Changes in neuronal markers in a mononeuropathic rat model: relationship between neuropeptide Y, pre-emptive drug treatment and long-term mechanical hyperalgesia

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Abstract
Using the chronic constriction model (CCI) of Bennett and Xie (1988), changes in the lumbar spinal cord in neuropeptides and lectin IB4 were examined at 28 days post-nerve constriction and were compared with the degree of mechanical hyperalgesia. Animals following nerve ligation were significantly more hyperalgesic than sham-operated animals (P < 0.0001). Lectin IB4, a marker of primary afferent C fibres, showed a qualitative decrease in staining intensity in laminae 1-2 with ligation compared with both the unoperated contralateral side and with sham animals. Using fluorescent immunohistochemistry to quantify changes in neuropeptides in the dorsal horn we found that substance P showed significant decreases with ligation compared to sham operation (P < 0.002). CGRP and galanin showed no significant changes in laminae 1-2 compared to sham-operated animals. Neuropeptide Y (NPY) showed no significant changes in intensity in laminae 1-2; however, in laminae 3-4 there was a significant increase with nerve ligation compared to sham (P < 0.005). We examined how pre-emptive drug treatment affected these neuronal markers at 28 days. We used (1) clonidine, an alpha 2-adrenoreceptor agonist (1 mg/kg, i.p.), (2) morphine, a mu-opioid agonist (5 mg/kg, i.p.) or (3) MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist (0.3 mg/kg, s.c.) administered 30 min prior and 6 h following nerve ligation or sham-operation. Hyperalgesia in the ligated group at 28 days was suppressed by treatment with pre-emptive clonidine (P = 0.003) or MK-801 (P = 0.003) but not morphine. With the exception of NPY there was no effect of pre-emptive drug treatment on any neuronal marker examined. Pre-emptive MK-801 reduced the magnitude of the increase in NPY in laminae 3-4 in the ligated group (P < 0.005) and clonidine showed a similar trend but this did not reach significance. Morphine had no effect on NPY staining. There was a significant correlation between the increase in NPY staining in laminae 3-4 and the degree of hyperalgesia (r = 0.6, P < 0.001). These results suggest that the increased NPY expression in laminae 3-4 of the spinal cord (the territory of the myelinated sensory input) may be crucial to the development of hyperalgesia in this model.