TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury (original) (raw)
TRPA1, but not TRPM8, expression increases in trkA-expressing DRG neurons following peripheral inflammation. We first investigated the role of TRPA1 and TRPM8 in cold hyperalgesia after inflammation induced by CFA. The CFA injection clearly increased the number of paw lifts on a cold plate at 5°C for the 5-minute testing period (Figure 1A). The number of paw lifts increased from 1.8 ± 0.5 before CFA injection to 10.6 ± 2.7 at day 1 and 10.3 ± 2.2 at day 3; cold hyperalgesia gradually resolved by day 7. In contrast, contralateral paw lifts were remarkably few (data not shown).
Time course of the exaggerated response to cold after peripheral inflammation and nerve injury. (A and B) The number of paw lifts on a cold plate at 5°C for the 5-minute testing period was examined at days 1, 3, 5, and 7 after CFA injection (A) and L5 SNL (B). Data represent mean ± SEM; n = 8 per group. BL, baseline. *P < 0.05 compared with the naive control.
In situ hybridization histochemistry (ISHH) revealed that most of the TRPA1 and TRPM8 mRNA-labeled neurons in the DRG were small or medium in size (Figure 2A), consistent with previous studies (9–11). Using a computerized image analysis, we found that 32.4% ± 1.8% and 29.1% ± 2.7% of DRG neurons were positively labeled for TRPA1 and TRPM8 mRNA in naive control rats, respectively (Figure 2B). There was no change in the percentage of TRPA1 mRNA–positive neurons on the contralateral side (data not shown); however, inflammation induced a significant increase in the percentage of TRPA1 mRNA–positive neurons in the ipsilateral DRG at days 1 and 3 (44.1% ± 2.9% and 42.0% ± 2.2%, respectively); the levels gradually declined, returning to normal by day 7 (Figure 2C). This upregulation corresponded well with the development and maintenance of CFA-induced cold hyperalgesia. In contrast, there was no significant difference in the percentages of TRPM8 mRNA–positive neurons over 7 days (Figure 2D). These changes in TRPA1 and TRPM8 mRNA were also confirmed by RT-PCR (Figure 2E). In the spinal cord, neither TRPA1 nor TRPM8 mRNA were detected (data not shown).
Marked upregulation of TRPA1, but not TRPM8, mRNA in DRG neurons after peripheral inflammation induced by CFA. (A) Bright- and dark-field photomicrographs of ISHH showing expression of TRPA1 and TRPM8 mRNA in the naive DRG and the ipsilateral DRG at day 1 after inflammation. Scale bars: 100 μm. (B) Scatterplot diagrams made by plotting the individual cell profiles at day 1 after CFA injection. The gray lines represent the borderlines between the negatively and positively labeled neurons (signal/noise [S/N] ratio = 10). (C and D) Time course of the mean percentages of TRPA1 (C) and TRPM8 (D) mRNA–positive neurons after CFA injection. (E) mRNA expression of TRPA1 and TRPM8 in the DRG after inflammation, as detected by RT-PCR. Quantification of RT-PCR data is shown at right. Data represent mean ± SD; n = 4 per group. *P < 0.05 compared with the naive control.
Several studies have demonstrated an increase in levels of TRPV1 protein, but not in levels of mRNA, after peripheral inflammation (23, 24). Therefore, we examined the changes of TRPM8 expression in the DRG at the protein level using immunohistochemistry (Figure 3A). Consistent with the data obtained from ISHH, there was no difference in the percentages of TRPM8-immunoreactive neurons at day 3 after inflammation (Figure 3B). We found that inflammation increased TRPA1 expression in small- and medium-size neurons (Figure 2, A and B). Cutaneous nociceptors can be divided into 2 broad groups: NGF-responsive/trkA-expressing neurons and GDNF-responsive/c-ret–expressing neurons (25). Double labeling showed that both TRPA1 and TRPM8 heavily colocalized with trkA after inflammation (Figure 3C). However, only TRPA1-expressing neurons coexpressed SP, CGRP, and TRPV1 (Table 1). Furthermore, approximately 10% of TRPA1-labeled neurons were also labeled for TRPM8, indicating that inflammation increased TRPA1 in TRPM8-negative peptidergic neurons.
