RESEARCH Open Access Retracted: Comparison of central versus … · 2017. 8. 28. · Dura Dorsal...

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RESEARCH Open Access Jose A Martinez 1 , Manami Kasamatsu 1 , Alma Rosales-Hernandez 1 , Leah R Hanson 2 , William H Frey 2,3 and Cory C Toth 1* Abstract Background: Although pregabalin therapy is beneficial for neuropathic pain (NeP) by targeting the CaVa 2 δ-1 subunit, its site of action is uncertain. Direct targeting of the central nervous system may be beneficial for the avoidance of systemic side effects. Results: We 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 spinal nerve ligation. As well, radiolabelled pregabalin was administered to determine localization with different deliveries. We evaluated tactile allodynia and thermal hyperalgesia at multiple time points, and then analyzed harvested nervous system tissues for molecular and immunohistochemical changes in CaVa 2 δ-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 CaVa 2 δ-1 protein within peripheral nerve, dorsal root ganglia (DRG), and dorsal spinal cord, but not brain. Pregabalins effect was limited to suppression of CaVa 2 δ-1 protein (but not CACNA2D1 mRNA) expression at the spinal dorsal horn in neuropathic pain states. Dorsal root ligation prevented CaVa 2 δ-1 protein trafficking anterograde from the dorsal root ganglia to the dorsal horn after neuropathic pain initiation. Conclusions: Either intranasal or intrathecal pregabalin relieves neuropathic pain behaviours, perhaps due to pregabalins effect upon anterograde CaVa 2 δ-1 protein trafficking from the DRG to the dorsal horn. Intranasal delivery of agents such as pregabalin may be an attractive alternative to systemic therapy for management of neuropathic pain states. Keywords: neuropathic pain, pregabalin, diabetic peripheral neuropathy, spinal nerve ligation Background Neuropathic pain is a consequence of nerve damage or disease in the central and/or peripheral nervous system such 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 animal models 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 CaVa 2 δ-1 [5-7], particularly at the dorsal horn [8,9]. This is of importance for the clini- cal utility and potential mechanism of two different pharmacotherapies, gabapentin and pregabalin, both of which are indicated for the management of neuropathic pain due to multiple etiologies. There is uncertainty about the anatomical location of pregabalins beneficial effect, as CaVa 2 δ-1 subunit upregulation is multifocal [10]. In addition to the known expression within the DRG, CACNA2D1 mRNA is also localized to brain regions known to be involved in cortical processing, sensory conduction, and arousal [11]. Also, there is also marked CaVa 2 δ-1 subunit expression in spinal cord regions where DRG projections occur. As a result, the localization of pregabalins greatest therapeutic effect is * Correspondence: [email protected] 1 Department of Clinical Neurosciences and the University of Calgary, Calgary, AB, Canada Full list of author information is available at the end of the article Martinez et al. Molecular Pain 2012, 8:3 http://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 Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Retracted: Comparison of central versus peripheral delivery of pregabalin in neuropathic pain states A retraction article was published for this article. It is available from the following link; http://www.molecularpain. 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RETRACTED 2 nd April 2014 doi:10.1186/1744-8069-10-20

Transcript of RESEARCH Open Access Retracted: Comparison of central versus … · 2017. 8. 28. · Dura Dorsal...

  • 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.

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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, 8:3http://www.molecularpain.com/content/8/1/3

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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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    pathy and spinal nerve ligation models. Medium (0.51mg/kg/d) doses of pregabalin decreased thermal hyperal-

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    gesia due to diabetic peripheral neuropathy and spinalnerve ligation without impact upon tactile allodynia.

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    or thermal hyperalgesia in either model. Near-nervedelivery of pregabalin had no impact upon tactile allody-RE

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    nia or thermal hyperalgesia with any dose (data notshown) in either model of neuropathic pain.RE

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    shown) in either model of neuropathic pain.mRNA Expression for CACNA2D1 and Other Voltage GatedRE

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  • 42

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    Intranasal Pregabalin (2.04 mg/kg)Intranasal Saline

    Control Rat

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    Thermal Hyperalgesia Measurements in DPN with Intranasal Pregabalin

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    Tactile Allodynia Measurements in SNL with Intranasal Pregabalin50

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    Intranasal Saline

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    Thermal Hyperalgesia Measurements in SNL with Intranasal Pregabalin

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    Thermal Hyperalgesia Measurements in SNL with Intrathecal Pregabalin

    Intrathecal Pregabalin (2.04 mg/kg)Intrathecal Pregabalin (0.51 mg/kg)Intrathecal Saline

