Effect of a Single Dose of Oxaliplatin on the Induction of Peripheral Neuropathy in a Rat Model: An in Vivo Electrophysiological Study

Abstract

The anticancer drug oxaliplatin is associated with peripheral neuropathy as a side effect accompanied by mechanical and cold allodynia. Although the superficial layer of the spinal cord dorsal horn is known to receive information primarily from peripheral pain nerves, to our knowledge, no in vivo electrophysiological analyses have been conducted to determine whether oxaliplatin administration increases the excitability of superficial layer neurons. Therefore, in vivo extracellular recordings were performed to measure action potentials in the deep and superficial layers of the spinal cord dorsal horn in rats treated with a single dose (6 mg/kg) of oxaliplatin. Action potentials were produced by mechanical stimulation with von Frey filaments to the hindlimb receptive fields. The results revealed that the firing frequency of action potentials increased relative to the intensity of mechanical stimulation, and that both deep and superficial layer neurons in the spinal cord dorsal horn increased significantly in oxaliplatin-treated compared with vehicle-treated rats, especially in the superficial layer. Several superficial layer neurons showed spontaneous firing that was not seen in vehicle-treated rats. In addition, a clear increase was seen in the firing frequency of neurons in the superficial layer of oxaliplatin-treated rats in response to a cold stimulus (here, the addition of acetone to the hindlimb receptive field). This study suggests that the superficial spinal cord dorsal horn strongly reflects the pain pathophysiology in peripheral neuropathy induced by oxaliplatin administration, and that the superficial layer neurons are useful for in vivo electrophysiological analysis using this pathological model.

INTRODUCTION

Drugs used to treat cancer often damage peripheral nerves. This common side effect of anticancer drug-induced peripheral neuropathy can worsen the QOL of patients with cancer and even lead to treatment interruption or discontinuation, and in turn, has a significant adverse impact on life expectancy. Chemotherapy-induced peripheral neuropathy (CIPN) is a common type of peripheral neuropathy caused by anticancer drugs.¹⁾

Oxaliplatin (OXA) is a third-generation, platinum-based, chemotherapy drug used to treat metastatic colorectal,^2,3) breast, lung, and ovarian cancers.⁴⁾ Its effectiveness has also been reported for treating advanced esophagogastric⁵⁾ and pancreatic cancers.⁶⁾ However, an association has also been reported between OXA and peripheral neuropathy, accompanied by symptoms such as cold and mechanical allodynia as side effects,^4,5,7) and this has become a problem in the clinical setting. However, the pathogenesis of the disease remains unclear, and effective prevention and treatment methods have not yet been established.⁸⁾

Thin myelinated Aδ and unmyelinated C afferents are responsible for the transmission of nociceptive information from the periphery to the superficial (lamina I and II) and deep (lamina V) layers of the spinal cord dorsal horn.⁹⁾ For the most part, C fibers terminate in lamina II, while the obtained nociceptive information is transmitted from interneurons in lamina II to wide dynamic range (WDR) and nociceptive-specific neurons in laminae V and I, respectively. This nociceptive information is then modified and integrated by interneurons in lamina II, which also control the output.¹⁰⁾ Thus, lamina II is a pain-specific region that processes pain-related information and strongly reflects functional changes in neurons and glial cells when nerves are damaged and pain pathology occurs.^11,12) For this reason, much of the research on neuropathic pain has been conducted in lamina II, and the same is true of electrophysiological approaches.^13,14)

However, in vivo electrophysiological evaluation of OXA-induced CIPN models has so far been based on the response of WDR neurons.^15,16) As noted above, the responses exhibited by WDR neurons can be modified by interneurons in lamina II, and the responses do not necessarily reflect pain. Therefore, the electrophysiological changes in lamina II need to be investigated to elucidate the pathogenesis of OXA-induced CIPN.

Given the background, the present study aimed to record and compare the firing of neurons in the deep and superficial layers of the spinal cord dorsal horn in rats treated with a single dose of OXA (6 mg/kg), which is known to induce cold and mechanical allodynia.^15,16)

MATERIALS AND METHODS

All experiments in this study were performed in accordance with the Physiological Society of Japan’s “Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences” and approved by the University of Toyama Animal Experiment Committee and the Pharmaceutical Research Center of Asahi Kasei Pharma Corporation’s Institutional Animal Care Committee. Every effort was made to minimize animal suffering and the number of animals used in the studies.

