Abstract Increased expression of the transient receptor potential vanilloid 1 (TRPV1) channels, following nerve injury, may facilitate the entry of QX-314 into nociceptive neurons in order to achieve effective and selective pain relief. In this study we hypothesized that the level of QX-314/capsaicin (QX-CAP) - induced blockade of nocifensive behavior could be used as an indirect in-vivo measurement of functional expression of TRPV1 channels. We used the QX-CAP combination to monitor the functional expression of TRPV1 in regenerated neurons after inferior alveolar nerve (IAN) transection in rats. We evaluated the effect of this combination on pain threshold at different time points after IAN transection by analyzing the escape thresholds to mechanical stimulation of lateral mental skin. At 2 weeks after IAN transection, there was no QX-CAP mediated block of mechanical hyperalgesia, implying that there was no functional expression of TRPV1 channels. These results were confirmed immunohistochemically by staining of regenerated trigeminal ganglion (TG) neurons. This suggests that TRPV1 channel expression is an essential necessity for the QX-CAP mediated blockade. Furthermore, we show that 3 and 4 weeks after IAN transection, application of QX-CAP produced a gradual increase in escape threshold, which paralleled the increased levels of TRPV1 channels that were detected in regenerated TG neurons. Immunohistochemical analysis also revealed that non-myelinated neurons regenerated slowly compared to myelinated neurons following IAN transection. We also show that TRPV1 expression shifted towards myelinated neurons. Our findings suggest that nerve injury modulates the TRPV1 expression pattern in regenerated neurons and that the effectiveness of QX-CAP induced blockade depends on the availability of functional TRPV1 receptors in regenerated neurons. The results of this study also suggest that the QX-CAP based approach can be used as a new behavioral tool to detect dynamic changes in TRPV1 expression, in various pathological conditions.

Citation: Zakir HM, Mostafeezur RM, Suzuki A, Hitomi S, Suzuki I, Maeda T, et al. (2012) Expression of TRPV1 Channels after Nerve Injury Provides an Essential Delivery Tool for Neuropathic Pain Attenuation. PLoS ONE 7(9): e44023. https://doi.org/10.1371/journal.pone.0044023 Editor: Joao B. Calixto, Universidad Federal de Santa Catarina, Brazil Received: April 11, 2012; Accepted: August 1, 2012; Published: September 4, 2012 Copyright: © Zakir et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) Grants-in-Aid for Scientific Research Kakenhi 23592730 and 22659367. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Neuropathic pain (NP), which may arise as a result of injury, inflammation, or disease of the peripheral or central nervous systems, is characterized by spontaneous pain (i.e. ongoing, paroxysmal) and evoked sensitization in the form of hyperalgesia or allodynia. The TRPV1 channel, which is classically associated with transduction of painful stimuli such as hot temperature, low pH and application of vanilloid substances [1], [2], [3], [4] has been shown to change its expression profile under neuro-pathological conditions. Such changes have been implicated in neuropathic pain, by underlying changes in neuronal excitability [5], [6], [7], [8], [9], [10]. Several reports have described changes in TRPV1 expression levels in neuropathic pain models. Decrease of TRPV1 levels in injured and increased expression of TRPV1 in uninjured or spared neurons, was reported to occur after nerve ligation/transection [5], [6], [7], [8], [9], [10], however, the dynamics of functional TRPV1 expression during regeneration of transected nerves in this respect is still elusive. This information is highly important when exploring therapeutically relevant avenues in which TRPV1 may play an essential role. In naïve animals, TRPV1 is exclusively expressed in peripheral C- and Aδ- fibers [1]. Contrary to its role as a transducer in