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Background: Resiniferatoxin (RTX) is a potent analog of capsaicin and activates transient receptor potential ( TRP) vanilloid type ( TRPV ) 1. In the current study, we investigated the preventive effect of perineural RTX on the develop ‑ ment of cold hypersensitivity induced by spinal nerve ligation (SNL) in rats. Furthermore, we examined the association between the expression level of TRPV1, TRP ankyrin type ( TRPA) 1 and TRP melastatin type ( TRPM) 8 in the dorsal root ganglion (DRG) and cold hypersensitivity after SNL. Results: RTX pretreatment prevented the development of SNL‑ induced hypersensitivity to mechanical, thermal, and cold stimuli. Western blot analysis 4 weeks after RTX pretreatment showed that RTX pretreatment decreased the protein expression level of SNL‑ induced TRPM8, but not TRPV1 or TRPA1, in the DRG of SNL rats. Immunofluorescent analysis revealed that up‑ regulated TRPM8‑ stained neurons after SNL co‑ localized with neurofilament 200 ‑ positive neurons located in the DRG. Conclusions: Pretreatment with perineural RTX significantly inhibits SNL ‑ induced mechanical, thermal, and cold hypersensitivity. The antinociceptive effect of perineural RTX, especially on cold hypersensitivity, may be related to the suppression of TRPM8 expression in DRG. Keywords: Hypersensitivity, Resiniferatoxin, Spinal nerve ligation, Transient receptor potential TRPV1 channel is considered a target for pain control, Background and many TRPV1 agonists and antagonists are ongoing The capsaicin receptor was first cloned in 1997 [ 1], which preclinical and clinical trials as analgesics [4, 5]. was later named the transient receptor potential (TRP) Resiniferatoxin (RTX) is a TRPV1 agonist isolated vanilloid type 1 (TRPV1). TRPV1 is a non-selective cat- from Euphorbia resinifera and an ultrapotent capsaicin ion channel sensitive to noxious heat, pH, putative endo- analog . It is more potent than capsaicin by 34-fold in vanilloids, and pungent plant products such as capsaicin. its effects on thermoregulation and neurogenic inflam - TRPV1 channels are found in some A-delta and unmy- mation . Unlike capsaicin, binding of RTX to TRPV1 elinated C-fibers of peripheral nerves [ 2, 3], well known is irreversible leading to a sustained influx of sodium and as part of the pain conduction pathway. Therefore, the calcium through the channels, thereby desensitize the TRPV1 expressing neurons at the dorsal root ganglion *Correspondence: email@example.com (DRG) . Furthermore, RTX has advantages over cap- Won Uk Koh and Seong‑ Soo Choi contributed equally to this work and saicin in that it induces less initial irritation and produces also co‑ first authors Department of Anesthesiology and Pain Medicine, Asan Medical Center, less systemic toxicity . RTX has been used for perineu- University of Ulsan, College of Medicine, 88 Olympic‑ro 43 gil, Songpa‑Gu, ral injection like a local anesthetic in animal models, and Seoul 05505, Korea © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Koh et al. BMC Neurosci (2016) 17:38 Page 2 of 9 has been shown to produce conduction analgesia without suppressing motor and other sensory function . There - fore, perineural RTX may hold promise for preventing postoperative pain and consequent chronic neuropathic pain. Indeed, numerous studies revealed that pretreat- ment with perineural RTX, rather than posttreatment, has a preventive effect on the development of neuro - pathic pain in various animal pain models. For example, perineural RTX prevents mechanical and heat hyper- sensitivity after carrageenan injection , and in sciatic Fig. 1 Eec ff t of resiniferatoxin (RTX) pretreatment on the withdrawal nerve ligation model of rats, perineural RTX attenuated threshold to mechanical stimuli. Perineural administration of vehicle the development of mechanical and heat hypersensitivity or RTX (0, 0.1, or 1 µg) was performed before spinal nerve ligatoin [11, 12]. However, the effectiveness of perineural RTX on (SNL). Behavioral tests were performed before surgery and weekly for 4 weeks after SNL. To test mechanical hyperalgesia, a von Frey preventing cold hypersensitivity is unclear. filament was used to stimulate the plantar surface of the ipsilateral Since the characterization of TRPV1, many other families left hind foot. The data are presented as the mean ± standard error. of transient receptor potential channel have been discov- **P < 0.01 and ***P < 0.001 compared to the RTX 0 µg group ered. Among them, transient receptor potential melastatin type 8 (TRPM8) and transient receptor potential ankyrin type 1 (TRPA1) are known as ‘cold sensitive’ channels . 