Animals

Male Sprague Dawley rats (age 7 weeks) obtained from Charles River Laboratories Japan (Yokohama, Japan) were used in the experiments. All animals were housed in a room under controlled conditions (temperature 22–23 °C; relative humidity 45–65%; 12-h light/dark cycle) and given ad libitum access to food and water.

Animal Model

Neuropathic pain model rats were induced with OXA, as described elsewhere.¹⁷⁾ Each rat was intravenously injected with OXA (6 mg/kg; Yakult Honsha, Tokyo, Japan) or water (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) after being anesthetized with isoflurane. At 3–7 d after OXA or water injection, in vivo extracellular recordings were obtained.

Extracellular Recording from Spinal Dorsal Horn (SDH) Neurons in Vivo

The in vivo recordings of SDH neurons were carried out as described previously.^14,18) Briefly, after being anesthetized with urethane (1.2–1.5 g/kg, intraperitoneally (i.p.)), lumbar laminectomy was carried out to expose the spinal column from lumbar 1 (L1) to 5 (L5). The animals were then placed in a stereotaxic apparatus (Fig. 1A). Next, the dura mater was removed and the arachnoid membrane was cut away to create a sufficiently large window for an electrode (tungsten microelectrode; tip diameter 25 µm; tip impedance 0.9–1.2 and 9–12 MΩ for the deep and superficial layers, respectively; FHC, Bowdoin, ME, U.S.A.), and then the spinal cord surface was irrigated with 95% O₂–5% CO₂-equilibrated Krebs solution containing 117 mM NaCl, 3.6 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM NaH₂PO₄, 11 mM glucose, and 25 mM NaHCO₃ at 37 ± 1 °C for 10–15 mL/min through glass pipettes. Extracellular single-unit recordings were made of SDH neurons, and spikes were selected based on amplitude discrimination, as previously described.^14,18) A tungsten microelectrode was then inserted into the ipsilateral side of the spinal cord at a 20–30° angle (latero-medial), and recordings were made from neurons 20–800 µm below the surface, corresponding to laminae I–V (Fig. 1B). The Clampfit software package (version 10.2; Molecular Devices, Union City, CA, U.S.A.) or Sutter patch software (Sutter Instrument, Novato, CA, U.S.A.) was used to amplify (EX1; Dagan Corporation, Minneapolis, MN, U.S.A.), digitize (Digidata 1550A; Molecular Devices, or IPA; Sutter Instrument), and display unit signals online. Next, regions on the skin where a noxious pinch with forceps or a touch with a cotton wisp and/or light brush produced a neural response were identified. Neurons were classified as WDR-type or nociceptive-specific if they responded to innocuous mechanical stimulation and a noxious pinch in a graded manner, or to a noxious pinch, but not the cotton wisp or brush stimuli, respectively. Then, mechanical stimulation was applied to the skin for 10 s using von Frey filaments (vFFs; North Coast Medical, Morgan Hill, CA, U.S.A.),¹⁴⁾ followed by a drop of acetone (50 µL) to the identified receptive field for cold stimulation.

Fig. 1. In Vivo Extracellular Recording of the Spinal Dorsal Horn Neurons

(A) Schematic diagram of the in vivo preparation of rats. The rats were fixed using stereotaxic instruments under urethane anesthesia and inhaled oxygen gas throughout the experiment. Mechanical stimuli (i.e., a soft brush, pinching by forceps, or poking with von Frey filaments [vFFs]) or acetone drops were administered to the identified receptive field. (B) A tungsten electrode was inserted to the superficial (left, 20–150 µm) or deep layer (right, 150–800 µm) of spinal dorsal horn neurons from the lumbar spinal cord (L4–L5).

Statistical Analysis

All measured values were expressed as the mean ± standard error of the mean. The Wilcoxon rank-sum test was used for all statistical testing, and SAS (ver. 9.4; SAS Institute, Cary, NC, U.S.A.) and EXSUS (ver. 10.0; CAC EXICARE, Tokyo, Japan) were used for all statistical analyses. For all tests, values of p < 0.05 were considered statistically significant.