    Control Rat

    * **

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    Thermal Hyperalgesia Measurements in DPN with Intrathecal Pregabalin

    A

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    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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  • 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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    Dand assembly of channels at the cell surface [45]. CAC-NA2D1 is transcribed as a single mRNA from a single

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    gene, with the translated CaV

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    CACNA2D1 mRNA was upregulated in both dorsal spinal cord (

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    DA)

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    ) in the presence of either diabetic peripheral

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    D) in the presence of either diabetic peripheral

    neuropathy (DPN) or spinal nerve ligation (SNL). Dorsal horn mRNA

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    neuropathy (DPN) or spinal nerve ligation (SNL). Dorsal horn mRNAexpression was unchanged by presence of high dose pregabalin

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    expression was unchanged by presence of high dose pregabalin(2.04 mg/kg/d) provided either through intrathecal or intranasal

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    (2.04 mg/kg/d) provided either through intrathecal or intranasaldelivery when compared to saline delivery (

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    delivery when compared to saline delivery (A

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    A). There was no impact

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    ). There was no impactof pregabalin delivered in any manner upon mRNA expression in

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    of pregabalin delivered in any manner upon mRNA expression in

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    lumbar DRGs when compared to saline delivery, although the

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    lumbar DRGs when compared to saline delivery, although thepresence of spinal nerve ligation or diabetic peripheral neuropathy

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    presence of spinal nerve ligation or diabetic peripheral neuropathywas associated with elevated CACNA2D1 mRNA expression when

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    was associated with elevated CACNA2D1 mRNA expression whencompared to control rat specimens (

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    compared to control rat specimens (B

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    B), indicated by the * above

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    ), indicated by the * abovethe Control rat data (non-matched ANOVA tests,

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    the dorsal root but not spinal nerve at 7 days after pro-RETR

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    the dorsal root but not spinal nerve at 7 days after pro-cedures were performed (Figure 7). Dorsal horn expres-RE

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    between 0.42-1.00 for indicated groups and time points,

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    between 0.42-1.00 for indicated groups and time points,< 0.0125) [n = 6-8 specimens for each cohort].

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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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    . Western blotting identified increased levels of CaVexposed to either diabetic peripheral neuropathy (DPN) or spinal nerve ligation (SNL) when compared to control rats (RE

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    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

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    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

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    B). The near nerve delivery of pregabalin failed to impact upon CaVRETR

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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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    blishment of neuropathic

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    blishment of neuropathicpain, confirming the results of Bauer et al [23]. These

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    pain, confirming the results of Bauer et al [23]. Thesesame unmyelinated DRG sensory neurons important for

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    same unmyelinated DRG sensory neurons important fornociception (unmyelinated C-fibers for transmission of

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    nociception (unmyelinated C-fibers for transmission ofthermal hyperalgesia and large-diameter A

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    thermal hyperalgesia and large-diameter Aδ

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    δ afferent

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    afferentfibers for transmission of tactile allodynia [48,49])

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    fibers for transmission of tactile allodynia [48,49])synapse in laminae I and II of the dorsal horn or at Lis-

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    synapse in laminae I and II of the dorsal horn or at Lis-s tract. We determined that CaV

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    s tract. We determined that CaVa

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    aeactivity was elevated unilaterally with spinal nerve

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    eactivity was elevated unilaterally with spinal nerveligation and bilaterally with diabetic peripheral neuropa-

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    ligation and bilaterally with diabetic peripheral neuropa-thy in the dorsal column at thoracolumbar levels aboveRE

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    thy in the dorsal column at thoracolumbar levels abovethe spinal nerve ligation injury, as well as in DRG neu-RE

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    the spinal nerve ligation injury, as well as in DRG neu-rons [23]. Most importantly, decreases in CaVRE

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    rons [23]. Most importantly, decreases in CaVimmunoreactivity at the dorsal horn were associatedRE

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    immunoreactivity at the dorsal horn were associatedwith successful analgesia of neuropathic pain with preg-RE

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    with successful analgesia of neuropathic pain with preg-

    -1 protein [55]. There has been no

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    D-1 protein [55]. There has been no

    2

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    D-1 protein or CAC-

    NA2D1 mRNA elsewhere, though, such as at the DRG

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    DNA2D1 mRNA elsewhere, though, such as at the DRG(despite the presence of all three CaV

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    D(despite the presence of all three CaVa

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    Dδ subunits at

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    Dsubunits atthe DRG) [11] and in the peripheral nerve. This may

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    Dthe DRG) [11] and in the peripheral nerve. This maysignify that trafficking, bu

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    Dsignify that trafficking, but not expression or endocy-

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    Dt not expression or endocy--1 from DRG to presynaptic terminals

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    D-1 from DRG to presynaptic terminalsis impacted upon by pregabalin [23]. Other potential

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    Dis impacted upon by pregabalin [23]. Other potentialmechanisms for gabapentinoids include a more acute

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    Dmechanisms for gabapentinoids include a more acuteinhibitory effect upon neurotransmitter release [56]

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    Dinhibitory effect upon neurotransmitter release [56]which may relate to upregulation of protein kinase C

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    Dwhich may relate to upregulation of protein kinase Cactivation [57,58].