2 weeks compared to the RTX 0 µg group. In contrast, pre- These thermo-sensitive channels (TRPV1, TRPM8, and treatment with RTX 0.1 µg did not produce a protective effect TRPA1) are known to be involved in the development of on SNL-induced cold hypersensitivity (Fig. 3). The results of neuropathic pain, and many researchers have reported the mechanical, cold and hot hypersensitivity are also pro- that expression of these channels in sensory neurons are vided as an additional file (Additional files 1 , 2 and 3). increased in various neuropathic pain models [14–20]. In our current study, we investigated the preventive effect TRPV1, TRPA1, and TRPM8 protein expression levels in the of perineural RTX on the development of cold hyper- DRG 4 weeks after SNL sensitivity induced by spinal nerve ligation (SNL) in rats. Immunoblot analysis of the ipsilateral L5 and L6 DRG Furthermore, we examined the association between the 4 weeks after SNL revealed that the protein expression level expression level of TRPV1, TRPM8, and TRPA1 in the of TRPV1 in the DRG significantly increased 4 weeks after DRG and cold hypersensitivity after SNL. SNL, which was not attenuated by pretreatment with RTX 1 µg (Fig. 4). As shown in Fig. 5, TRPA1 protein expres- Results sion in the DRG was not affected by either SNL or RTX Behavioral tests The baseline paw withdrawal threshold to mechanical stimuli in all rats before SNL surgery was 15 g (maximal mechanical stimulus). As shown in Fig. 1, SNL significantly reduced the paw withdrawal threshold to mechanical stimuli after 1 week, and this effect was sustained for 4 weeks after SNL. Perineu - ral administration of RTX 1 µg inhibited the development of SNL-induced mechanical hypersensitivity throughout the whole experimental period, with a significant difference noted compared to the RTX 0 µg group from 1 to 3 weeks after SNL. The paw withdrawal response to heat stimuli (44 °C) is shown in Fig. 2. Compared to baseline values, paw Fig. 2 Eec ff t of resiniferatoxin (RTX) pretreatment on the withdrawal withdrawal latency to heat stimuli significantly decreased latency to heat stimuli. Perineural administration of vehicle or RTX (0, from 1 to 2 weeks after SNL. Pretreatment of both RTX 1 µg 0.1, or 1 µg) was performed before spinal nerve ligation (SNL). Behav‑ and 0.1 µg resulted in a significant protective effect against ioral tests were performed before surgery and weekly for 4 weeks after SNL. To test heat hyperalgesia, the rats were placed on the 44 °C SNL-induced heat hyperalgesia. In the cold-plate test, SNL hot‑plate apparatus (Ugo ‑Basile, Comerico, Italy). The reaction time resulted in significant cold hypersensitivity throughout the starting from the placement of the rat on the hot‑plate to the time of whole experimental period (1–4 weeks). In the RTX 1 µg licking or withdrawing the hind paw was measured. The cut‑ off time group, the threshold to cold stimuli was higher than in the for the hot‑plate test was set at 30 s. The data are presented as the RTX 0 µg group throughout the experimental period. This mean ± standard error. **P < 0.01, ***P < 0.001 and ****P < 0.0001 compared to the RTX 0 µg group protective effect showed statistical significance at 1 and Koh et al. BMC Neurosci (2016) 17:38 Page 3 of 9 Fig. 3 Eec ff t of resiniferatoxin (RTX) pretreatment on the withdrawal latency to cold stimuli. Perineural administration with vehicle or RTX (0, 0.1, and 1 µg) was performed before spinal nerve ligation (SNL). Behavioral tests were performed before surgery and weekly for 4 weeks after SNL. To test cold allodynia, response to cold stimuli was examined using the 10 °C cold‑plate (Ugo ‑Basile, Varese, Italy). The reaction time starting from the placement of the rat on the cold‑plate to the time of licking or withdrawing the hind paw was measured. Fig. 5 Eec ff t of resiniferatoxin (RTX) pretreatment on transient recep ‑ The cut‑ off time for the cold‑plate test was set at 100 s. The data tor potential