Neuroscience Letters 571 (2014) 39–44

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A reversible functional sensory neuropathy model Aurore Danigo a , Laurent Magy a,b , Laurence Richard a,b , Franck Sturtz a , Benoît Funalot a,b , Claire Demiot a,∗ a b

EA 6309 – Schools of Medicine and Pharmacy – University of Limoges, France Service de Neurologie, Centre de référence national “neuropathies périphériques rares” – CHU Limoges, 87042 Limoges Cedex, France

h i g h l i g h t s • • • • •

Systemic resiniferatoxin induced reversible thermal and mechanical hypoalgesia. Nociception behaviors were restored three weeks after resiniferatoxin injection. Neuropeptides were depleted in cell bodies and terminals of primary sensory neurons. Resiniferatoxin did not induce nerve degeneration in this model. We developed a purely functional small-fiber neuropathy model.

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Article history: Received 27 January 2014 Received in revised form 7 April 2014 Accepted 19 April 2014 Available online 2 May 2014 Keywords: Small-fiber neuropathy Resiniferatoxin Intraepidermal nerve fiber Calcitonin-gene related peptide Substance P Nociception

a b s t r a c t Small-fiber neuropathy was induced in young adult mice by intraperitoneal injection of resiniferatoxin (RTX), a TRPV1 agonist. At day 7, RTX induced significant thermal and mechanical hypoalgesia. At day 28, mechanical and thermal nociception were restored. No nerve degeneration in skin was observed and unmyelinated nerve fiber morphology and density in sciatic nerve were unchanged. At day 7, substance P (SP) was largely depleted in dorsal root ganglia (DRG) neurons, although calcitonin gene-related peptide (CGRP) was only moderately depleted. Three weeks after, SP and CGRP expression was restored in DRG neurons. At the same time, CGRP expression remained low in intraepidermal nerve fibers (IENFs) whereas SP expression had improved. In summary, RTX induced in our model a transient neuropeptide depletion in sensory neurons without nerve degeneration. We think this model is valuable as it brings the opportunity to study functional nerve changes in the very early phase of small fiber neuropathy. Moreover, it may represent a useful tool to study the mechanisms of action of therapeutic strategies to prevent sensory neuropathy of various origins. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Painful neuropathy is a major cause of discomfort and disability and has a wide range of causes, among which are diabetes and chemotherapy for cancer treatment [1,2]. Longstanding neuropathic pain from peripheral origin is difficult to treat, partly because central sensitization may occur sometime after the peripheral nerves are damaged [3]. The cellular and molecular events

Abbreviations: CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion; IENFs, intraepidermal nerve fibers; PGP 9.5, protein gene product 9.5; RTX, resiniferatoxin; SP, substance P. ∗ Corresponding author at: EA 6309 «Maintenance myélinique et neuropathies périphériques», Faculté de Médecine et Pharmacie, 2, rue du Docteur Marcland, 87025 Limoges Cedex, France. Tel.: +33 5 55 43 59 15; fax: +33 0555435912. E-mail address: [email protected] (C. Demiot). http://dx.doi.org/10.1016/j.neulet.2014.04.026 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

that take place in the early stages of sensory neuropathy, before irreversible axonal damage, are not well characterized. However, these changes that may be reversible are probably important to understand, as they may constitute a window of opportunity to develop therapeutic strategies that prevent axon loss and chronic pain. Small fiber neuropathy is caused by selective damage to C and A␦ fibers (that are the main peripheral transmitters of nociception) and is an increasingly recognized cause of neuropathic pain [4]. Transient receptor potential vanilloid type 1 (TRPV1) is a non selective cation channel mainly expressed in sensory C- and A␦fibers. C- and A␦-fibers innervate dermis and epidermis, and respond to noxious stimuli [5]. Resiniferatoxin (RTX), an ultrapotent capsaicin analog that acts on TRPV1, which mediates nociceptive pathways, has been shown to induce neuropathies affecting only small-diameter unmyelinated sensory nerve fibers expressing TRPV1, by neuropeptide depletion and/or by nerve

