The results of the present study demonstrate the attenuation of TH and reduction of injury discharges following CCI on median nerves by lidocaine pretreatment. Correspondingly, both the level of the injury-induced NPY fibers and the number of injury-induced c-Fos-LI cells in the CN at four weeks after medina nerve CCI were also dose-dependently reduced by lidocaine pretreatment. These results provide a possible mechanism in that the suppression of injury discharges by lidocaine pretreatment not only relieves neuropathic pain but also attenuates the NPY and c-Fos expressions in the CN after CCI.
Following median nerve CCI, signs of mechanical allodynia were detected three days after CCI and lasted throughout the experiment period of 28 days in the present study. Similar results have been reported, where hyperalgesia responses to noxious radiant heat were observed on the second postoperative day and lasted for over two months after sciatic nerve CCI [3
]. Another study also indicated that mechanical allodynia was found three to five days following CCI [28
], whereas this neuropathic sign was detected one day after CCI in our recent study [22
]. There are discrepancies in the time points of neuropathic pain initiating after CCI between various studies. The reason for these discrepancies may be simply due to different time points being examined. We focused on the role of lidocaine pretreatment in the paw withdrawal threshold of the CCI rats. The reductions in the paw withdrawal threshold after median nerve CCI were reversed by lidocaine pretreatment in a dose-dependent manner. A previous study [6
] reported that lidocaine pretreatment relieved thermal hyperalgesia for a long postoperative period (up to three weeks) after sciatic nerve CCI. However, another study showed that prior to spinal nerve ligation (SNL), lignocaine pretreatment increased the paw withdrawal threshold for only 24
]. These discrepancies may be related to differences in the injury models used in the above-mentioned studies (CCI versus SNL). The lesion site to the DRG in the CCI model was more distal than that in the SNL model. The injury level caused by the former was less severe than that by the latter, so the neuropathic pain induced by the CCI model could be prevented by lidocaine pre-treatment.
Furthermore, four weeks after CCI (29 days postinjury), a significant increase in the number of spikes in all CCI groups, which was regarded as ectopic discharges induced after nerve injury. Then, our results also demonstrated that the ectopic discharges were suppressed by lidocaine pre-treatment in a dose-dependent manner. The suppression in ectopic discharges was considered as one of the contributing factors in relieving neuropathic pain induced by median nerve CCI. Our recent study reported that ectopic discharges evoked by median nerve transection (MNT) were suppressed by pre-treatment with 5% and 10% lidocaine, but not 1% lidocaine [15
]. However, in the present study, ectopic discharges induced by CCI were significantly attenuated by 1% lidocaine pre-treatment. This discrepancy may also be explained by the difference in injury model employed between these two studies. The injury severity induced by CCI was milder than that by MNT. For this reason, the rate of injury discharges in the CCI rats, but not MNT, could be significantly reduced by low-dose (1%) lidocaine pre-treatment.
The present study further demonstrated that a significant increase in NPY in the CN at four weeks (29 days) post-CCI was also dose-dependently reduced by lidocaine pre-treatment. Previous studies have reported that intense afferent discharges and depolarization enhanced NPY induction [30
]. Furthermore, the NPY induction in the CN exclusively originated from injured DRG neurons, particularly medium- and large-size neurons, via primary afferent fibers [26
]. It is reasonable to infer that the reduction of NPY expression in the CN would result from the suppression in injury discharges following CCI with lidocaine pre-treatment. This is consistent with NPY reduction in the CN after MNT [15
] with lidocaine pre-treatment and in the spinal cord laminae 3-4 following sciatic nerve CCI with MK-801 and clonidine pre-treatment [30
]. Taken together, these findings suggest that the magnitude of nerve discharges may be one of the most important factors to induce NPY synthesis.
In the rats with bilateral median nerve CCI, c-Fos-LI cells were found only in the CN with electrical stimulation, but not in the unstimulated side of the CN; the level of NPY-LI fibers in the stimulated side of the CN was also significantly lower than that in the unstimulated side. One possible explanation for this is that NPY is released from the injured median nerve in the stimulated side of the CN resulting in NPY reduction and induced c-Fos expression in the same region. This is compatible with previous studies where NPY reduction and c-Fos induction were detected in the stimulated side of the CN following electrical stimulation with the transected median nerve; injection of an NPY receptor antagonist into the CN coupled with electrical stimulation to the injured nerve resulted in a dramatic decrease in the number of c-Fos-LI cells in the ipsilateral CN [15
]. In the present study, we also found that the number of c-Fos-LI cells in the CN after electrical stimulation of the injured nerve was dose-dependently reduced by lidocaine pre-treatment. Statistical analysis further demonstrated that the number of c-Fos-LI cells in the stimulated side of the CN was significantly correlated to the level of NPY reduction. Taken together, these results suggest that the amount of NPY release (NPY reduction level), following electrical stimulation of the injured nerve, directly modulates c-Fos expression in the CN.
Although the function of c-Fos induction in the CN remains uncertain, the expression of c-Fos immunoreactivity in the spinal cord has been considered as a convincing marker of pain [19
]. Our results showed that the number of c-Fos-LI cells in the CN coincided with the reduction in paw withdrawal thresholds, regarded as mechanical allodynia. This is in agreement with a previous study which reported that the number of c-Fos-LI cells was positively associated with the magnitude of mechanical allodynia [22
]. Earlier studies have also clarified that after electrical stimulation of the injured median nerve, about 78% of c-Fos-LI cells in the middle CN were cuneothalamic projection neurons (CTNs) [17
]. This study further showed that the number of c-Fos-LI cells was dose-dependently reduced by lidocaine pre-treatment. Injury discharges have been reported to be implicated in the increase of c-Fos LI cell expression in the spinal cord dorsal horn [32
], while lidocaine pre-treatment attenuates the discharges to prevent c-Fos induction [21