsal horn
regions of ratssubjected to spinal nerve ligation or diabetes (A).
Rats exposed to high dose (2.04 mg/kg/d) pregabalin provided
intrathecally or intranasally hadless expression of CaVa2δ-1 at the
dorsal horn as compared to rats receiving near-nerve delivery of
pregabalin or with saline delivery for bothmodels of diabetic
peripheral neuropathy (DPN) (B) and spinal nerve ligation (SNL)
(C). As compared to control rat specimens, the presence ofeither
spinal nerve ligation or diabetes uniformly led to upregulation of
CaVa2δ-1 at the dorsal horn. Unmatched ANOVA tests were
performedbetween cohorts receiving pregabalin and saline for each
intervention and condition, and between intervention locations,
with * indicatingsignificant difference (p < 0.0125 after
Bonferroni corrections) between cohorts [n = 3-5 specimens for each
cohort].
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impact of pregabalin with lowering of elevated CaVa2δ-1 protein
levels at the dorsal horn suggests a pregabalin-mediated prevention
of an anterograde trafficking pro-cess [23]. However, no such
distal anterograde traffick-ing of CaVa2δ-1 protein towards the
periphery could bedetected, and there was no accumulation of
CaVa2δ-1
protein at the spinal nerve ligation as occurred at theligature
around the dorsal nerve root. The presence of adorsal root ligature
prevented the upregulation ofCaVa2δ-1 protein levels at the dorsal
horn, leading toaccumulation of CaVa2δ-1 protein proximal to the
dor-sal root ligature, implicating CaVa2δ-1 protein
Figure 5 Protein Quantification. Western blotting identified
increased levels of CaVa2δ-1 protein in the dorsal horn and lumbar
DRGs for ratsexposed to either diabetic peripheral neuropathy (DPN)
or spinal nerve ligation (SNL) when compared to control rats (A).
In the dorsal spinalcord, either intrathecal or intranasal delivery
of high dose (2.04 mg/kg/d) pregabalin was associated with less
upregulation of CaVa2δ-1 proteinthan with saline delivery (B). The
near nerve delivery of pregabalin failed to impact upon CaVa2δ-1
protein expression in the dorsal spinal cordfollowing SNL or DPN
(A, B). Finally, pregabalin delivered in any manner was not
associated with impact upon CaVa2δ-1 protein expression inthe
lumbar DRGs exposed to either DPN or SNL when compared to saline
delivery (intrathecal delivery results shown) (A). Multiple
unmatchedANOVA tests were performed between cohorts receiving
pregabalin and saline for each intervention and condition, with *
indicating significantdifference (p < 0.0125 after Bonferroni
corrections) between cohorts. b-actin protein levels were used for
quantification of ratios of CaVa2δ-1/b-actin demonstrated in B.
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D
RETR
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D
Protein QuantificationRETR
ACTE
D
Protein Quantification. Western blotting identified increased
levels of CaVRETR
ACTE
D
. Western blotting identified increased levels of CaVexposed to
either diabetic peripheral neuropathy (DPN) or spinal nerve
ligation (SNL) when compared to control rats (RE
TRAC
TED
exposed to either diabetic peripheral neuropathy (DPN) or spinal
nerve ligation (SNL) when compared to control rats (cord, either
intrathecal or intranasal delivery of high dose (2.04 mg/kg/d)
pregabalin was associated with less upregulation of CaVRE
TRAC
TED
cord, either intrathecal or intranasal delivery of high dose
(2.04 mg/kg/d) pregabalin was associated with less upregulation of
CaVthan with saline delivery (RE
TRAC
TED
than with saline delivery (BRETR
ACTE
D
B). The near nerve delivery of pregabalin failed to impact upon
CaVRETR
ACTE
D
). The near nerve delivery of pregabalin failed to impact upon
CaVfollowing SNL or DPN (RE
TRAC
TED
following SNL or DPN (A, BRETR
ACTE
D
A, B). Finally, pregabalin delivered in any manner was not
associated with impact upon CaVRETR
ACTE
D
). Finally, pregabalin delivered in any manner was not
associated with impact upon CaVthe lumbar DRGs exposed to either
DPN or SNL when compared to saline delivery (intrathecal delivery
results shown) (RE
TRAC
TED
the lumbar DRGs exposed to either DPN or SNL when compared to
saline delivery (intrathecal delivery results shown) (