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degeneration in rodents [6–8]. Sensory nerve terminals in the skin are the peripheral processes of small-diameter dorsal root ganglion (DRG) neurons. Recently, to investigate pathophysiology of sensory small-fiber neuropathy induced in a rodent model by intraperitoneal RTX administration, thermal nociception, expression of SP and CGRP in DRG neurons and in intraepidermal nerve fibers (IENFs) were evaluated [9–12]. Authors have shown that intraperitoneal injection of RTX induces small-fiber degeneration with thermal and mechanical hypoalgesia and tactile allodynia. Sensory behavior dysfunctions in RTX-induced neuropathy were reversible in the course of time after toxin injection in Sprague-Dawley rats [10,13] and in ICR male mice [14,15] despite irreversible nerve damage. However, sensory small fiber neuropathy in the long term is responsible for persistent neuropathic pain and allodynia, so it may be of importance in order to develop strategies to prevent small fiber neuropathy, to understand the changes that occur early its course. In the present work, we induced a reversible, purely functional neuropathy, by a single injection of RTX in young mice. This new model of sensory neuropathy is characterized by functional changes that are reversible in the course of experiments, without evidence of sensory nerve fiber degeneration at the peripheral and root level. Our systemic RTX mice model could mimic the early stage of diabetic neuropathy [16] and is reminiscent of what might be observed in chemotherapy-induced neuropathy with normal IENFs density [17]. This model may be valuable as it gives the opportunity to improve our knowledge about early mechanisms underlying sensory neuropathy. Moreover, it may allow in future studies, to test pharmacological substances to prevent the early sensory neuropathy development before irreversible degeneration. 2. Methods 2.1. Animals and RTX treatment Young (4–5 weeks) adult male Swiss mice (20–25 g) were randomly assigned in two groups of 6 each. One group received RTX (50 ␮g/kg, i.p., Sigma–Aldrich, Lyon, France) and other an equal volume of vehicle (10% DMSO, i.p.), under light isofluran anesthesia for avoiding unnecessary pain. Experiments were performed 7 days and 28 days after injection. Days of experiments were chosen in accordance with thermal nociception results (day 7: thermal hypoalgesia; day 28: thermal nociception recovery). Mice were housed in plastic cages and maintained on a 12-h light/dark cycle with food and water available ad libitum. The current investigation was conformed to the guidelines for ethical care of experimental animals of the European Community and was approved by the French Agriculture Ministry (authorization n◦ 87-019, Comité Régional d’Ethique sur l’Expérimentation Animale du Limousinn◦ 1-2013-1, 2).

grams. Three tests separated by at least 15 min were performed for each animal, and the mean value of these tests was calculated [18]. 2.3. Immunohistochemistry and quantification of intraepidermal nerve fibers (IENFs) in footpad skin and DRG neurons To assess for footpad skin innervation, animals were sacrificed by transcardiac perfusion with Phosphate Buffer Saline (PBS), followed by buffered 4% paraformaldehyde solution. Then, footpads were removed with a punch biopsy (diameter of 3 mm), post-fixed 6 h, cryoprotected (30% sucrose) and frozen at −20 ◦ C. Sections were cut on a cryostat at 20 ␮m and were incubated overnight with primary antibody to Protein gene product 9.5 (PGP 9.5, 1:600; UltraClone, Isle of Wight, UK), Substance P (SP, 1:100; Millipore, Molsheim, France) or Calcitonin gene-related peptide (CGRP, 1:1000; Abcam, Paris, France). Sections were then incubated with appropriate secondary antibodies Cy3-conjugated (1:500; Jackson Immunoresearch, Suffolk, UK) or AF488-conjugated (1:500; Life Technologies, Saint-Aubin, France). Epidermal nerve fibers were blindly counted under 400× magnification (Eclipse 50i, Nikon Instruments), according to established guidelines for Human [19]. The length of the dermo-epidermal junction was determined with NIS-Elements BR 2.30 software (Nikon) and was defined as the epidermal length. Epidermal nerve density was defined as the number of epidermal nerves divided by the epidermal length. To assess for the expression of the same markers in DRG neurons, two lumbar (L4-L5) DRG per mice were collected, and processed as described above except that 8 ␮m sections were sampled. Doublelabeling with PGP 9.5 and SP or CGRP was performed. Each DRG section was photographed at 200× under fluorescence microscope in a systematic fashion. Immunoreactive DRG neurons were counted and only the area containing neurons was measured with NIS-Elements BR 2.30 software (Nikon). The density of PGP 9.5(+) neurons was expressed as neurons/mm2 . The density of peptidergic neurons was expressed as CGRP(+) or SP(+) neurons/PGP 9.5(+) neurons. 2.4. Assessment of unmyelinated nerve fibers in sciatic nerves To assess for the presence and morphology of unmyelinated nerve fibers, sciatic nerves were dissected after transcardiac perfusion with 2.5% glutaraldehyde diluted in Sorensen buffer, dehydrated and embedded in Epon 812 resin (Euromedex, France). Semi-thin sections were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under an electron microscope (Jeol 1011). Photographs were taken at 25,000× magnification. Numbers of unmyelinated fibers enclosed within the basal lamina of single Schwann cells (i.e. a Remak bundle) were counted for comparison. 2.5. Data analysis