ankyrin type 1 ( TRPA1) expression. Perineural adminis‑ are presented as the mean ± standard error. *P < 0.05, **P < 0.01, tration with vehicle or RTX (0 or 1 µg) was performed prior to spinal ***P < 0.001 and ****P < 0.0001 compared to the RTX 0 µg group nerve ligation (SNL). To evaluate the protein expression level of TRPA1 located in the dorsal root ganglion (DRG), the left L5 and L6 DRG were isolated 4 weeks after SNL. Western blot analysis was performed using 40 μg of total protein extracted from the isolated DRG (a). The specific signal for TRPA1 was quantified and plotted (b). β‑actin was used as internal loading control Fig. 4 Eec ff t of resiniferatoxin (RTX) pretreatment on transient recep ‑ tor potential vanilloid type 1 ( TRPV1) expression. Perineural admin‑ istration with vehicle or RTX (0 or 1 µg) was performed before spinal nerve ligation (SNL). To evaluate the protein expression level of TRPV1 located in the dorsal root ganglion (DRG), the left L5 and L6 DRG were isolated 4 weeks after SNL. Western blot analysis was performed Fig. 6 Eec ff t of resiniferatoxin (RTX) pretreatment on transient using 40 μg of total protein extracted from the isolated DRG (a). The receptor potential mellastatin type 8 ( TRPM8) expression. Perineural specific signal for TRPV1 was quantified and plotted (b). β‑actin was administration with vehicle or RTX (0 or 1 µg) was performed before used as an internal loading control. *P < 0.05, **P < 0.01 compared to spinal nerve ligation (SNL). To evaluate the protein expression level the control (sham) group of TRPM8 located in the dorsal root ganglion (DRG), the left L5 and L6 DRG were isolated 4 weeks after SNL. Western blot analysis was performed using 40 μg of total protein extracted from the isolated treatment. On the other hand, TRPM8 protein expression DRG (a). The specific signal for TRPM8 was quantified and plotted (b). in the DRG significantly increased 4 weeks after SNL and β‑actin was used as an internal loading control. **P < 0.01 compared perineural pretreatment with RTX 1 µg before SNL pre- †† to the control (sham) group. P < 0.01 compared to the SNL group vented the increase in TRPM8 protein expression (Fig. 6). Koh et al. BMC Neurosci (2016) 17:38 Page 4 of 9 Immunofluorescence analysis of TRPM8 expression Our experiments focused on the effect and relationship As shown in Figs. 7 and 8, immunofluorescence analy - of perineural RTX on the expression of TRP channels sis revealed that SNL surgery (RTX 0 µg group) led to after SNL. Various neuropathic pain models have shown an increased trend in the number of TRPM8-positive that TRPV1 expression in the peripheral nerve increases neurons in the DRG compared with the sham-operated following nerve injury [15–17, 19]. Nerve injury induces control group. In the RTX 1 µg group, this increase in neuropathic pain and especially heat hypersensitiv- the number of TRPM8-expressing neurons was not ity, which is known to be closely related with increased observed. The results of TRPM8 immunoreactive neu - levels of TRPV1 expression. For instance, in two experi- ronal cell counts are further provided in additional file 4. ments using an SNL model of rats, ligation of the L5 nerve was associated with increased TRPV1 expression Discussion in the L4 DRG [15, 16]. TRPV1 is known to be related In our current study using a rat model, we found that not only to the development of heat hypersensitivity but pretreatment with perineural RTX significantly inhib - also to mechanical hypersensitivity , but the role of ited mechanical, heat, and cold hypersensitivity after TRPV1 in the development of mechanical hypersensitiv- SNL. Western blot analysis demonstrated that TRPV1 ity is controversial. Previous studies have demonstrated and TRPM8 protein expression levels in the DRG had that administration of TRPV1 antagonists in a neuro- increased 4 weeks after SNL. Perineural pretreatment pathic pain model did not attenuate the development of with RTX reduced the expression level of TRPM8, but mechanical hypersensitivity . Furthermore, in a paw not of TRPV1 in the DRG 4 weeks after SNL. incision pain model of TRPV1 knockout mice, the devel- The protective effect of perineural RTX pretreatment opment of heat hypersensitivity was suppressed, but on the development of heat and mechanical hypersen- in