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trafficking away centrally from the dorsal root ganglia asan
important factor in establishment of neuropathicpain, confirming
the results of Bauer et al [23]. Thesesame unmyelinated DRG sensory
neurons important fornociception (unmyelinated C-fibers for
transmission ofthermal hyperalgesia and large-diameter Aδ
afferentfibers for transmission of tactile allodynia
[48,49])synapse in laminae I and II of the dorsal horn or at
Lis-sauer’s tract. We determined that CaVa2δ-1 immunor-eactivity
was elevated unilaterally with spinal nerveligation and bilaterally
with diabetic peripheral neuropa-thy in the dorsal column at
thoracolumbar levels abovethe spinal nerve ligation injury, as well
as in DRG neu-rons [23]. Most importantly, decreases in
CaVa2δ-1immunoreactivity at the dorsal horn were associatedwith
successful analgesia of neuropathic pain with preg-abalin. As a
result, we hypothesize that pregabalin actsto slow or prevent the
trafficking of the CaVa2δ-1 pro-tein from the dorsal root ganglia
to the dorsal horn.The upregulation of CaVa2δ-1 within the
spinal
cord leads to increased presynaptic Ca2+ influx as wellas
neurotransmitter release, contributing to sensitiza-tion and
neuropathic pain [50]. In models of neuro-pathic pain, the
gabapentinoids gabapentin andpregabalin reduce neurotransmitter
release and excita-tory synaptic transmission at the spinal cord
[51,52].Controversies regarding the mechanism of action of
the gabapentinoids have occurred, with some studiesdemonstrating
no acute effects of gabapentin uponspontaneous synaptic currents in
lamina II neurons[53], while other studies have shown gabapentin’s
abil-ity to reduce both inhibitory and excitatory
neuro-transmission at the dorsal horn by preferentiallyblocking
only P/Q-type Ca2+ channels [54] andthrough prevention of
intracellular endosome recy-cling of CaVa2δ-1 protein [55]. There
has been noeffect upon the levels of CaVa2δ-1 protein or CAC-NA2D1
mRNA elsewhere, though, such as at the DRG(despite the presence of
all three CaVa2δ subunits atthe DRG) [11] and in the peripheral
nerve. This maysignify that trafficking, but not expression or
endocy-tosis, of CaVa2δ-1 from DRG to presynaptic terminalsis
impacted upon by pregabalin [23]. Other potentialmechanisms for
gabapentinoids include a more acuteinhibitory effect upon
neurotransmitter release [56]which may relate to upregulation of
protein kinase Cactivation [57,58].Chronic, and not acute,
pregabalin delivery reduces
the neuronal expression of CaVa2δ-1 in vitro withoutimpact upon
constitutive endocytosis, leading to highlevels of CaVa2δ-1 being
present in intracellular vesicles[23]. This effect may be a
consequence of calcium chan-nel trafficking through the Von
Willebrand factor-Adomain in the a2 subunit of CaVa2δ-1 [59], as
well asblocked CaVa2δ-1 trafficking [23,60]. Transgenicmutants for
CaVa2δ-1 unable to bind gabapentinoidshave reduced trafficking,
supporting this hypothesis[60,61]. This hypothesized mechanism of
disrupted traf-ficking would be unique, distinct from that of
otherpharmacotherapies with a direct neuronal surface action,and
may relate to CaVa2δ-1 recycling at the endosome[55]. We postulate
that the effects of pregabalin uponCaVa2δ-1 trafficking occurs
peripherally, either at thedorsal root ganglia or the dorsal
root.There was no measureable impact of pregabalin deliv-
ered by any method upon the activation and accumula-tion of
microglia in the spinal cord either. Ectopicactivity contributing
to sensitization due to excessiveactivity in voltage-gated calcium
channels and voltage-gated sodium channel may occur within both A
or Cfibres [62-64], relating to neuronal hyperexcitability
[14].Microglia and their activation, leading to cytokine
pro-duction important in neuropathic pain, are apparent andplay a
key role in pathophysiological pain due to bothnerve injury [15-17]
and diabetes [18]. Pregabalin’s lackof impact upon these other
established mechanisms forinduction of neuropathic pain suggest its
specificity atthe CaVa2δ-1 subunit of the voltage-dependent
calciumchannel. However, other pathological changes associatedwith
neuropathic pain continue abated despite pregaba-lin delivery.