RESULTS

All results showed in this paper were obtained using rats 3 to 7 d after a single dose of OXA or water for injection.

In Vivo Extracellular Recordings in the Deep Layer of the Spinal Cord

To study whether OXA model rats had altered neural excitability, single-unit recordings of deep SDH neurons in the in vivo preparation were performed. Spontaneous neuronal firing was observed in the deep SDH neurons of the sham and OXA model rats (Figs. 2A, B). The SDH neurons were then subjected to a series of vFFs and mechanically evoked firing was recorded. Mechanical stimulus intensity-dependent increases in the firing rate were observed in the deep SDH neurons in the sham and OXA model rats (Figs. 2A, C). The firing rate was significantly increased at most stimulation intensities in the OXA compared with the sham rats (Figs. 2A, C). The recording depth (measured from the dorsal surface of the spinal cord to the point of contact with the neuron recorded) of the deep SDH neurons was 150–800 µm. No significant difference in depth was seen between the sham and OXA model rats (421 ± 14.3 µm, n = 44 vs. 415 ± 14.7 µm, n = 49, respectively; p = 0.732).

Fig. 2. Spontaneous and vFF-Evoked Firing of Deep Spinal Dorsal Horn Neurons in Sham and OXA Model Rats

(A) Representative trace of spontaneous and vFF (1.0, 8.0, and 26.0 g)-evoked firing recorded from the sham (upper) and the OXA model (lower) rats. (B) Frequency of spontaneous neuronal firing in the sham (n = 44) and OXA model rats (n = 49). (C) Frequency of vFF-evoked firing in the sham (n = 44) and OXA model rats (n = 49). * p < 0.05, ** p < 0.01, *** p < 0.001.

In Vivo Extracellular Recordings in the Superficial Layer of the Spinal Cord

In the superficial SDH neurons of the sham rats, spontaneous neuronal firing was seldom observed. By contrast, compared to the sham rats, significantly more superficial SDH neurons of the OXA model rats showed spontaneous firing (Figs. 3A, B). In addition, mechanically evoked firing recorded after a series of vFFs revealed that the superficial SDH neurons showed mechanical stimulus intensity-dependent increases in the firing rate in both the sham and OXA model rats (Figs. 3A, C). The firing rate was significantly increased in the OXA model compared with the sham rats at all stimulation intensities (Figs. 3A, C).

Fig. 3. Increased Spontaneous and vFF-Evoked Firing of the Superficial Spinal Horn Neurons in the Sham and OXA Model Rats

(A) Representative trace of spontaneous and vFF (1.0, 8.0, and 26.0 g)-evoked firing recorded from the sham (upper) and the OXA model (lower) rats. (B) Frequency of spontaneous firing in the sham (n = 27) and OXA model rats (n = 37). (C) Summary showing the frequency of vFF (1.0, 4.0, 8.0, 26.0 and 60.0 g)-evoked firing in sham (n = 27) and OXA model (n = 37) rats. *** p < 0.001.

The recording depth (measured from the dorsal surface of the spinal cord to the point of contact with the neuron recorded) of the superficial SDH neurons was 20–150 µm. No significant difference in depth was observed between the sham and OXA model rats (69.7 ± 6.2 µm, n = 27 vs. 72.7 ± 5.4 µm, n = 37, respectively; p = 0.698).

Effects of the Topical Application of Acetone on the Frequency of Spontaneous Neuronal Firing in Superficial SDH Neurons

Cold dysesthesia is recognized as a characteristic symptom of OXA-induced peripheral neuropathy in both humans⁷⁾ and animals.^15,16) Immediately after the administration of a drop of acetone on the receptive field, the vast majority of superficial SDH neurons in the sham rats showed transient firing (less than 5 min) in response to the stimulus (Fig. 4, n = 22). By contrast, most of the superficial SDH neurons in the OXA model rats showed a longer duration of firing (more than 5 min) in response to the acetone stimulus (Fig. 4, n = 37). In addition, the frequency of acetone-evoked firing was significantly higher in the OXA model than in the sham rats (Fig. 4).

