Differential Role Of CGRP And IB4Afferent Pathway In Mediating Positive Or Negative Neuropathic Pain Syndroms Induced By A Rat’s Sciatic Nerve Crush Model

ObjectivesHypoalgesia denotes a decreased or increased threshold to thermal, cold or mechanical stimuli. Hypoalgesia occurs after nerve injury, diabetes mellitus or other central or peripheral neuropathies, which accompany with positive or negative sign of neuropathic pain. In our experiment, negative and positive signs are induced by crushing the rat scaitic nerve with different pressures measured by FSR sensor. Furthermore, DRG and spine immunofluerescence staining showed that transient loss of IB4positive non-peptidergic C fiber occurred in the CCI and light crush groups with signs of hyperalgesia, while longer loss of CGRP positive peptidergic C fiber occurred in the heavy crush group with signs of hypoalgesia. The different phenotype indicated that these two kinds of C fiber had distinct functions in the pain signaling pathways: thermal hypoalgesia after nerve injury was resulted by the loss of peptidergic C fiber; non peptidergic C fiber played an important role in the central modulation of pain signaling and the onset of positive signs. These data provided new insights into the comprehensive therapy of neuropathic pain.Methods1. Twenty four male SD rats were randomly divided into four group Sham (n=6),100g (n=6),400g(n=6)and group CCI (n=6). In group CCI, left sciatic nerve CCI model were established according to Bennett and Xie, in group Sham, left sciatic nerves were only exposed without ligation. In100g group and400g group, left sciatic nerves were crushed by100g and400g force respectively. Paw withdrawal threshold to mechanical stimuli(MWT) and paw withdrawal latency to a thermal nociceptive stimulus(TWL) were measured at1day before (baseline) and3,7,10,14,21,28days after operation in the lateral and medial part of both left and right paw. Evaluation of the cold allodynia, touch allodynia and pain-related behavior were performed at1day before (baseline) and3,7,10,14,21,28days after operation in both left and right paw. The animals were then sacrificed and the left sciatic nerve, L5DRG and lumbar enlargement of the spinal cord were harvested at7,10,21,28days after operation and immersed in4%formaldehyde for a night.2. The harvested DRG and Spine were embedded with OCT and stained with double labeling immunofluorescence method. In DRG, the segments at200micron,250micron and300micron were chose to calculate positive cells. In Spine, the area of positive nerve sprouting in ipilateral and contralateral lamina2were calculated. The sciatic nerve were performed with bodian silver staining method and the fiber density were compared in four group.Results1. Compared to DO, both the100g group and CCI groups showed thermal and mechanical hyperalgesia (P<0.05), and the behavior test scores of dynamic allodynia, cold allodynia and pain-related behavior were higher than DO. On the contrary, the400g group showed obvious hypoalgesia, which presented as higher thresholds of thermal and mechanical pain (P<0.05). Although there were a slight increase of the behavior test scores on dynamic allodynia, cold allodynia and pain-related beavior, but without statistical significance. 2. In dorsal root ganglion (DRG), compared to the Sham group, the number of IB4and CGRP positive neurons showed temporary decrease in the100g group and CCI group, while presented as long lasting, unrestorable decrease in the400g group. In spinal cord, the decrease of IB4fibers in Lamina2of the lumbar enlargement comparing to the contra side was shown in all of the100g,400g and CCI groups, while the decrease of CGRP fibers was only detected in the400g group (P<0.05). In sciatic nerves, Bodian Silver Staining showed that the density of fibers on D3in the400g group was lower comparing with that in the other two groups.Conclusions1、100g crush generated a transient pressure on rat sciatic nerve, led to signs of hyperalgesia that similar to CCI model.400crush resulted to cold and touch allodynia, as well as negative signs presented as mechanical and heat hypoalgesia2、Compare the dynamic changes of IB4and CGRP positive neurons and fibers in DRG and spinal cord between groups, IB4fibers may take part in the central regulatory mechanisms of pain signaling, while CGRP fibers directly transduct the thermal pain signal.