2.2. Nociception behaviors To assess thermal nociception, mice were placed on a 52 ◦ C hot plate (Bioseb, France), enclosed in a Plexiglas cage for less than 25 s to avoid potential tissue damage. Each test session consisted of three trials separated by 15 min. The criteria of withdrawal included shaking, licking, or jumping on the hot plate. The mean latency was expressed as the threshold of an individual animal to the thermal stimulation [9]. To assess mechanical nociception, tail pressure thresholds were registered with the Paw/Tail Pressure Analgesia meter for the Randall–Sellito test (Bioseb, Vitrolles, France). Pressure increasing at a linear rate of 16 g s−1 was applied to the base of the tail with a cut off of 250 g to avoid tissue injury. The applied tail pressure that evoked biting or licking behaviors was registered and expressed in

Data were expressed as means ± SEM. They were compared using an unpaired t test with a Gaussian distribution and a nonparametric Mann–Whitney test, which did not follow a Gaussian distribution. Significance was defined at p < 0.05. For comparison of thermal withdrawal latencies, two-way repeated measures ANOVAs was performed followed by Bonferroni’s post hoc test. 3. Results 3.1. Thermal and mechanical nociceptions in RTX mice Typical thermal hypoalgesia as measured by hot-plate withdrawal latencies was developed in the RTX group compared with the vehicle group (p < 0.0001; two-way ANOVA test). Before RTX

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Fig. 1. Effects of resiniferatoxin (RTX) on thermal and mechanical nociceptions. (a) Hot plate test. Withdrawal latencies to thermal stimuli (52 ◦ C) in vehicle and RTX mice. (b) Randall–Sellito tail pressure test. Mechanical withdrawal thresholds in vehicle and RTX mice. ***p < 0.001, **p < 0.01, *p < 0.05 vs. respective vehicle mice; n = 6 in each group.

treatment, the hot-plate test results were similar between RTX group and vehicle group (9.8 ± 1.0 vs. 10.6 ± 1.2 s, p > 0.05). Persistent thermal hypoalgesia was induced by RTX from day 7 (18.8 ± 3.1 vs. 10.4 ± 1.1 s, p < 0.05) to day 21 (16.2 ± 0.4 vs. 9.4 ± 1.8 s, p < 0.05) but was restored at day 28 (12.0 ± 2.0 vs. 9.0 ± 1.7 s, p > 0.05) (Fig. 1a). Similarly, at day 7, the tail pressure nociceptive threshold was significantly increased by 48% in RTX mice compared with vehicle mice (177.0 ± 12.3 vs. 119.9 ± 5.4 g, p < 0.001) and the mechanical withdrawal threshold was normalized at day 28 (143.3 ± 9.8 g, p > 0.05) (Fig. 1b). 3.2. No unmyelinated nerve degeneration after RTX administration Density of IENFs positive for protein gene product 9.5 (PGP 9.5) was unchanged in RTX mice compared with vehicle mice at day 7