Figure 6 Microglia Assessment. Microglia accumulation
wasassessed in the dorsal regions of thoracic and lumbar spinal
cordfor control rat spinal cord (A), and with either spinal nerve
ligation(B) or diabetic peripheral neuropathy (C) in rats.
Greaterimmunohistochemically-identified accumulation and activation
ofmicroglia in the dorsal spinal cord of rats was seen with
spinalnerve ligation or diabetic peripheral neuropathy (D) without
impactof pregabalin delivery.* indicates significant differences
betweencontrol (sham SNL surgery or citrate-injected non-diabetic
ratrespectively) and all SNL and DPN rat values using
individualunmatched ANOVA testing (p < 0.0125 after Bonferroni
corrections).Bar = 50 μm.
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Page 9 of 20
RETR
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Dtrafficking away centrally from the dorsal root ganglia as
RETR
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Dtrafficking away centrally from the dorsal root ganglia as
blishment of neuropathic
RETR
ACTE
D
blishment of neuropathicpain, confirming the results of Bauer et
al [23]. These
RETR
ACTE
D
pain, confirming the results of Bauer et al [23]. Thesesame
unmyelinated DRG sensory neurons important for
RETR
ACTE
D
same unmyelinated DRG sensory neurons important fornociception
(unmyelinated C-fibers for transmission of
RETR
ACTE
D
nociception (unmyelinated C-fibers for transmission ofthermal
hyperalgesia and large-diameter A
RETR
ACTE
D
thermal hyperalgesia and large-diameter Aδ
RETR
ACTE
D
δ afferent
RETR
ACTE
D
afferentfibers for transmission of tactile allodynia
[48,49])
RETR
ACTE
D
fibers for transmission of tactile allodynia [48,49])synapse in
laminae I and II of the dorsal horn or at Lis-
RETR
ACTE
D
synapse in laminae I and II of the dorsal horn or at Lis-s
tract. We determined that CaV
RETR
ACTE
D
s tract. We determined that CaVa
RETR
ACTE
D
aeactivity was elevated unilaterally with spinal nerve
RETR
ACTE
D
eactivity was elevated unilaterally with spinal nerveligation
and bilaterally with diabetic peripheral neuropa-
RETR
ACTE
D
ligation and bilaterally with diabetic peripheral neuropa-thy in
the dorsal column at thoracolumbar levels aboveRE
TRAC
TED
thy in the dorsal column at thoracolumbar levels abovethe spinal
nerve ligation injury, as well as in DRG neu-RE
TRAC
TED
the spinal nerve ligation injury, as well as in DRG neu-rons
[23]. Most importantly, decreases in CaVRE
TRAC
TED
rons [23]. Most importantly, decreases in CaVimmunoreactivity at
the dorsal horn were associatedRE
TRAC
TED
immunoreactivity at the dorsal horn were associatedwith
successful analgesia of neuropathic pain with preg-RE
TRAC
TED
with successful analgesia of neuropathic pain with preg-
-1 protein [55]. There has been no
RETR
ACTE
D-1 protein [55]. There has been no
2
RETR
ACTE
D2δ
RETR
ACTE
Dδ-1 protein or CAC-
RETR
ACTE
D-1 protein or CAC-
NA2D1 mRNA elsewhere, though, such as at the DRG
RETR
ACTE
DNA2D1 mRNA elsewhere, though, such as at the DRG(despite the
presence of all three CaV
RETR
ACTE
D(despite the presence of all three CaVa
RETR
ACTE
Da2
RETR
ACTE
D2δ
RETR
ACTE
Dδ subunits at
RETR
ACTE
Dsubunits atthe DRG) [11] and in the peripheral nerve. This
may
RETR
ACTE
Dthe DRG) [11] and in the peripheral nerve. This maysignify that
trafficking, bu
RETR
ACTE
Dsignify that trafficking, but not expression or endocy-
RETR
ACTE
Dt not expression or endocy--1 from DRG to presynaptic
terminals
RETR
ACTE
D-1 from DRG to presynaptic terminalsis impacted upon by
pregabalin [23]. Other potential
RETR
ACTE
Dis impacted upon by pregabalin [23]. Other potentialmechanisms
for gabapentinoids include a more acute
RETR
ACTE
Dmechanisms for gabapentinoids include a more acuteinhibitory
effect upon neurotransmitter release [56]
RETR
ACTE
Dinhibitory effect upon neurotransmitter release [56]which may
relate to upregulation of protein kinase C
RETR
ACTE
Dwhich may relate to upregulation of protein kinase Cactivation
[57,58].