No change of TRPM8 protein in the DRG and no overlap between TRPM8- and TRPA1-expressing neurons after inflammation. (A) Protein expression of TRPM8 in the naive DRG and the ipsilateral DRG at day 3 after inflammation, as detected by immunohistochemistry. TRPM8-immunoreactive (TRPM8-IR) neurons were invariably small or medium in size (arrows). (B) Quantification of the percentage of TRPM8-IR neurons at day 3 after CFA injection. (C) Double labeling by a combined method of ISHH and immunohistochemistry for TRPA1 or TRPM8 mRNA and trkA-IR, SP-IR, CGRP-IR, and TRPV1-IR in the DRG at day 3 after CFA injection. Double labeling for TRPA1 and TRPM8 mRNA by dual ISHH is shown at right. TRPA1- and TRPM8-expressing neurons were clearly distinguishable. Open arrows indicate double-labeled neurons. Scale bars: 50 μm.
Percentages of trkA-IR, SP-IR, CGRP-IR, or TRPV1-IR neurons in TRPA1 or TRPM8 mRNA–positive neurons in the DRG
Anti-NGF and a p38 MAPK inhibitor alleviate inflammation-induced cold hyperalgesia and TRPA1 upregulation. NGF injection in a peripheral target induces p38 MAPK activation in trkA-expressing DRG neurons; activation of p38 MAPK in DRG neurons contributes to persistent inflammatory pain via the transcriptional regulation of key gene products (23). To determine whether alterations of the endogenous NGF and p38 MAPK pathway might be involved in inflammation-induced cold hyperalgesia, anti-NGF or a p38 MAPK inhibitor, SB203580, was delivered intrathecally before CFA injection and maintained for 3 days via a catheter whose tip was positioned close to the L4/5 DRG. Intrathecal administration of anti-NGF or SB203580 into naive rats produced no significant changes in basal pain sensitivity (data not shown). We found that both anti-NGF (1 μg/μl–1/h–1) and SB203580 (0.5 μg/μl–1/h–1) infusion significantly inhibited inflammation-induced cold hyperalgesia at days 1 and 3 (Figure 4A). Furthermore, anti-NGF reduced inflammation-induced heat hyperalgesia and mechanical allodynia whereas SB203580 attenuated heat hyperalgesia but not mechanical allodynia at days 1 and 3, consistent with previous reports (23).
Anti-NGF and a p38 MAPK inhibitor reverse cold hyperalgesia and TRPA1 upregulation induced by inflammation. (A) Cold and heat hyperalgesia were tested using the cold plate test and the plantar test, respectively, at days 1 and 3 after CFA injection. Mechanical allodynia was determined with a Dynamic Plantar Aesthesiometer. Data represent mean ± SEM; n = 8 per group. (B and C) Quantification of the percentages of TRPA1 (B) and TRPM8 (C) mRNA–positive neurons at day 3 after CFA injection. Data represent mean ± SD; n = 4 per group. (D) Double labeling for TRPA1 or TRPM8 mRNA and p-p38–IR at day 3 after CFA injection. Open arrows indicate double-labeled neurons. SB, SB203580. *P < 0.05 compared with the naive control; #P < 0.05 compared with the vehicle group. Scale bar: 50 μm.
We then assessed the effects of anti-NGF and SB203580 on inflammation-induced TRPA1 upregulation in the DRG. CFA induced a substantial increase in the percentage of TRPA1-positive neurons in the vehicle group at day 3, but pretreatment with either anti-NGF or SB203580 prevented this increase (Figure 4B). However, there was no apparent change in the expression of TRPM8 (Figure 4C). Furthermore, double labeling showed that phosphorylated p38 (p-p38) was predominantly expressed in TRPA1- but not TRPM8-positive neurons at day 3 after inflammation (Figure 4D).
NGF, but not GDNF, increases TRPA1 expression in DRG neurons through the p38 MAPK pathway. To further investigate the effects of NGF and the p38 MAPK pathway on TRPA1 expression in the DRG neurons, we injected NGF (1 μg or 10 μg in 10 μl saline) intrathecally into naive rats. Three days after the 10-μg NGF injections, 46.8% ± 5.7% of the neurons were TRPA1 positive; this percentage was higher than that of TRPA1-positive neurons in the saline group (32.6% ± 1.5%). This increase, however, was significantly reversed by cotreatment with SB203580 (Figure 5A). Additionally, TRPA1 levels did not change after the 10-μg GDNF injection, and neither NGF nor GDNF significantly altered TRPM8 expression (Figure 5B).
NGF, but not GDNF, induces an increase of TRPA1 expression in DRG neurons via p38 MAPK activation. Quantification of the percentage of TRPA1 (A) and TRPM8 (B) mRNA–positive neurons at day 3 after the injection. Data represent mean ± SD; n = 4 per group. *P < 0.05 compared with the naive control. #P < 0.05 compared with the NGF 10-μg group.