and day 28 (Fig. 2a). Density of DRG neurons positive for PGP 9.5 was not affected either by RTX injection (Fig. 2b). Moreover, ultrastructural examination of sciatic nerves showed that unmyelinated fiber morphology was unchanged by RTX injection (Fig. 2c). At day 7 the number of unmyelinated fibers enclosed by Schwann cell in RTX group was similar to those of vehicle group (13.45 ± 1.5 vs. 14.2 ± 2.1, p > 0.05). 3.3. At day 7, RTX induced a large depletion of SP(+) and a mild depletion of CGRP(+) DRG neurons and IENFs At day 7, RTX induced a total depletion of SP (0 vs. 2.45 ± 0.3 SP(+) nerve fibers/mm, p < 0.01) (Fig. 3a and b) and a mild depletion of CGRP in IENFs of footpad skin (5 ± 0.5 vs. 7.6 ± 0.5 CGRP(+) nerve fibers/mm, p < 0.001) compared with vehicle mice (Fig. 3a and c). We observed a large depletion of SP (6.2 ± 0.5 vs. 0.1 ± 0.07 SP(+) neurons/PGP 9.5(+) neurons, p < 0.001) (Fig. 3a and d) and a

Fig. 2. Effects of RTX on unmyelinated nerve fiber and DRG neurons on Day 7 and Day 28. Foot pad skin and DRG neurons of vehicle and RTX mice were immunostained for protein gene product 9.5 (PGP 9.5) (a) Quantification of intraepidermal nerve fiber positive for PGP 9.5. n = 6 in each group. (b) Quantification of DRG neurons positive for PGP 9.5. The density of neurons was expressed as neurons/square millimeter. n = 12 in each group. (c) Unmyelinated fiber morphology in sciatic nerve was examined by electron microscopy. Scale bar = 1 ␮m.

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Fig. 3. Effect of RTX on intraepidermal nerve fibers (IENF) and dorsal root ganglion (DRG) neurons positive for substance P (SP) or calcitonin gene-related peptide (CGRP), 7 days and 28 days after injection. (a) Footpad skin of vehicle and RTX mice was immunostained for SP or CGRP and DRG neurons of vehicle and RTX mice were doubleimmunostained for SP or CGRP and PGP 9.5. Scale bar = 50 ␮m. ep, epidermis; d, dermis; sc, stratum corneum. (b and c) Quantification of intraepidermal nerve fiber positive for SP and CGRP. n = 6 in each group. (d and e) The density of neurons is expressed as SP(+) or CGRP(+) neurons/PGP 9.5(+) neurons. n = 12 in each group. ***p < 0.001, **p < 0.01, *p < 0.05 vs. vehicle. ††p < 0.01, †††p < 0.001 RTXd7 vs. RTXd28.