Fig. 4. Increased Acetone-Evoked Firing of the Superficial Spinal Horn Neurons in the Sham and OXA Model Rats

Frequency of acetone-evoked firing in the sham (n = 22) and OXA model rats (n = 37). *** p < 0.001.

DISCUSSION

In this study, we recorded the firing of neurons in the deep and superficial layers of the spinal cord dorsal horn in rats administered a single dose of OXA (6 mg/kg). In deep layer neurons, mechanical stimulation with vFFs increased the firing frequency, replicating previous reports.^15,16) The hypersensitive response was more intense in the superficial than in the deep layer neurons, including spontaneous firing, which did not occur in the sham group, and a substantial increase in the firing frequency in response to a mechanical stimulus. Moreover, a significant increase in the firing frequency was seen immediately after cold stimulation with acetone. There are also reports of behavioral evaluations in which mechanical and cold hypersensitivity persisted for about 10 d in rats administered a single dose of 6 mg/kg OXA, which is consistent with these results.^19–21) Although previous studies utilizing neuropathic pain models have reported the hypersensitivity responses of neurons in the superficial layer observed in in vivo electrophysiological evaluations,^13,14) to our knowledge, this is the first study to use a single dose of OXA in a CIPN model. These results suggest that superficial layer neurons are suitable for in vivo electrophysiological assessment of OXA-induced adverse effects.

The hypersensitive response of superficial layer neurons may be explained by various factors, such as the increased release of glutamate from primary nerve endings,¹⁴⁾ the decreased synaptic transmission of γ-aminobutyric acid (GABA)ergic neurons,^14,22) and the activation of glial cells within the spinal cord dorsal horn.¹¹⁾ However, whether these changes occur in OXA-induced neuropathy remains unclear. In the clinical setting, diabetic neuropathy and CIPN are characterized by similar symptoms.²³⁾ In a rat diabetic neuropathy model induced with streptozotocin (STZ), electrical stimulation of the dorsal root attached spinal cord slices resulted in an increased frequency of glutamatergic miniature excitatory postsynaptic currents (EPSCs), as well as an increased amplitude of monosynaptic EPSCs in lamina II neurons.²⁴⁾ In addition, in STZ models, activated microglia have been shown to be increased within the spinal cord dorsal horn,²⁵⁾ and synapsin II, a synaptic vesicle protein associated with the glutamate regulation and GABA release in the spinal cord, has been shown to be upregulated in the dorsal horn surface of the spinal cord,^26,27) which suggests that the development of neuropathic pain may be caused by an imbalance between glutamatergic and GABAergic synaptic transmission. Whether these changes occur in rats treated with a single dose of OXA should be examined in the future.

In previous studies, cold allodynia was observed in a rat model of OXA-induced neuropathy, and a significant increase in the firing frequency of WDR neurons after cold stimulation using a drop of acetone to the receptive field was seen in an in vivo electrophysiological evaluation of a rat model.^15,16) When a drop of acetone was tested in neurons in the superficial layer, a marked increase was observed in the firing frequency after the addition of acetone among rats treated with a single dose of OXA (Fig. 4). Although the mechanism of OXA-induced cold allodynia has not yet been fully explained, there are reports that OXA or its metabolites activate TRPA1 expressed in DRGs²⁸⁾ and induce increases in TRPM8 mRNA^29,30) and protein levels.³⁰⁾ The expression of these channels on C fibers^28,31) suggests the increased firing frequency of superficial layer neurons observed with the drop of acetone.

In conclusion, the comparison of neuronal firing frequencies carried out in the present study of a CIPN rat model treated with a single dose of OXA revealed that pain pathology is reflected more strongly by the superficial than by the deeper layer of the SDH. Therefore, the superficial layer would be useful for in vivo electrophysiological analysis of the CIPN model. Furthermore, as dynamic plastic changes in the superficial layer might lead to a strong hypersensitive neuronal response, detailed analysis of the effects of OXA on the superficial layers necessitates the recording of synaptic-level responses, and changes in the upper centers (e.g., descending inhibitory system) should also be considered.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Nos. 22K09020 and 19K09323 to D.U., and in part by the Adaptable and Seamless Technology transfer Program through Target-driven R&D (A-STEP) from the Japan Science and Technology Agency (JST) (to D.U.; JPMJTM20DN). This study was also supported in part by Asahi Kasei Pharma Corp.