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    Dactivation [57,58].Chronic, and not acute, pregabalin delivery reduces

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    DChronic, and not acute, pregabalin delivery reduces

    the neuronal expression of CaV

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    [23]. This effect may be a consequence of calcium chan-

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    D[23]. This effect may be a consequence of calcium chan-nel trafficking through the Von Willebrand factor-A

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    mutants for CaV

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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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  • 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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    [23]. Rats received isoflurane anesthesia prior to intra-

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    D[23]. Rats received isoflurane anesthesia prior to intra-nasal delivery, but were tested for neuropathic pain at

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    Dnasal delivery, but were tested for neuropathic pain attimes that were many half lives after isoflurane discon-

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    Dtimes that were many half lives after isoflurane discon-tinuation to avoid anesthetic effects upon behavioural

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    Dtinuation to avoid anesthetic effects upon behaviouraltesting. Finally, we designed this study to compare dif-

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    Dferent modes of delivery of pregabalin upon neuro-pathic pain behaviours; however, our results cannot be

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    Dpathic pain behaviours; however, our results cannot beused to interpret the site of action of pregabalin due to

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    may be demonstrated, as

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    shown in prior studies [36,69,70]. For example, in a

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    ral neuropathy with daily

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    gabapentin delivery over 5 days followed by immediate

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    tissue harvesting, amelioration of spinal microglial acti-vation occurred [71]. The levels of pregabalin achieved

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    vation occurred [71]. The levels of pregabalin achievedwithin the lumbar spinal cord and dorsal root ganglia

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    within the lumbar spinal cord and dorsal root gangliawere measured to be low after 74 hours-we hypothesize

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    were measured to be low after 74 hours-we hypothesizes effects may be summated over time

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    s effects may be summated over timerather than related to an absolute concentration

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    D

    rather than related to an absolute concentrationachieved at one time point, although we cannot verify

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    D

    achieved at one time point, although we cannot verifythis. We did not study a systemic application such as

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    D

    this. We did not study a systemic application such aswith gastric/oral or intravenous forms of delivery due

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    with gastric/oral or intravenous forms of delivery dueto the more widespread distribution of gabepentinoids

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    D

    to the more widespread distribution of gabepentinoidswith these methods and theRE

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    with these methods and the presence of prior dataRETR

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    D

    presence of prior dataassessing these delivery methods [72,73]. The timing ofRE

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    assessing these delivery methods [72,73]. The timing ofpregabalin/saline delivery in the spinal nerve ligationRE

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    pregabalin/saline delivery in the spinal nerve ligationexperiments was initiated 7RE

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    experiments was initiated 7ligation occurred in order to provide intervention dur-RE

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    ligation occurred in order to provide intervention dur-

    significant overlap in pregabalin

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    Dsignificant overlap in pregabalintions with the intranasal and intrathecal delivery

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    Dtions with the intranasal and intrathecal deliverymodes. However, we propose that a non-invasive form

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    Dmodes. However, we propose that a non-invasive formof pharmacologically targeting the dorsal horn and dor-

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    Dof pharmacologically targeting the dorsal horn and dor-sal root ganglia, such as with intranasal delivery, may

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    Dsal root ganglia, such as with intranasal delivery, maybe a consideration for the management of human neu-

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    Dbe a consideration for the management of human neu-ropathic pain.

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    Dropathic pain.

    Conclusions

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    DConclusionsThe present data confirm the efficacy of pregabalin in

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    DThe present data confirm the efficacy of pregabalin inthe modulation of acute thermal and tactile hypersensi-

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    D

    the modulation of acute thermal and tactile hypersensi-

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    D 2

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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

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    daily for 3 days prior to a single delivery on day 4 as per

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    Ddaily for 3 days prior to a single delivery on day 4 as pera prior schedule [30]. For intrathecal delivery,

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    Da prior schedule [30]. For intrathecal delivery,abalin was delivered using a placed intrathecal catheter

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    Dabalin was deli