pain fibers, TRPV1 has been shown to serve also as a carrier for selective blockers of excitability. Blocking pain fibers specifically can be achieved by exploiting the selective TRPV1 expression in these fibers and the ability to use TRPV1 as a carrier of neuronal excitability blockers such as the non-permeable sodium channel blocker N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314). QX-314 is a permanently positively charged sodium channel blocker, which is unable to readily cross the cell membrane in a passive manner [11], [12], [13], [14]. However, when opening the TRPV1 channel by capsaicin, QX-314 can enter and thereby block nociceptive sodium channels from the inside of the cell, producing a long-lasting, pain-specific local anesthesia, devoid motor or tactile deficits [15], [16], [17], [18]. Based on the fact that TRPV1 plays a major role in this strategy, we explored whether the combination of QX-314 together with capsaicin (QX-CAP) could be used, not only to understand the dynamic functional expression of TRPV1 during regeneration of injured nerves, but also to block nerve injury mediated hyperalgesia. We further examined where (i.e. cell types) TRPV1 is expressed following IAN transection and nerve regeneration. This information is essential for better understanding mechanisms of pain, and thereby allowing development of novel strategies to manage pain. In this study we used the combination of QX-314 and capsaicin that was developed for selective blocking of pain [15], [16], [17] to understand the functional expression of TRPV1 in conjunction with profiling TRPV1 expression by immunohistochemistry. We show that starting 3 weeks after nerve transection, the QX-CAP combination reduces the hypersensitivity in the area of nerve regeneration and that this is dependent upon the amount of nerve regeneration in the injured area and the level of TRPV1 expression in these nerves. We further show a shift in the expression of TRPV1 from non-myelinated regenerated nerves to myelinated regenerated nerves and compare this data between those animals which underwent hyperalgesia and those which did not, as result of induced nerve transection (as a model for nerve injury induced neuropathic pain) hinting to the fact that other fibers besides nociceptive fibers participate in inducing pain sensation. Finally we propose using the behavioral testing as a tool to qualitatively relay relative TRPV1 expression levels after nerve injury is initiated. For the first time we can give an accurate account not only of changes in TRPV1 expression (both in time and cell type) but cross correlate this to behavioral testing and blockade of painful sensation. This novel approach for targeted painful stimuli in a neuropathic pain model can serve as a platform to be developed into clinically relevant strategies for pain management.

Methods The experiments were carried out in accordance with the guidelines of National Institute of Health Guide for the Care and Use of Laboratory (NIH Publication no. 80–23) revised 1996 and the International Association for the Study of Pain in conscious Animals, and were approved by the intramural Animal Care and Veterinary Science Committee of Niigata University [19]. Surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. A total of 120 male rats (Sprague-Dawley), weighing 150–200 grams at the start of the experiment, were used. The rats were exposed to a light dark cycle of 12 hours. Food and water were available ad libitum. IAN Transection and Sham Operation Rats were anesthetized with sodium pentobarbital (50 mg/kg, administered intraperitoneally (IP)), which was proceeded with left IAN transection. In this procedure, the rats were placed on a warm mat (to control for normal bodily temperature) and a small incision was made in the facial skin over the masseter muscle. The muscle was dissected to expose the surface of the alveolar bone. The bone covering IAN was removed using a dental drill. The exposed IAN was