RETR
ACTE
Dactivation [57,58].Chronic, and not acute, pregabalin delivery
reduces
RETR
ACTE
DChronic, and not acute, pregabalin delivery reduces
the neuronal expression of CaV
RETR
ACTE
Dthe neuronal expression of CaVimpact upon constitutive
endocytosis, leading to high
RETR
ACTE
Dimpact upon constitutive endocytosis, leading to highlevels of
CaV
RETR
ACTE
Dlevels of CaVa
RETR
ACTE
Dalevels of CaValevels of CaV
RETR
ACTE
Dlevels of CaValevels of CaV 2
RETR
ACTE
D2δ
RETR
ACTE
Dδ
[23]. This effect may be a consequence of calcium chan-
RETR
ACTE
D[23]. This effect may be a consequence of calcium chan-nel
trafficking through the Von Willebrand factor-A
RETR
ACTE
Dnel trafficking through the Von Willebrand factor-Adomain in
the
RETR
ACTE
Ddomain in theblocked CaV
RETR
ACTE
Dblocked CaVmutants for CaV
RETR
ACTE
D
mutants for CaV
RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
D
RETR
ACTE
Dunmatched ANOVA testing (p < 0.0125 after Bonferroni
corrections).
RETR
ACTE
Dunmatched ANOVA testing (p < 0.0125 after Bonferroni
corrections).
RETR
ACTE
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Figure 7 Dorsal and Spinal Root Ligation Experiments. A cartoon
diagram of the procedures performed in spinal nerve ligation and
dorsalroot ligation is provided. Tissues obtained after 7 days of
spinal nerve ligation and/or dorsal root ligation (or neither in
control rats) wereexamined using immunohistochemistry for CaVa2δ-1
protein and with Western blotting for CaVa2δ-1 protein and b-actin,
used as a loadingcontrol. At the level of the dorsal horn (1), the
presence of a dorsal root ligature prevented upregulation of
CaVa2δ-1 protein regardless ofdelivery of intrathecal pregabalin or
intrathecal saline. At the dorsal root proximal to dorsal root
ligature (2), there was a similar lack ofupregulation of CaVa2δ-1
protein regardless of delivery of intrathecal pregabalin or
intrathecal saline, unless dorsal root ligature was absent. Atthe
level of the dorsal root distal to spinal nerve ligation (3), there
was an accumulation of CaVa2δ-1 protein, with less significant
CaVa2δ-1protein expression when intrathecal pregabalin was
delivered or if spinal nerve ligation was absent. Distal to the
dorsal root ganglia andproximal to spinal nerve ligation (4) or at
the spinal root distal to spinal nerve ligation (5), there was no
change in expression of CaVa2δ-1protein irregardless of delivery of
intrathecal pregabalin or intrathecal saline, or even if spinal
nerve ligation was absent. Multiple unmatchedANOVA tests were
performed between cohorts receiving intrathecal pregabalin and
saline as compared to control rat samples and sampleswithout
placement of either spinal or dorsal root ligatures, with *
indicating significant difference (p < 0.017 after Bonferroni
corrections)between cohorts. Bars = 10 μm.