contrast, mechanical hypersensitivity was preserved sitivity has been demonstrated in previous studies using . However, there are evidences that TRPV1 contrib- neuropathic models. For example, Kissin et al. reported utes in the development of mechanical hypersensitivity, that pretreatment with perineural RTX reduced the especially in the chronic phase. Previous studies dem- development of mechanical and heat hypersensitiv- onstrated that antisense oligonucleotide against TRPV1 ity after carrageenan injection and sciatic nerve ligation reduced mechanical hypersensitivity in SNL rats and [10, 11]. Other authors also reported reduced mechani- selective antagonists of TRPV1 attenuated the develop- cal and heat hypersensitivity in RTX-pretreated rats after ment of mechanical hypersensitivity [25–27]. Our cur- carrageenan injection and common peroneal/tibial nerve rent study results also showed that TRPV1 expression ligation [12, 21]. Our findings are consistent with these levels were increased after SNL, which is in agreement previous experiments and strengthen the evidence for the with previous study results. protective role of RTX on the development of mechanical In the present study, we further attempted to eluci- and heat hypersensitivity. date the relationship between cold hypersensitivity and Following nerve injury, cold hypersensitivity may TRP receptors. The TRPM8 is well known to be a cold develop in addition to mechanical and heat hypersensi- receptor and a potential transducer for cold tempera- tivity. Indeed, cold allodynia and hyperalgesia are com- ture mediated allodynia and hyperalgesia. Several studies mon symptoms in patients presenting with neuropathic have shown that TRPM8 expression may be associated pain. Therefore, the ideal medication to prevent neu - with the development of neuropathic pain. For example, ropathic pain must have protective effects against cold Frederick et al. reported that the RNA level of TRPM8 hypersensitivity along with the protective effects against significantly increased after sciatic nerve chronic con - mechanical and heat hypersensitivity. However, whether striction injury (CCI) . Other reports showed that perineural RTX exerts a protective effect on cold hyper - CCI increased TRPM8 expression in the DRG [18, 20]. sensitivity remains unclear. Recently, Cruz et al. reported Furthermore, nocifencive behavior of mice to cold stim- that intraganglionic RTX prevents cold hypersensitivity uli has been shown to be related to TRPM8 expression induced by carrageenan injection into the upper lip and [28, 29]. In our study results, the TRPM8 expression was constriction of the infraorbital nerve . In our pre- increased after SNL observed through both Western blot sent study, perineural RTX treatment before nerve injury analysis and immunofluorescence analysis. This increase significantly reduced the development of cold hyper - of TRPM8 expression was prevented by RTX pretreat- sensitivity throughout the entire study period. Taken ment. Therefore, together with our present results, the together, these results indicate that perineural RTX treat- preventive effects of perineural RTX on SNL-induced ment may be an effective method of preventing cold cold hypersensitivity appear to be closely related to the hypersensitivity. expression of TRPM8 in the DRG. Another interesting Koh et al. BMC Neurosci (2016) 17:38 Page 5 of 9 Fig. 7 Immunofluorescence view of transient receptor potential mel‑ lastatin type 8 ( TRPM8) expression after resiniferatoxin (RTX) pretreat‑ ment. Perineural administration with vehicle or RTX (0 or 1 µg) was performed before spinal nerve ligation (SNL). To evaluate the protein expression level of TRPM8 located in the DRG, the ipsilateral (left) L5 and L6 DRG were isolated 4 weeks after SNL and confocal immuno‑ fluorescence analysis was performed. Mounting medium containing 4′,6‑ diamidino‑2‑phenylindole (DAPI; blue) counterstain was dis‑ persed over the sections before mounting. a TRPM8 expression (red; arrows) in the DRG of sham‑ operated control group rats. b TRPM8 expression (red; arrows) in the DRG of RTX 0 µg group rats. TRPM8 expression in the DRG was up‑regulated and the co ‑localization of TRPM8 expressing neurons with NF200‑positive neurons (green) was increased (arrow heads). c TRPM8 expression (red; arrows) in the