lifted, transected, and then placed back in the mandibular canal without any discernible gap between the cut ends [20], [21]. Rats with a similar facial skin dissection but without IAN exposure and transection were categorized as the sham-operated group (QX-CAP administration: n = 15, CAP administration: n = 15) in all experiments performed. After surgery, all animals received penicillin G potassium (20,000 units) intramuscularly, to prevent infection. Behavioral Testing and Division of Rats into Groups In daily sessions, rats were trained to stay in a plastic cage and keep their snout protruding through a hole in the cage wall during mechanical stimulation of the mental skin, using von Frey filaments (Touch-Test Sensory Evaluators; North Coast Medical, Inc., CA, USA). Touching and rubbing the rat’s mental skin without painful stimuli, by the shaft of von Frey filaments every day for 5–7 days (when the rats kept their snout protruded), allowed training the rats to keep their snout protruding through the hole for a long period of time. The force used, which brought upon escape behavior was determined and defined as the escape threshold (Fig 1). After successful training, the escape threshold was determined for the mental skin area, before and after IAN transection. The rats were free to escape following von Frey stimulus. Such escape behavior was defined as nocifensive. To determine the escape threshold, von Frey mechanical stimuli were applied to the mental skin in ascending and descending series of trials. The von Frey stimulus was applied 5 times in each series of trials. Escape threshold intensity was determined when the rats moved their heads away from the hole in at least one of the 5 stimuli. The average threshold intensity was calculated from the values after 2 ascending and 1 descending series of trials. Mechanical escape thresholds were measured at pre and 3 days, 2, 3, and 4 weeks post IAN transection. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. The effect of QX-CAP application on the escape threshold of NP and non-NP group at different time points after IAN transection. The changes in escape threshold following subcutaneous application of QX-CAP in sham-operated group (A); Only CAP injected sham-operated group (B); 2-weeks NP group (C); 2-weeks non-NP group (D); 3-weeks NP group (E); 3-weeks non-NP groups (F); 4-weeks NP group (G); 4-weeks non-NP group (H). The measurement were performed before the transection, 3 days after transection, 2, 3, and 4 weeks after transection/sham operation (depending on groups) and at various time points after injection of QX-CAP or CAP (n = 15 for each group, ANOVA followed by Dunnett’s test, *p<0.05). QX: QX-314; CAP: Capsaicin; Preop.: Preoperation; Preinj.: Preinjection; Pretrans.: Pretransection. https://doi.org/10.1371/journal.pone.0044023.g001 The IAN-transected rats (IANx) were divided into neuropathic pain (NP) and non-neuropathic pain (non-NP) groups according to the following criteria: the rats that showed a mechanical escape threshold of ≤8 gram (g) after IAN transection were considered to have developed NP [20], [21], [22]. Each group was further divided into 3 subgroups, according to the time (in weeks) elapsed after IAN transaction and the escape threshold before QX-CAP administration. QX-CAP administration was performed at 2, 3 and 4 weeks following IAN transection. The rats that showed an escape threshold of ≤8 g at 2 weeks after IAN transection were defined as the 2-week NP group (n = 15). The rats that showed an escape threshold of ≤8 g at 2 weeks after IAN transection, which remained constant also at 3 weeks, were named the 3-week NP group (n = 15). The rats that showed an escape threshold of ≤8 g at 2 weeks after IAN transection, which remained constant also at 3 and 4 weeks, were defined as the 4-week NP group (n = 15). In the non-NP group (escape threshold following IAN >8 g), the rats were similarly divided into the 2-week non-NP (n = 15), 3-week non-NP (n = 15), and 4-week non-NP (n = 15) groups respectively, in order to evaluate behavioral responses in an extended period of between 2–4 weeks following transection similar to the NP groups. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Photomicrographs of immunohistochemistry of TG cells labeled for TRPV1, NF200 and FG in sham-operated group and in 2-; 3-and 4-week NP groups and in 2-; 3- and 4 weeks non-NP groups. Expanded view of TG in the sham-operated group (D1–D4). Arrow points on an example of TRPV1++FG++NF- cell. Arrowhead points on an example of TRPV1++FG++NF+ cell. Note that TRPV1-positive cells increased with time after transection. Scale bar: 50 µm. https://doi.org/10.1371/journal.pone.0044023.g002 Drugs and Chemicals N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) (Sigma-Aldrich, St. Louis, USA) was used as a 2% solution in normal saline (0.9% NaCl in distilled water). Capsaicin solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was prepared with Tween 20 (10%), ethanol (10%), and normal saline (80%). QX-314 was freshly prepared on the day of the experiment. Capsaicin solution was prepared every 15 days and kept in the refrigerator (4°C). Before commencement of individual experiments, capsaicin solution was equilibrated at room temperature for 30 min. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. IAN transection both in NP and non-NP groups changes the expression profile of TRPV1 to myelinated neurons of a larger diameter. The total number of TG cells labeled for the fluoro-gold (FG+) (A: NP group, B: Non-NP group); TG cells that labeled for TRPV1 and FG (TRPV1++FG+) in 2-; 3-and 4-week NP groups and in sham-operated group (C: NP group, D: Non-NP group). The ratio of TRPV1++FG+ to all FG+ positive cells (E: NP group, F: Non-NP group). n = 5 for each group, (ANOVA followed by the Student–Newman–Keuls test, *p<0.05). The number of cells positive for TRPV1, FG and NF200 (TRPV1++FG++NF+) (G: NP group, H: Non-NP group); positive for TRPV1 and FG but not for NF200 (TRPV1++FG++NF-) (I: NP group, J: Non-NP group); positive for NF200 and FG (NF++FG+) (K: NP group, L: Non-NP group) in 2-; 3-and 4-week NP groups and in sham-operated group revealed by immunohistochemistry. ANOVA followed by the Student–Newman–Keuls test. # indicates non-significant difference. TRPV1++FG++NF- and TRPV1++FG++NF+ positive cells between the same groups are compared by paired t-test and the statistical significances are shown in the figure (G and H). 1*−4* indicate significant difference. 1: Sham TRPV1++FG++NF+ Vs TRPV1++FG++NF-, 2: 2-wk non-NP TRPV1++FG++NF+ Vs TRPV1++FG++NF-, 3: 3-wk non-NP TRPV1++FG++NF+ Vs TRPV1++FG++NF-, 4: 4-wk non-NP TRPV1++FG++NF+ Vs TRPV1++FG++NF-. p<0.05. n = 5 for each group. https://doi.org/10.1371/journal.pone.0044023.g003 Injection of Drugs and Behavioral Testing During each experimental session, preinjection mechanical escape thresholds of the mental skin area were measured ipsilateral to IAN transection. QX-314 (2%, 50 µl) with capsaicin (1 µg/µl, 30 µl) solution was subcutaneously injected into the mental skin area on the side ipsilateral to IAN transection, using a Hamilton microsyringe. In the sham-operated group, the solution of QX-314 with capsaicin or capsaicin was injected into the left mental skin area. Following injection, the escape threshold from the ipsilateral side was measured at 15 and 30 min, 1, 2, 3, 4, 5, 6, 7, and 24 h after injection. In the sham-operated group, the escape threshold from the left mental skin area was measured. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. The pattern of distribution of TRPV1 was altered in non-NP groups. The distribution area of TRPV1++FG++NF+ positive cells for all experimental groups. A cell area >1000 µm2 was considered large, while that <1000 µm2 was considered medium. Note that most of the cells were in the medium range, and the peak distribution shifted to the right in the transected groups. n = 5 for each group. https://doi.org/10.1371/journal.pone.0044023.g004 PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. The distribution analysis of cell area of TRPV1++FG++NF+ in different groups. https://doi.org/10.1371/journal.pone.0044023.t001 Immunohistochemistry Five rats were chosen randomly from each group and used for the immunohistochemical experiments. Fluoro-Gold (FG) (2%, 10 µl) was subcutaneously injected into the mental skin area under sodium pentobarbital (50 mg/kg, administered intraperitioneally) anesthesia, 2 days before perfusion and used for retrograde labeling of neurons in order to give an estimate of the extent of regeneration following the IAN procedure. Then the rats were deeply anesthetized with sodium pentobarbital and perfused with 200 ml of normal saline followed by 500 ml of 4% paraformaldehyde. The trigeminal ganglion (TG) was removed and post-fixed in 4% paraformaldehyde, for 2 days and the tissue was then transferred to a solution of 20% sucrose in phosphate-buffered saline (PBS) for several days for cryoprotection. Sections (16 µm in thickness) were cut using a cryostat, and every fifth section was mounted on MAS-coated glass slides (Matsunami Glass Ind., Ltd., Osaka, Japan). After washing with PBS, the sections were incubated at room temperature with 3% normal goat serum (NGS) in 0.01 M PBS with 0.3% Triton X-100, for 1.5 hours. They were then coincubated overnight at 4°C with a combination of rabbit anti-TRPV1 antibody (1∶200; Alomone Labs Ltd., Israel), which was diluted with 3% NGS in 0.01 M PBS with 0.3% Triton X-100, and mouse monoclonal anti-neurofilament 200 (NF200) antibody (1∶1000; Sigma-Aldrich), which was diluted with 3% NGS in 0.01 M PBS with 0.3% Triton X-100. The sections were washed 3 times with PBS and then incubated with goat anti-rabbit IgG (Alexa Fluor 488, 1∶1000; Invitrogen, USA) and goat anti-mouse IgG (Alexa Fluor 568, 1∶1000; Invitrogen) for 2 h at room temperature. After washing with PBS, the slides were coverslipped with Vectashield mounting medium (Vector Laboratories, Inc., USA). The stained slides were viewed and imaged using a camera attached to a Biozero BZ-8000 fluorescent microscope (Keyence Corp., Japan). The area viewed at 100x (700×850 µm) at the root of the third branch of TG was used for counting labeled cells. For each rat, three sections (one with the largest number of labeled cells and the next two sections) were selected for counting. The cell area was measured using ImageJ software (NIH Image, USA) for cells expressing TRPV1, NF200, and FG. A cell area >1000 µm2 was considered large, while that <1000 µm2 was considered medium and <400 µm2 was considered small [20]. Statistical Analysis For the analysis of the last significant time point of the behavioral effect of QX-CAP, one way analysis of variance (ANOVA) followed by Dunnett’s test were used. For comparison of the magnitude of the effect, the area under curve (AUC) was calculated and compared using a t-test. In addition, the comparison between different groups was tested statistically using two-way ANOVA. Immunohistochemical data were analyzed using one-way ANOVA followed by the Student–Newman–Keuls test. To compare TRPV1 expressing regenerated myelinated and non-myelinated neurons between the same groups paired t-test was used. To compare changes in distribution of the area of cells expressing TRPV1 channels, 3 slices from each rat for each group were compared. Since the distribution of cell areas did not follow Gaussian distribution (analyzed by Shapiro-Wilk and Kolmogorov Smirnov test), the cumulative probability of the areas of the examined cells was calculated. Data were then fit by a Boltzmann relationship: y = (A 1 – A 2 /(1+ exp [(x-x(0))/dx] = A 2 ), and the x(0) (which is the cell area at which of 50% of examined cells express TRPV1 channels), was calculated for each group and compared using one-way ANOVA with post-hoc Bonferroni. p value <0.05 was considered as statistically significant. Data are expressed as mean ± standard deviation.