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RETR
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D
RETR
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D
Dorsal and Spinal Root Ligation ExperimentsRETR
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D
Dorsal and Spinal Root Ligation Experiments
RETR
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There are limitations to the present study thatrequire
discussion. There is overlap between the tissuesthat receive
pregabalin from intranasal and intrathecaldelivery-it is probable
that both forms of deliveryimpacted upon the dorsal horn region,
but there mayhave been some undefined impact of intranasal
prega-balin delivery at supraspinal locations as well (evenwith the
lack of impact upon CaVa2δ-1 expression inbrain). The absence of
heightened CaVa2δ-1 expressionat the supraspinal locations does not
exclude the possi-bility of pregabalin possessing a role in
supraspinalpain processing-it is possible that pregabalin may
haveimportant effects at the medulla and cortex [65-68];this may
explain the effects of intranasal pregabalindelivery, which only
resulted in small concentrations ofpregabalin in the lumbar spinal
cord and dorsal rootganglia. Pregabalin’s effects within the
cerebrum maybe contributory, especially after demonstrations of
highcerebral concentratons of pregabalin following its intra-nasal
delivery. Although we did not demonstrateimpact of forms of
delivery of pregabalin upon micro-glia accumulation in the spinal
cord, it is possible thata longer duration of pregabalin delivery,
or earlieronset of pregabalin delivery, is required before an
anti-neuroinflammatory effect may be demonstrated, asshown in prior
studies [36,69,70]. For example, in amodel of diabetic peripheral
neuropathy with dailygabapentin delivery over 5 days followed by
immediatetissue harvesting, amelioration of spinal microglial
acti-vation occurred [71]. The levels of pregabalin achievedwithin
the lumbar spinal cord and dorsal root gangliawere measured to be
low after 74 hours-we hypothesizethat pregabalin’s effects may be
summated over timerather than related to an absolute
concentrationachieved at one time point, although we cannot
verifythis. We did not study a systemic application such aswith
gastric/oral or intravenous forms of delivery dueto the more
widespread distribution of gabepentinoidswith these methods and the
presence of prior dataassessing these delivery methods [72,73]. The
timing ofpregabalin/saline delivery in the spinal nerve
ligationexperiments was initiated 7 days after the spinal
nerveligation occurred in order to provide intervention dur-ing the
time of greatest neuropathic pain behavior;however, we selected the
first 7 days after double liga-tion to study CACNA2D1 mRNA and
CaVa2δ-1 pro-tein expression during the time period of
neuropathicpain behavior initiation-this difference in timing
mayhave led to inconsistencies in molecular test results.Although
we, and others [23], have hypothesized thatpregabalin’s mechanism
of action is the disruption ofCaVa2δ-1 trafficking, it remains
unclear why expres-sion of CACNA2D1 mRNA and CaVa2δ-1 protein
isunaffected at the dorsal root ganglia. Together, these
findings suggest that pregabalin’s action is independentof
translation of CaVa2δ-1 or transcription of CAC-NA2D1. As well, it
would be anticipated that disruptedtrafficking of CaVa2δ-1 would
result in CaVa2δ-1upregulation at the dorsal root ganglia, but this
wasnot identified. However, pregabalin-mediated effectsupon the
presence of CaVa2δ-1 protein at the moreproximal spinal nerve root
and dorsal horn is indicativeof pregabalin-induced suppression of
the central traf-ficking of CaVa2δ-1 protein, as previously
identified[23]. Rats received isoflurane anesthesia prior to
intra-nasal delivery, but were tested for neuropathic pain attimes
that were many half lives after isoflurane discon-tinuation to
avoid anesthetic effects upon behaviouraltesting. Finally, we
designed this study to compare dif-ferent modes of delivery of
pregabalin upon neuro-pathic pain behaviours; however, our results
cannot beused to interpret the site of action of pregabalin due
tosignificant overlap in pregabalin’s anatomical destina-tions with
the intranasal and intrathecal deliverymodes. However, we propose
that a non-invasive formof pharmacologically targeting the dorsal
horn and dor-sal root ganglia, such as with intranasal delivery,
maybe a consideration for the management of human neu-ropathic
pain.