Conflict of Interest

D.U. has no conflict of interest to declare. K.T., K.F., H.K., and A.I. are employees of Asahi Kasei Pharma Corp.

REFERENCES

• 1) Flatters SJL, Dougherty PM, Colvin LA. Clinical and preclinical perspectives on chemotherapy-induced peripheral neuropathy (CIPN): a narrative review. Br. J. Anaesth., 119, 737–749 (2017).
• 2) de Gramont A, Figer A, Seymour M, Homerin M, Hmissi A, Cassidy J, Boni C, Cortes-Funes H, Cervantes A, Freyer G, Papamichael D, Le Bail N, Louvet C, Hendler D, de Braud F, Wilson C, Morvan F, Bonetti A. Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J. Clin. Oncol., 18, 2938–2947 (2000).
• 3) André T, Boni C, Mounedji-Boudiaf L, Navarro M, Tabernero J, Hickish T, Topham C, Zaninelli M, Clingan P, Bridgewater J, Tabah-Fisch I, de Gramont A. Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N. Engl. J. Med., 350, 2343–2351 (2004).
• 4) Belani CP. Recent updates in the clinical use of platinum compounds for the treatment of lung, breast, and genitourinary tumors and myeloma. Semin. Oncol., 31 (Suppl. 14), 25–33 (2004).
• 5) Cunningham D, Starling N, Rao S, Iveson T, Nicolson M, Coxon F, Middleton G, Daniel F, Oates J, Norman AR. Capecitabine and oxaliplatin for advanced esophagogastric cancer. N. Engl. J. Med., 358, 36–46 (2008).
• 6) Suker M, Beumer BR, Sadot E, Marthey L, Faris JE, Mellon EA, El-Rayes BF, Wang-Gillam A, Lacy J, Hosein PJ, Moorcraft SY, Conroy T, Hohla F, Allen P, Taieb J, Hong TS, Shridhar R, Chau I, van Eijck CH, Koerkamp BG. FOLFIRINOX for locally advanced pancreatic cancer: a systematic review and patient-level meta-analysis. Lancet Oncol., 17, 801–810 (2016).
• 7) Quasthoff S, Hartung HP. Chemotherapy-induced peripheral neuropathy. J. Neurol., 249, 9–17 (2002).
• 8) Wolf S, Barton D, Kottschade L, Grothey A, Loprinzi C. Chemotherapy-induced peripheral neuropathy: prevention and treatment strategies. Eur. J. Cancer, 44, 1507–1515 (2008).
• 9) Yoshimura M. Slow synaptic transmission in the spinal dorsal horn. Prog. Brain Res., 113, 443–462 (1996).
• 10) Braz JM, Nassar MA, Wood JN, Basbaum AI. Parallel “pain” pathways arise from subpopulations of primary afferent nociceptor. Neuron, 47, 787–793 (2005).
• 11) Inoue K, Tsuda M. Microglia in neuropathic pain: cellular and molecular mechanisms and therapeutic potential. Nat. Rev. Neurosci., 19, 138–152 (2018).
• 12) Gao YJ, Zhang L, Samad OA, Suter MR, Kawasaki Y, Xu ZZ, Park JY, Lind AL, Ma Q, Ji RR. JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain. J. Neurosci., 29, 4096–4108 (2009).
• 13) Uta D, Yoshimura M, Koga K. Chronic pain models amplify transient receptor potential vanilloid 1 (TRPV1) receptor responses in adult rat spinal dorsal horn. Neuropharmacology, 160, 107753 (2019).
• 14) Uta D, Tsuboshima K, Nishijo H, Mizumura K, Taguchi T. Neuronal sensitization and synaptic facilitation in the superficial dorsal horn of a rat reserpine-induced pain model. Neuroscience, 479, 125–139 (2021).
• 15) Kim W, Chung Y, Choi S, Min BI, Kim SK. Duloxetine protects against oxaliplatin-induced neuropathic pain and spinal neuron hyperexcitability in rodents. Int. J. Mol. Sci., 18, 2626 (2017).