TRPA1 induced in spared DRG neurons contributes to cold hyperalgesia following nerve injury. The L5 spinal nerve ligation (SNL) model (26), which is one of the most popular models of neuropathic pain, is unique because the L4 DRG neurons are clearly separated from the axotomized L5 neurons (20). Recent studies have shown increased expressions of SP, CGRP, BDNF, and TRPV1 in the spared L4 DRG following L5 SNL, which indicates that the molecular phenotypic changes in the spared L4 DRG are just like those produced by peripheral inflammation (18–20). Therefore, we investigated the role of TRPA1 and TRPM8 in nerve injury–induced cold hyperalgesia using the L5 SNL model. The L5 SNL clearly increased the number of paw lifts on a cold plate, and cold hyperalgesia lasted for more than 7 days after surgery (Figure 1B). The contralateral side of nerve-ligated rats and both sides of sham-operated rats did not show cold hyperalgesia (data not shown). We then examined the changes of TRPA1 and TRPM8 expression in the L4 and L5 DRG after L5 SNL. Seven days after surgery, the L5 SNL decreased both TRPA1 and TRPM8 mRNA expression in the L5 DRG (Figure 6A). In contrast, the percentage of TRPA1 mRNA–positive neurons significantly increased in the L4 DRG at day 7 after L5 SNL (43.1% ± 5.9%), mainly in small- to medium-diameter neurons. However, there was no change in the expression of TRPM8 in the L4 DRG.
Anti-NGF and a p38 MAPK inhibitor suppress cold hyperalgesia and TRPA1 upregulation in the spared L4 DRG neurons caused by injury to the L5 spinal nerve. (A) Dark-field photomicrograph of ISHH showing the expression of TRPA1 and TRPM8 mRNA in the naive DRG, the injured L5 DRG, and the intact L4 DRG at day 7 after L5 SNL. (B) Cold hyperalgesia was determined using the cold plate test at days 3 and 7 after L5 SNL. Data represent mean ± SEM; n = 8 per group. (C and D) Quantification of the percentage of TRPA1 (C) and TRPM8 (D) mRNA–positive neurons at day 7 after surgery. Data represent mean ± SD; n = 4 per group. *P < 0.05 compared with the naive control; #P < 0.05 compared with the vehicle group. Scale bar: 100 μm.
To ascertain whether NGF-induced p38 activation in the uninjured L4 DRG regulates TRPA1 expression and contributes to cold hyperalgesia after L5 SNL, we administered anti-NGF or SB203580 into the intrathecal space. The treatment of anti-NGF and SB203580 diminished L5 SNL-induced cold hyperalgesia at days 3 and 7 (Figure 6B). Furthermore, we found that both anti-NGF and SB203580 application blocked the L5 SNL–induced increase in TRPA1 but not TRPM8 expression in the spared L4 DRG at day 7 (Figure 6, C and D).
Knockdown of the TRPA1 gene prevents and reverses inflammation- and nerve injury–induced cold hyperalgesia. Our results suggest that cold hyperalgesia after inflammation and nerve injury is critically dependent on functional TRPA1 in DRG neurons. We therefore predicted that a selective knockdown of TRPA1 expression should prevent inflammation- and nerve injury–induced cold hyperalgesia. To test this, rats with peripheral inflammation or nerve injury were intrathecally treated with either an antisense oligodeoxynucleotide (AS-ODN) targeting TRPA1 or a mismatch ODN (MM-ODN) beginning 12 hours before CFA injection or L5 SNL. We found that the CFA- and SNL-induced increase in the number of paw lifts on a cold plate was significantly less in the TRPA1 AS-ODN group (0.5 nmol/μl–1/h–1) than in the MM-ODN group (0.5 nmol/μl–1/h–1) (Figure 7, A and B). The number of paw lifts in the MM-ODN and AS-ODN groups (0.05 nmol/μl–1/h–1) groups was not different from that of the vehicle control rats. However, AS-ODN had no effect on CFA- and SNL-induced mechanical allodynia and heat hyperalgesia.