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mild decrease of CGRP in DRG neurons of RTX mice compared with vehicle mice (11.1 ± 0.6 vs. 14.9 ± 0.6 CGRP(+) neurons/PGP 9.5(+) neurons, p < 0.001) (Fig. 3a and e). 3.4. At day 28, improvement of SP(+) and of CGRP(+) IENFs and restoration of SP(+) and CGRP(+) DRG neurons At day 28, RTX mice had a significant improvement of SP(+) IENFs (1 ± 0.2 nerve fibers/mm; p = 0.0074) (Fig. 3a and b) and had a significant restoration of SP(+) DRG neurons (4.8 ± 0.7 SP(+) neurons/PGP 9.5(+) neurons; p < 0.001) (Fig. 3a and d) compared with RTX day 7 mice. SP expression in DRG neurons was restored at day 28 compared with vehicle mice (p > 0.05) (Fig. 3a and d). After 3 weeks, CGRP(+) IENFs remained mildly depleted (5.7 ± 0.7 IENFs/mm; p < 0.05) compared with RTX day 7 mice (Fig. 3a and c), but the number of CGRP(+) DRG neurons was restored compared with vehicle mice (12.6 ± 1.1 CGRP(+) neurons/PGP 9.5(+) neurons; p > 0.05) (Fig. 3a and e). 4. Discussion and conclusion We have shown in this report that a single injection of RTX may induce a purely transient functional small-fiber neuropathy, without overt nerve fiber loss or morphological alteration. RTXinduced small-fiber neuropathy model in mice was described by Hsieh et al. [9,14,7]. Single intraperitoneal RTX (50 ␮g/kg) injection to 8-week-old ICR mice lead to a degeneration of IENFs and DRG neurons, particularly cell bodies and terminals of peptidergic C-fibers. These authors described that RTX effects were stable throughout time from day 7 to day 56 after toxin injection. In our study, seven days after RTX injection, mice showed a classical thermal and mechanical hypoalgesia as Hsieh et al. studies [9,14,7]. However, IENFs positive for PGP 9.5 and total DRG neuron densities were not modified by RTX, suggesting that our model does not express a degenerative sensory neuropathy, confirmed by a normal morphological aspect and number of unmyelinated fibers in sciatic nerve. The neurotoxicity of vanilloids may differ dramatically depending on doses, administration modalities, animal species and method by which unmyelinated fibers are visualized [6]. Avelino and Cruz examined RTX-induced axonal degeneration in the rat bladder with the parallel use of immunohistochemistry and electron microscopy. They showed that RTX caused a profound reduction in SP and CGRP immunoreactivity without causing significant ultramorphometry changes [20]. Moreover, PGP 9.5 immunoreactivity, used in several RTX studies as an index of axon loss, could be lost because vanilloids cause axonal transport blockade that slows the arrival of PGP 9.5 to peripheral axon terminals, so the loss of PGP 9.5 immunoreactivity can be misinterpreted as neurodegeneration. This was obviously not the case in our model, as we did not observe a loss of PGP 9.5 immunoreactivity in footpad skin or signs of neurodegeneration. At day 7, RTX mice displayed a functional deficiency associated with a relatively minor effect on CGRP expression, and a preferential effect on SP expression in neurons, in accordance with the findings of Hsieh et al. [9]. Moreover, thermal withdrawal latencies were significantly higher than those of vehicle group from day 7 to day 21 and reached vehicle values at day 28. This deficiency induced by RTX was transient in our model, with a more quickly recovery of nociception behaviors, 28 days after toxin injection instead of 56 days [9]. This difference could be explained at day 7 by a mild sensory nerve alteration in our model in contrast to a severe neuropathy with nerve degeneration [9]. Recovery of functional nociceptive responses was not associated with a total restoration of SP and CGRP in IENFs, even if the number of DRG neurons positive for CGRP or SP was restored at day

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28. These findings are in accordance with the recent data of Hsieh et al. [9], who demonstrated that thermal sensation can be restored, while SP in IENFs was still depleted. Here, CGRP and SP content in IENFS were not fully restored. Recovery of functional nociceptive responses at day 28 might be due to compensatory mechanisms. Upregulation of neuropeptide receptors at spinal level could explain this phenomenon. Receptor upregulation could be due to prolonged TRPV1 stimulation by RTX as occurs during chronic inflammatory condition, or nerve injury [21,22]. Neuropeptides are synthesized in cell bodies of dorsal root ganglia neurons, and drive to peripheral afferent by antidromic axonal transport. To explain difference of SP expression between DRG neurons and IENFs at day 28, we suppose that SP stock in DRG cell bodies was recovered by synthesis, but that transport was uncompleted at the skin level. RTX might impair axonal transport by TRPV1 alteration. In summary, we have described a new model of mild smallfiber neuropathy in mouse. This model is characterized by a purely functional sensory nerve deficit induced by RTX with a mild CGRP depletion and a large SP depletion in sensory neurons, without overt nerve degeneration. Our model can mimic what is observed in some patients who experiment transient neuropathic pain in the course of chemotherapy for example. Loss of IENFs cannot explain pain in all cases, suggesting that different mechanism underpin the genesis of pain at various stages of sensory neuropathy [16]. During chemotherapy for example, results from patients revealed different evolutionary patterns of IENF density, but symptoms and IENF density were not related [23]. This model may be valuable in order to study the molecular and cellular events that take place in the early stages of sensory nerve fiber degeneration. Moreover, it may bring the opportunity to investigate the mechanisms by which several drug of interest exert their neuroprotective effects. Acknowledgement A. Danigo was supported by a grant from the “Conseil Régional du Limousin”.

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A reversible functional sensory neuropathy model.

Small-fiber neuropathy was induced in young adult mice by intraperitoneal injection of resiniferatoxin (RTX), a TRPV1 agonist. At day 7, RTX induced s...
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