DRG of RTX 1 µg group rats. TRPM8 expression was suppressed compared to that of the RTX 0 µg group finding was that increased co-localization of TRPM8 and NF 200 positive myelinated neurons were detected after SNL surgery. Previous study by Ji et al. reported that in the spinal nerve ligated neuropathic rat presenting cold hypersensitivity, the number of small myelinated A-delta fibers were significantly increased and the number of C-fibers were relatively constant in the plantar skin of the affected hind limb . The authors have concluded that the A-delta fibers provide major contribution in the development of cold hypersensitivity in neuropathic pain. Although the results in this study were electro physiological quantification of the A-delta and C fibers in the peripheral nerve, the results of this study support our findings of increased expressions of TRPM8 in the myelinated neurons at the DRG in the SNL group dem- onstrating cold hypersensitivity. The result of our study gives further information that the A-delta fibers present - ing TRPM8 may regulate cold hypersensitivity in neuro- pathic rats. In contrast to TRPM8, the expression level of TRPA1, another cold receptor, in the DRG did not differ between the three groups at 4 weeks after SNL. In fact, the asso- ciation between the expression level of TRPA1 and cold hypersensitivity remains controversial. First, many previ- ous studies suggested that TRPA1 is related to the devel- opment of cold allodynia and hyperalgesia [14, 31–33], which is not supported by our current data. This dis - crepancy may be explained by the time course of TRPA1 expression. In a previous study of a sciatic nerve injury model in rats, TRPA1 RNA levels in the DRG increased 1 and 2 weeks after nerve injury, while TRPM8 RNA lev- els increased 2 weeks after nerve injury . In our pre- sent study, the TRPA1 protein level in the DRG was only measured 4 weeks after SNL, while the expression level of TRPA1 at 1–3 weeks were not investigated. Second, some studies have reported inconsistent results on the association between TRPA1 and cold hypersensitivity. Koh et al. BMC Neurosci (2016) 17:38 Page 6 of 9 Methods This study was reviewed and approved by the Institu - tional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences, Asan Medical Center. The committee follows the guidelines of the Institute of Laboratory Animal Resources (ILAR). Behavioral test- ing and analgesiometry were performed according to the ethical guidelines set by the International Association for the Study of Pain (IASP) , and the animals were euth- anized after completion of planned tests. Experimental animals Male Sprague–Dawley rats (160–180 g) were housed three per cage in a room maintained at 21 ± 1.0 °C with an alternating 12 h light–dark cycle. Water and food were provided ad libitum and permitted to acclimate for at least 3 days prior to experiments. The animals were allowed to adapt to the laboratory environment for at least 2 h before testing, and they were used only once. To reduce variation related to diurnal rhythm, all experi- ments were performed during the light phase of the cycle (10:00–16:00). Group assignment and surgical procedure for animal model The rats were randomly assigned into four groups: (1) control group (no treatment, no SNL, n = 5), (2) RTX Fig. 8 Number of immunoreactive neurons in the ipsilateral L5 0 µg group (vehicle before SNL, n = 7), (3) RTX 0.1 µg dorsal root ganglion (DRG) positive for transient receptor potential group (0.0001 % RTX 0.1 mL before SNL, n = 7), and melastatin type 8 ( TRPM8). Compared with sham operated animals, (4) RTX 1 µg group (0.001 % RTX 0.1 mL before SNL). spinal nerve ligation (SNL) demonstrated an increased trend in Spinal nerve exposure and ligation were performed as number of neurons positive for TRPM8 4 weeks after surgery (a). Pretreatment with 1 µg of resiniferatoxin (RTX) decreased the number previously described . In brief, the rats were anesthe- of neurons presenting TRPM8 compared with the SNL group. The tized by intraperitoneal injection of zoletil (12.5 mg) and number of neurons coexpressing TRPM8 and neurofilament 200 were xylazine (3 mg). An approximately 1.5-cm midline inci- also increased in the SNL group compared with the sham group or sion above the left lateral lumbar spine was made, and