Discussion In the current study, we measured, for the first time, the functional dynamic expression of TRPV1 during the regeneration process of transected IAN nerve in rats. Using the facilitated entry of QX-314 through the TRPV1 channel activated by capsaicin, we demonstrated that increasing amounts of TRPV1 expression would allow for functional and selective blockade of painful sensation following neuropathic based nerve injury (i.e. IAN). For the first time, we could interpret the amount of TRPV1 expression as an indication of successful anesthetic effect for QX-CAP and vise versa. We are now able in this model to predict the effectiveness of such a strategy at different time points following nerve injury. We can now also use the behavioral tests as a bio assay to predict relative TRPV1 expression levels. Peripheral nerve transection followed by close apposition of the cut ends leads to axon regeneration and subsequent re-innervations of the target tissue. Depending on the regeneration distance, this process can take several weeks to months [23], [24], [25], [26] and involves various molecular and biophysical changes in sensory neurons [27], [28], [29], [30], [31], [32], [33], [34], [35]. For example, nerve injury is known to induce alteration in receptors, ion channels, neuropeptides, signal transduction molecules, and growth related proteins, as well as to increase the spontaneous activity and receptive field of sensory neurons [22], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48]. These changes could be attributed to the injury itself and/or the altered environment encountered by regenerating axons at the injury site and/or the target tissue, and lead to a neuropathic condition characterized by allodynia or hyperalgesia [22], [28], [39], [49]. The effectiveness of QX-CAP injection may differ under these abnormal conditions. In our study, we evaluated the effectiveness of this combination in the sham-operated group at 2 weeks after operation, which can be considered as the control condition, and in various IAN-transected groups. In the IAN-transected groups, we evaluated the effectiveness under NP (indicated by a decrease in mechanical escape thresholds) and non-NP conditions (where the mechanical escape threshold did not decrease). We also evaluated the effectiveness of QX-CAP at various time points after transection as the underlying environment may change with time and such a study has not yet been performed. Evaluating the effect of QX-CAP injection in various conditions allowed us to comprehend the functional expression of TRPV1 in those conditions and to evaluate the outcome of such manipulations. In agreement with this view, QX-CAP injection showed variable local anesthetic effects under different conditions. In the sham-operated group, QX-CAP injection caused a significant increase in the mechanical escape threshold for 3.5 h (Fig 1A). The escape threshold reduction lasted over 6 hours when only CAP was injected the sham-operated group. This result implies that capsaicin-induced sensitization does not evoke in the capsaicin concentration we used. The escape threshold increased to more than 2 times the preinjection level and in many cases, the mental skin was insensitive to even the 60 g von Frey stimulus. Under non-NP conditions at 4 weeks after IAN transection, the threshold was found to be similar to that of the sham-operated group (Fig 1H). These findings indicate that QX-CAP injection was highly effective in these 2 groups. Under NP conditions, at 3 and 4 weeks after transection, a significant increase was observed in the escape thresholds, indicating that QX-CAP produced an analgesic effect in these groups, similar to the non-NP groups. We observed that the effectiveness of QX-CAP injection depends on time elapsed from transection and are positively correlated to increasing expression levels of TRPV1. These observations are true for both NP and non-NP groups. These results are in line with previous studies performed on naive animals, whereby QX-CAP injection was shown to produce an effective, nociceptor-specific local anesthesia effect? [15], [16], [17]. QX-CAP injection into rat hind paws resulted in a long-lasting increase in mechanical and thermal nociceptive thresholds [16]. In a recent study, the co-application of these drugs was observed to be effective in blocking pain signals in the rat trigeminal system [17]. These studies showed that QX-314 entered through the activated TRPV1 channel. In the current study we also evaluated the type of regenerated neurons in which TRPV1 is expressed after nerve transection. As mentioned above, we used NF200 as a marker for myelinated neurons (including Aβ and Aδ) and also injected FG (a retrograde tracer) into the mental skin area to identify the regenerated neurons. We found that the regenerated neurons expressing TRPV1 gradually increased over time after IAN transection (Fig 3C, E and 3D, F). However, in rats that developed NP, the number of regenerated neurons expressing TRPV1 was smaller compared to those with non-NP at the same time points. In the 4-week non-NP group, the number of regenerated neurons expressing TRPV1 was similar to that in the sham-operated group. Comparison