ConclusionsThe present data confirm the efficacy of pregabalin
inthe modulation of acute thermal and tactile hypersensi-tivity as
features of neuropathic pain. The site of provi-sion is critical,
as exclusive delivery to the peripheralnerve has no impact; only
delivery using intranasal orintrathecal delivery was associated
with impact uponneuropathic pain behaviours and molecular
outcomes.Furthermore, delivery using either intranasal or
intrathe-cal methods led to diminished CaVa2δ-1 expression atthe
dorsal horn, suggesting that pregabalin inhibits cen-tral
trafficking of the CaVa2δ-1 subunit from the dorsalroot ganglia as
previously shown by Bauer et al [23].Future studies should examine
the in vivo and in vitrotrafficking of CaVa2δ-1 and the mechanisms
by whichpregabalin can influence the cell surface expression
ofCaVa2δ-1; another gabapentinoid, gabapentin, has beenshown to
prevent the recycling of the related proteinCaVa2δ-2 from endosomes
and the subsequent insertionin the plasma membrane without
influencing internali-zation of CaVa2δ-2 [55]. The different
impacts of prega-balin upon the central and peripheral axons of
thedorsal root ganglia neurons are not yet understood.Finally, the
intranasal delivery of agents such as prega-balin in order to
directly target the nervous system maybe a realistic method in
managing neuropathic pain inhumans, and may assist in the avoidance
of systemicadverse effects.
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RETR
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Dof pregabalin-induced suppression of the central traf-
RETR
ACTE
Dof pregabalin-induced suppression of the central traf-
-1 protein, as previously identified
RETR
ACTE
D-1 protein, as previously identified
[23]. Rats received isoflurane anesthesia prior to intra-
RETR
ACTE
D[23]. Rats received isoflurane anesthesia prior to intra-nasal
delivery, but were tested for neuropathic pain at
RETR
ACTE
Dnasal delivery, but were tested for neuropathic pain attimes
that were many half lives after isoflurane discon-
RETR
ACTE
Dtimes that were many half lives after isoflurane
discon-tinuation to avoid anesthetic effects upon behavioural
RETR
ACTE
Dtinuation to avoid anesthetic effects upon behaviouraltesting.
Finally, we designed this study to compare dif-
RETR
ACTE
Dtesting. Finally, we designed this study to compare dif-ferent
modes of delivery of pregabalin upon neuro-
RETR
ACTE
Dferent modes of delivery of pregabalin upon neuro-pathic pain
behaviours; however, our results cannot be
RETR
ACTE
Dpathic pain behaviours; however, our results cannot beused to
interpret the site of action of pregabalin due to
RETR
ACTE
Dused to interpret the site of action of pregabalin due to
RETR
ACTE
Da longer duration of pregabalin delivery, or earlier
RETR
ACTE
Da longer duration of pregabalin delivery, or earlieronset of
pregabalin delivery, is required before an anti-
RETR
ACTE
Donset of pregabalin delivery, is required before an anti-
may be demonstrated, as
RETR
ACTE
Dmay be demonstrated, as
shown in prior studies [36,69,70]. For example, in a
RETR
ACTE
Dshown in prior studies [36,69,70]. For example, in a
ral neuropathy with daily
RETR
ACTE
Dral neuropathy with daily
gabapentin delivery over 5 days followed by immediate
RETR
ACTE
Dgabapentin delivery over 5 days followed by immediatetissue
harvesting, amelioration of spinal microglial acti-
RETR
ACTE
D
tissue harvesting, amelioration of spinal microglial acti-vation
occurred [71]. The levels of pregabalin achieved
RETR
ACTE
D
vation occurred [71]. The levels of pregabalin achievedwithin
the lumbar spinal cord and dorsal root ganglia
RETR
ACTE
D
within the lumbar spinal cord and dorsal root gangliawere
measured to be low after 74 hours-we hypothesize
RETR
ACTE
D
were measured to be low after 74 hours-we hypothesizes effects
may be summated over time
RETR
ACTE
D
s effects may be summated over timerather than related to an
absolute concentration
RETR
ACTE
D
rather than related to an absolute concentrationachieved at one
time point, although we cannot verify
RETR
ACTE
D
achieved at one time point, although we cannot verifythis. We
did not study a systemic application such as
RETR
ACTE
D
this. We did not study a systemic application such aswith
gastric/oral or intravenous forms of delivery due
RETR
ACTE
D
with gastric/oral or intravenous forms of delivery dueto the
more widespread distribution of gabepentinoids
RETR
ACTE
D
to the more widespread distribution of gabepentinoidswith these
methods and theRE
TRAC
TED
with these methods and the presence of prior dataRETR
ACTE
D
presence of prior dataassessing these delivery methods [72,73].