SNL + RTX group (b). Three rats in each group were analyzed the paravertebral muscles were retracted, thereby par- tially exposing the L5 and L6 vertebral bodies. The left L5 For example, one study using TRPM8- and TRPA1- and L6 spinal nerves were isolated after partial removal knockout animals reported that noxious cold signaling of the left L6 transverse process. Vehicle or RTX (Sigma- may not be related to TRPA1 . The authors showed Aldrich Co., St. Louis, MO) of the above-indicated doses that nocifencive behavior to cold stimuli was absent in were administered perineurally distal to the DRG and TRPM8-knockout and TRPM8/TRPA1-double-knockout proximal to the formation of the sciatic nerve. The dose mice, but were retained in TRPA1-null mice. Our present of RTX was chosen based on a previous publication . study provides evidence that TRPM8 expression, but not After 30 min, tight ligation of L5 and L6 spinal nerves TRPA1 expression, is related to cold hypersensitivity at was done with 6-0 black silk distal to the DRG and proxi- 4 weeks after SNL. mal to the formation of the sciatic nerve at the site of RTX or vehicle injection. Conclusions In conclusion, pretreatment with perineural RTX inhibits Behavioral tests the development of SNL-induced mechanical, heat, and Behavioral responses to mechanical, heat, and cold cold hypersensitivity. The antinociceptive effect of peri - stimuli were performed before surgery and weekly for neural RTX against cold stimuli seems to be related to 4 weeks after SNL surgery. Animals were positioned indi- the TRPM8 expression in the DRG. vidually in plastic cages with wire-mesh bottoms and Koh et al. BMC Neurosci (2016) 17:38 Page 7 of 9 were acclimated for at least 20 min. To verify that the (5 mM) and bromophenol blue (0.1 % w/v) were further animal responses were normal, baseline behavioral tests added and boiled. The total protein samples (40 μg) were were performed before SNL surgery. separated by electrophoresis in 8–10 %-polyacrylamide Mechanical hypersensitivity was tested using a von gels, and transferred onto a polyvinylidene difluoride Frey filament (Stoelting, Wood Dale, IL). The plantar sur - membrane (Amersham Pharmacia Bioscience, Little face of the affected left hind foot was stimulated. Eight Chalfont, Buckinghamshire, UK). The membranes were calibrated von Frey filaments (0.41, 0.70, 1.20, 2.00, 3.63, immunoblotted with antibodies against TRPV1 (1:1000; 5.50, 8.50, and 15.10 g) were sequentially applied to the ab31895; Abcam, Cambridge, MA), TRPA1 (1:1000; plantar surface of the foot (in ascending or descend- ab68847; Abcam), TRPM8 (1:1000; ab104569; Abcam), ing order) and gently pressed against the foot. Rapid or β-actin (1:1000; A300-491A; Bethyl Laboratories INC., withdrawal or flinching was interpreted as a positive Montgomery, TX) and visualized with enhanced chemi- response. In the case of a positive response, the next- luminescence (ECL) -plus solution (Amersham Pharma- lightest filament was tested; if a negative response was cia Bioscience). The membranes were then exposed to observed, the next-heaviest filament was applied. The Hyperfilm-MP (Amersham Pharmacia Bioscience) for 50 % withdrawal threshold was determined by using the the detection of light emission. The specific signals for previously described up-down method . TRPV1, TRPM8, TRPA1, and β-actin were quantified Heat and cold hypersensitivity was assessed using the using the ImageJ freeware (NIH). hot and cold-plate test. To test heat hypersensitivity, the rats were individually placed on a hot-plate appara- Immunofluorescence analysis of TRPM8 expression tus (Ugo-Basile, Comerico, Italy) with the temperature Confocal immunofluorescence was performed to detect adjusted to 44 ± 0.1 °C , and the reaction time start- immunoreactivity in the DRG of SNL rats. TRPM8 and ing from the placement of the rat on the hot-plate to the neurofilament 200KDa (NF200; a marker of myelinated time of first nocifencive behavior (licking or withdraw - neurons) expression levels were analyzed using three ing the hind paw) was measured. The cut-off time for the rats in each group (total 9 rats) that were not used for hot-plate test was set at 30 s. Response to cold stimuli behavioral testing in each of the following groups: con- was examined using a cold-plate (Ugo-Basile, Comerico, trol