of these immunohistochemical findings with behavioral data suggested that the variable anesthetic effect of QX-CAP injection appears to be due to the availability of TRPV1 receptors on regenerated neurons. In rats with NP at 2 weeks after transection, the number of regenerated neurons expressing TRPV1 was smaller compared to sham and the corresponding non-NP group of 2 weeks, and the corresponding behavioral study showed that QX-CAP injection was not effective. We hypothesize that entry of QX-314 was limited due to reduced TRPV1 expression and therefore not sufficient to make the combination effective in the behavioral readout test. In the 3-and 4-week NP groups, TRPV1 was shown to be expressed in higher numbers in myelinated (medium-sized) neurons and the analgesic effect that we observed in the 3-and 4-week NP groups might have been mediated by entry of QX-314 via TRPV1 channels in these neurons, as well as through non-myelinated neurons with TRPV1. The smaller effect of QX-CAP injection in the non-NP group at 2 and 3 weeks after transection is also probably due to the reduced availability of TRPV1 in regenerated neurons. An interesting question arises as to why certain animals develop decreased threshold (the NP groups) and other do not (the non-NP groups) although they both underwent the same procedure and to the same extent? The results show that TRPV1 levels are generally higher in the non-NP groups even at 2 weeks post IANx. Many studies have associated painful sensation to TRPV1 expression as this channel is considered to be the transducer of painful stimuli. Therefore one would expect that higher levels of TRPV1 would convey higher sensitivity to painful stimuli. Could this difference in profile also indicate whether or not an animal will develop neuropathy based painful stimuli? Could it be related also to the extent of regeneration between the groups which is also higher in the non-NP groups? These points need further investigation. Insight into this subject has great relevance when crossing over to the clinic and will be of great importance in developing new strategies which distinguish between different groups but also offer the right protocol to diminish pain for those which unfortunately fall into the NP groups. The immunohistochemical study revealed that C-fibers (non myelinated TRPV1 expressing neurons) were slow to regenerate after transection, and this regeneration was even slower under NP conditions compared to non-NP conditions. This data is in conjunction with previous reports showing that the C-fibers of injured nerves take longer to regenerate than myelinated A-fibers [23], [25], [50], [51]. Saito and colleagues showed that regenerated TG neurons with small diameter were significantly reduced at 14 and 60 days after IAN transection [21], though it has been reported that TRPV1 function is upregulated in IB4-positive sensory neurons (small neurons) [52]. They demonstrated that IAN-transected rats showed a profound reduction to thermal stimuli. Thermal sensory information is predominantly conveyed by C-fibers [53], [54]. Therefore, reduced sensitivity to thermal stimuli indicates a reduced number of C-fibers after transection (see also [20]). Our study also showed that TRPV1 expression shifted to myelinated fibers after transection. In the sham-operated group, TRPV1 was mostly expressed in small size C-fibers. However, in the IAN-transected groups, TRPV1 was mostly expressed in medium sized-neurons, in both NP and non NP groups. These results fit well with previous studies which also showed TRPV1 expression shifting to myelinated neurons in the dorsal root ganglion of rats with chronic inflammatory conditions induced by Freund’s complete adjuvant [55], [56]. Similar shifting has also been reported in animal models for diabetic neuropathy and bone cancer pain [57], [58], [59]. Although medium sized myelinated fibers are traditionally not involved in pain detection, these fibers have been reported to be involved in NP after nerve injury [20], [21], [39], [48], [50], [60]. Background activity, mechanically evoked responses and discharge of Aδ-fibers increased significantly in IAN-transected rats compared with naïve rats [20]. In other studies, it has been reported that after nerve injury, A-fibers were able to produce substance P and calcitonin gene-related peptide (CGRP), usually released from C-fibers [61], [62], [63]. Central sprouting of myelinated fibers has also been postulated to be an underlying cause of NP after nerve injury [48], [60], [64], [65]. Therefore, it is certainly plausible that entry of QX-314 into myelinated fibers via TRPV1 activated by capsaicin, may block abnormal activity of those fibers under neuropathic conditions and that these fibers have a role in NP related behavior. A detailed knowledge of expression profiles together with profound understanding of myelinated and non-myelinated neurons contributing to neuropathy, will lead to successful development of strategies in attenuating neuropathic pain.

Author Contributions Conceived and designed the experiments: HMZ TM KS YY KY AMB KI JK. Performed the experiments: HMZ RMM AS SH IS JK. Analyzed the data: HMZ RMM AMB JK. Wrote the paper: HMZ SL AMB KI JK.