The timing ofRE
TRAC
TED
assessing these delivery methods [72,73]. The timing
ofpregabalin/saline delivery in the spinal nerve ligationRE
TRAC
TED
pregabalin/saline delivery in the spinal nerve
ligationexperiments was initiated 7RE
TRAC
TED
experiments was initiated 7ligation occurred in order to provide
intervention dur-RE
TRAC
TED
ligation occurred in order to provide intervention dur-
significant overlap in pregabalin
RETR
ACTE
Dsignificant overlap in pregabalintions with the intranasal and
intrathecal delivery
RETR
ACTE
Dtions with the intranasal and intrathecal deliverymodes.
However, we propose that a non-invasive form
RETR
ACTE
Dmodes. However, we propose that a non-invasive formof
pharmacologically targeting the dorsal horn and dor-
RETR
ACTE
Dof pharmacologically targeting the dorsal horn and dor-sal root
ganglia, such as with intranasal delivery, may
RETR
ACTE
Dsal root ganglia, such as with intranasal delivery, maybe a
consideration for the management of human neu-
RETR
ACTE
Dbe a consideration for the management of human neu-ropathic
pain.
RETR
ACTE
Dropathic pain.
Conclusions
RETR
ACTE
DConclusionsThe present data confirm the efficacy of pregabalin
in
RETR
ACTE
DThe present data confirm the efficacy of pregabalin inthe
modulation of acute thermal and tactile hypersensi-
RETR
ACTE
D
the modulation of acute thermal and tactile hypersensi-
RETR
ACTE
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MethodsAnimalsAll experiments were carried out using male
SpragueDawley rats (Charles River Laboratories), weighing 200-225
grams at initiation. Protocols were reviewed andapproved by the
University of Calgary Animal CareCommittee using the Canadian
Council of Animal Careguidelines. All attempts were made to
minimize animalnumbers and to maintain ethical standards.
Experimen-tal study groups were randomized and behavioural stu-dies
were performed by an experimenter who wasunaware of treatment
groups.In all cases, rats were housed in plastic sawdust cov-
ered, pathogen-free cages with a normal light-dark cycleand free
access to chow and water. Rats were anesthe-tized with
pentobarbital (57 mg/kg) prior to all surgeriesand terminal
endpoints, and with inhaled isoflurane pro-vided for all intranasal
delivery points.
Sample size calculationsA sample size calculation for
intervention groups wasbased upon an anticipated difference in
neuropathicpain behavioral changes observed in treated diabetic
ratsto date, with an a of 0.05 and b of 0.5 providing a mini-mal
sample size of n = 5 within each interventiongroup; this was
increased to 6 rats due to anticipatedminimal diabetes-related
mortality over the three weekstudy.
Radiolabeled Pregabalin Studies to DetermineLocalization of
DeliveryWe performed radiolabelled studies to determine
thedistribution of pregabalin reaching the central and per-ipheral
nervous system after intranasal or intrathecaldelivery. We examined
the distribution of pregabalin at1, 2, and 5 h after 72 hours of
chronic delivery of intra-nasal, intrathecal, or near nerve
delivery. A combinationof 125I labeled pregabalin and unlabeled
pregabalin wasdelivered during studies at the Alzheimer’s
ResearchCenter at Regions Hospital in St. Paul, MN, USA.