• 16) Chae HK, Kim W, Kim SK. Phytochemicals of cinnamomi cortex: cinnamic acid, but not cinnamaldehyde, attenuates oxaliplatin-induced cold and mechanical hypersensitivity in rats. Nutrients, 11, 432 (2019).
• 17) Minami T, Takeda M, Sata M, Kato H, Yano K, Sakai T, Tsujita R, Kawasaki K, Ito A. Thrombomodulin alfa prevents oxaliplatin-induced neuropathic symptoms through activation of thrombin-activatable fibrinolysis inhibitor and protein C without affecting anti-tumor activity. Eur. J. Pharmacol., 880, 173196 (2020).
• 18) Uta D, Koga K, Furue H, Imoto K, Yoshimura M. L-bupivacaine inhibition of nociceptive transmission in rat peripheral and dorsal horn neurons. Anesthesiology, 134, 88–102 (2021).
• 19) Ling B, Coudoré-Civiale MA, Balayssac D, Eschalier A, Coudoré F, Authier N. Behavioral and immunohistological assessment of painful neuropathy induced by a single oxaliplatin injection in the rat. Toxicology, 234, 176–184 (2007).
• 20) Aoki M, Mori A, Nakahara T, Sakamoto K, Ishii K. Effect of synthetic eel calcitonin, elcatonin, on cold and mechanical allodynia induced by oxaliplatin and paclitaxel in rats. Eur. J. Pharmacol., 696, 62–69 (2012).
• 21) Aoki M, Mori A, Nakahara T, Sakamoto K, Ishii K. Salmon calcitonin reduces oxaliplatin-induced cold and mechanical allodynia in rats. Biol. Pharm. Bull., 36, 326–329 (2013).
• 22) Moore KA, Kohno T, Karchewski LA, Scholz J, Baba H, Woolf CJ. Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord. J. Neurosci., 22, 6724–6731 (2002).
• 23) Didangelos T, Doupis J, Veves A. Painful diabetic neuropathy: clinical aspects. Handb. Clin. Neurol., 126, 53–61 (2014).
• 24) Wang XL, Zhang HM, Chen SR, Pan HL. Altered synaptic input and GABAB receptor function in spinal superficial dorsal horn neurons in rats with diabetic neuropathy. J. Physiol., 579, 849–861 (2007).
• 25) Tsuda M. Microglia in the spinal cord and neuropathic pain. J. Diabetes Investig., 7, 17–26 (2016).
• 26) He WY, Zhang B, Zhao WC, He J, Zhang L, Xiong QM, Wang J, Wang HB. Contributions of mTOR activation-mediated upregulation of synapsin II and neurite outgrowth to hyperalgesia in STZ-induced diabetic rats. ACS Chem. Neurosci., 10, 2385–2396 (2019).
• 27) Schmidtko A, Luo C, Gao W, Geisslinger G, Kuner R, Tegeder I. Genetic deletion of synapsin II reduces neuropathic pain due to reduced glutamate but increased GABA in the spinal cord dorsal horn. Pain, 139, 632–643 (2008).
• 28) Zhao M, Isami K, Nakamura S, Shirakawa H, Nakagawa T, Kaneko S. Acute cold hypersensitivity characteristically induced by oxaliplatin is caused by the enhanced responsiveness of TRPA1 in mice. Mol. Pain, 8, 55 (2012).
• 29) Kawashiri T, Egashira N, Kurobe K, Tsutsumi K, Yamashita Y, Ushio S, Yano T, Oishi R. L type Ca²+ channel blockers prevent oxaliplatin-induced cold hyperalgesia and TRPM8 overexpression in rats. Mol. Pain, 8, 7 (2012).
• 30) Ta LE, Bieber AJ, Carlton SM, Loprinzi CL, Low PA, Windebank AJ. Transient receptor potential vanilloid 1 is essential for cisplatin-induced heat hyperalgesia in mice. Mol. Pain, 6, 15 (2010).
• 31) Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A, Noguchi K. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with Trk receptors. J. Comp. Neurol., 493, 596–606 (2005).