group, the RTX 0 µg group, and the RTX 1 µg group. Italy) with the temperature adjusted to 10 ± 0.1 °C . Four weeks after SNL, the animals were anesthetized by The reaction time starting from the placement of the intraperitoneal injection of zoletil (12.5 mg) and xylazine rat on the cold-plate to the time of licking or withdraw- (3 mg), and a fixative containing 4 % buffered paraform - ing the hind paw was measured and the cut-off time for aldehyde was perfused through the left ventricle. The left the cold-plate test was set at 100 s. The latency to first L5 DRG was isolated and fixed immediately in the same nocifensive behavior in each rat was regarded as an index solution. The tissue samples were embedded in paraffin, to nociceptive threshold. Each rat was tested three times and the blocked sections were cut in 10-µm-thick cross with sufficient intervals between trials to avoid possible sections by the middle of the DRG with a microtome effects of anesthesia or tissue damage. and then mounted onto slides. The sections were depar - affinized with xylene and rehydrated sequentially with Total cellular protein extraction and western blot analysis 100, 95, and 70 % ethanol and then rinsed twice in dis- To evaluate the protein expression levels of TRPV1, tilled water. The slides were then immersed in 0.01 M TRPA1, and TRPM8 in the DRG, the left L5 and L6 DRG sodium citrate buffer (pH 6.0) and cooled in air. The sec - was isolated 4 weeks after SNL, immediately after the last tions were blocked with 5 % normal donkey serum, 0.3 % behavioral examination. After dissecting the left L5 and Triton X-100, and 1 % bovine serum albumin (BSA) in L6 DRG, DRG samples were washed twice with cold Tris phosphate buffered saline-Tween (PBS-T) for 1 h. The buffered saline (TBS; 20 mM Trizma base and 137 mM sections were incubated overnight at 4 °C with rabbit pol- NaCl, pH 7.5). Immediately after washing, the cells were yclonal antibodies to TRPM8 (1:1000; ab104569; Abcam) lysed with sodium dodecyl sulfate (SDS) lysis buffer with the IgG fraction of mouse polyclonal antibody to (62.5 mM Trizma base, 2 % w/v SDS, 10 % glycerol) con- NF200 (1:1000; n0142; Sigma, St Louis, MO). The sec - taining 0.1 mM Na VO , 3 mg/mL aprotonin, and 20 mM tions were subsequently incubated with Alexa Flour 546 3 4 NaF. Sonication was performed in brief to reduce vis- donkey anti-rabbit IgG (red; 1:1000; Invitrogen, Carls- cosity and shear DNA. Each protein concentration was bad, CA) and Alexa Flour 488 donkey anti-mouse IgG determined with the detergent compatible protein assay (green; 1:1000; Invitrogen). The sections were rinsed in reagent (Bio-Rad Laboratories, Richmond, CA) using PBS-T and 25 μL of mounting medium containing the bovine serum albumin as the standard. Dithiothreitol 4′,6-diamidino-2-phenylindole (DAPI; H-1200; Vector; Koh et al. BMC Neurosci (2016) 17:38 Page 8 of 9 Availability of data and materials Burlingame; CA) counterstain was dispersed over the We have provided the raw data which is attached in a separate file as a sections and then mounted onto slides. Immunofluores - Additional files. cence was assayed using a confocal fluorescent micro - However, we do not wish to open our raw data except for editorial issues. Although this study is an investigator owned experimental research, it scope (Olympus BX51 system microscope, Tokyo, Japan) was funded by the institutional research grant. Original data is also kept in with imaging software (Image Pro Plus ver.5.1, Media our institutional data base which is considered an intellectual property of our Cybernetics, Rockville, MD). The number of immuno - institute. reactive cells was counted manually in three slices of the DRG for each group. The total number of TRPM8 Ethics approval expressing neurons and doubly labeled neurons were This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences, Asan Medical counted and compared between each group. Center. IACUC 2010‑02‑039. Statistical analysis Funding This study was supported by an institutional research grant (2010‑331) from The data are presented as the mean ± standard error the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea. (SEM). 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BMC Neuroscience – Springer Journals
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