Reversal of hyperalgesia to cold by a selective blockade of TRPA1 expression. (A and B) Pretreatment with TRPA1 AS-ODN (0.5 nmol/μl–1/h–1) (AS 0.50) prevents cold hyperalgesia, but not heat hyperalgesia or mechanical allodynia, induced by peripheral inflammation (A) and injury to the L5 spinal nerve (B). Cold and heat hyperalgesia were tested using the cold plate test and the plantar test, respectively, at days 1 and 3 after CFA injection or at days 3 and 7 after L5 SNL. Mechanical allodynia was examined using a Dynamic Plantar Aesthesiometer. Data represent mean ± SEM; n = 8 per group. #P < 0.05 compared with the MM-ODN (0.5 nmol/μl–1/h–1) (MM 0.50) group.
We then confirmed that the level of TRPA1 mRNA in the DRG neurons of the AS-ODN–treated rats was significantly lower than that in the MM-ODN–treated rats (Figure 8A) whereas there was no difference in TRPM8 expression between TRPA1 AS-ODN–treated rats and control rats both in the CFA model and in the L5 SNL model (Figure 8B). The distribution of FITC-labeled ODN showed the progressive increase in the fluorescence associated with DRG cell bodies, indicating that the uptake of the ODN occurred in a time-dependent manner (Figure 8C). Furthermore, the treatment with AS-ODN, beginning 12 hours after CFA injection, reversed the inflammation-induced cold hyperalgesia at day 3 but not at day 1 (Figure 8D).
Confirmation of a selective blockade of TRPA1 expression in a time-dependent manner. (A and B) TRPA1 AS-ODN (0.5 nmol/μl–1/h–1) attenuates the induction of TRPA1, but not TRPM8, mRNA induced by peripheral inflammation and injury to the L5 spinal nerve. Quantification of the percentage of TRPA1 (A) and TRPM8 (B) mRNA–positive neurons at day 3 after CFA injection or in the L4 DRG at day 7 after L5 SNL. Data represent mean ± SD; n = 4 per group. (C) Fluorescence in the naive DRG and the DRG at 6 and 24 hours after intrathecal injection of FITC-labeled ODN. Scale bar: 50 μm. (D) Effect of treatment with TRPA1 AS-ODN (0.5 nmol/μl–1/h–1) following CFA injection on CFA-induced cold hyperalgesia at days 1 and 3. Data represent mean ± SEM; n = 8 per group. *P < 0.05 compared with naive control. #P < 0.05 compared with MM-ODN (0.5 nmol/μl–1/h–1) group.
Noxious cold stimulation induces very rapid phosphorylation of p38 MAPK through the TRPA1 channel. Recent work has failed to reproduce cold responsiveness in TRPA1 in vitro (27). To investigate how TRPA1 can be activated by noxious cold stimulation in vivo, we examined the phosphorylation of p38 MAPK in the DRG 2 minutes after noxious cold stimulation. We did this because activation of p38 in primary afferents is involved in acute nociceptive processing by a nontranscriptional mechanism and examination of p-p38 is very useful as an indicator of the activated DRG neurons after noxious stimulation in vivo (28). We applied thermal stimulation by immersion of the hind paw of naive rats into cool to cold water (28°C, 16°C, and 4°C) and found that at 28°C, there was no increase in p38 phosphorylation in the DRG (Figure 9, A and B). However, noxious cold stimulation at lower temperatures (16°C and 4°C) increased the phosphorylation of p38 in the DRG, mainly in small-size neurons 2 minutes after stimulation. Double labeling showed that at 4°C, the majority of the p-p38–labeled neurons also expressed TRPA1 (Figure 9C). Furthermore, we found that pretreatment with AS-ODN but not MM-ODN inhibited noxious cold stimulation–induced p38 activation (Figure 9D), which suggests that the TRPA1 channel is required for p38 activation after noxious cold stimulation.
Activation of p38 MAPK in TRPA1-containing neurons by noxious cold stimulation. (A) p-p38 labeling in naive DRG and ipsilateral DRG 2 minutes after cold stimulation at 4°C. (B) Quantification of the percentage of p-p38–IR neurons after innocuous and noxious cold stimulation. (C) Double labeling for TRPA1 mRNA and p-p38–IR in naive DRG and ipsilateral DRG 2 minutes after cold stimulation at 4°C. Open arrows indicate double-labeled neurons. (D) Effect of pretreatment with TRPA1 AS-ODN (0.5 nmol/μl–1/h–1) on the percentage of p-p38–IR neurons after cold stimulation at 4°C. Data represent mean ± SD; n = 4 per group. *P < 0.05 compared with naive control. #P < 0.05 compared with MM-ODN (0.5 nmol/μl–1/h–1) group. Scale bars: 100 μm.