Thisprocedure was approved by the Institutional AnimalCare and Use
Committee at Regions Hospital. Prior toexperimentation, 30 diabetic
rats (induced via streptozo-tocin injections, described below) were
sedated for intra-nasal or intrathecal delivery using
pentobarbitalanesthesia (60 mg/kg). 125I-labelled pregabalin was
pro-vided to 10 rats via intrathecal delivery, to 10 rats
viaintranasal delivery, and to 10 rats via near nerve delivery(see
below for procedure descriptions). Pregabalin (Pfi-zer Global, New
York, New York) with an initial con-centration 3.125 μg/μl was
dissolved in PBS and customlabeled with 125I (GE Healthcare,
Piscataway, New Jer-sey, USA). Synthesized radiolabelled pregabalin
solution
contained 266.7 μCi/μg. 125I-labeled pregabalin
delivery(intranasal or intrathecal) was performed in a fumehood
behind a lead impregnated shield, with anesthe-tized rats placed
supine. A mixture of 125I pregabalin(19.2 μCr) and unlabeled
pregabalin (30.0 μg) wereadministered intranasally or
intrathecally. 125I pregabalinwas intranasally administered over
alternating nares aseight 6-μL drops with an Eppendorf pipetter
every 2minutes, for a total volume of 48 μL, provided twicedaily
for 3 days prior to a single delivery on day 4 as pera prior
schedule [30]. For intrathecal delivery, 125I preg-abalin was
delivered using a placed intrathecal catheter(see below) attached
to an Alzet pump (describedbelow) with delivery of 2.04 mg/kg/d
over 72 hours con-tinually delivering a mixture of 125I pregabalin
(19.2μCr) and unlabeled pregabalin (30.0 μg) over each 24hour
period for 73, 74 and 75 hours prior to harvesting.For near nerve
delivery, 125I pregabalin was deliveredusing a placed T-chamber
around the proximal sciaticnerve (see below) attached to an Alzet
pump (describedbelow) with delivery of 2.04 mg/kg/d over 72 hours
con-tinually delivering a mixture of 125I pregabalin (19.2μCr) and
unlabeled pregabalin (30.0 μg) over each 24hour period for 73, 74
and 75 hours prior to harvesting.Each desired dose contained a
calculated radioactivedose of 40 μCi per day to best provide
concentrationssimilar to the long-term duration experiments
describedbelow.At each of 1, 2, and 5 hours after 72 hours of
initiat-
ing either 125I pregabalin intranasal delivery, intrathecalor
near-nerve delivery, cardiocentesis was performed forblood
extraction. Euthanasia was performed via trans-cardial perfusion
using 60 mL of saline, followed by 500mL of 4% paraformaldehyde
while the rat was main-tained under anesthesia. To quantify 125I
distribution,blood, urine, lymphatic (superficial
perimandibularlymph nodes and cervical lymph nodes) and
visceralorgan structures (quadriceps muscle, kidney, liver,
andlung), as well as portions of the central (olfactory
bulb,anterior olfactory cortex, frontal cortex, caudate puta-men,
parietal cortex, temporal cortex, hippocampus,septum, thalamus,
hypothalamus, midbrain, pons,medulla, cerebellum, cephalad cervical
spinal cord, cau-dal spinal cord, mid-thoracic spinal cord, and
lumbarspinal cord) and peripheral nervous systems (fifth lum-bar
dorsal root ganglia and proximal sciatic nerve) andassociated
tissues (ventral and dorsal cervical duramater) were harvested.
Gamma signal was recorded foreach body region with autoradiographic
imaging using aCOBRA II Auto-Gamma Counter (Perkin-Elmer, Wal-tham,
Mass., USA). Concentrations of 125I pregabalinwere calculated based
upon the gamma counting data,tissue weight, specific activity of
the drug administered
Martinez et al. Molecular Pain 2012,
8:3http://www.molecularpain.com/content/8/1/3
Page 12 of 20
RETR
ACTE
Dμ
RETR
ACTE
Dμ
daily for 3 days prior to a single delivery on day 4 as per
RETR
ACTE
Ddaily for 3 days prior to a single delivery on day 4 as pera
prior schedule [30]. For intrathecal delivery,
RETR
ACTE
Da prior schedule [30]. For intrathecal delivery,abalin was
delivered using a placed intrathecal catheter
RETR
ACTE
Dabalin was deli