The Experimental Study On The Analgesic Effect And Mechanisms Of Intrathecal Administration Of ω-conopeptide SO3 On The Chronic Neuropathic Pain

Part I The analgesic effect of intrathecal administration of ω-conopeptide SO3 on an animal model of neuropathic pain Experiment I The Experimental Study on CCI and lumbar intrathecalcatheterization in ratsObjective To investigate the extent of behavioral and morphological changes following right sciatic nerve chronic constriction injury (CCI), or chronic implantation with lumbar i.t. catheter in rats. Methods (1)24 adult, male, Sprague-Dawley rats were randomly divided into three groups. Six rats composed each group except the CCI group, which was composed of twelve rats. Rats in the N group served as unoperated controls; rats in the sham group received a sham injury, the sciatic nerve was surgically exposed but not ligated; rats in group CCI, a mononeuropathy were produced by loosely ligating the right sciatic nerve of the rat with chromic gut suture. Rats were tested for hyperalgesia and allodynia by measuring paw withdrawal latency using von-Frey filaments and thermal stimulation. (2)54 adult, male SD rats were randomly divided into nine groups of 6 animals each, the morphological changes at different time following the right sciatic nerve CCI were tested. (3)30 adult, male SD rats were randomly divided into two groups, the behavioral and morphological changes following chronic implantation with lumbar i.t. catheter were investigated. Results (1) Rats undergoing CCI surgery exhibited both thermal hyperalgesia and mechanical allodynia with onset at 3 days and maximal responses at 7-14 days , and keeped on all the time. (2)The pathological changes were axonal edema and segmental demyelination at different extent which were observed at the compression segment and the segment distal to compression in the CCI group. The changes were aggravated as the time of compression prolonged. (3) There were no difference in allodynia or hyperalgesia threshold between normal rats and thosewith lumbar intrathecal catheterization. Conclusion (1)The chronic constrictioninjury is an animal model of an experimental peripheral neuropathy which canlead to spontaneous pain, hyperalgesia and allodynia. (2) chronic lumbarintrathecal catheterization is feasible and safe in rats..Experiment II The analgesic effect of intrathecal bolus injection of ω-conopeptide SO3 on an animal model of neuropathic pain Objective To evaluate the effect of intrathecal bolus injection of 60 -conopeptide, N-type calcium-channel blocker SO3 on pain related behaviors in a rat model of chronic constriction injury (CCI). Methods 110 SD rats weighing 230g~270 g were randomly divided into 11 groups of 10 animals each. All rats were placed four loose ligatures around the right sciatic nerve. 10 days after CCI, the rats were implanted with chronic lumbar i.t. catheters. In the control group the animals were i.t. normal saline 20// L. In other groups the animals were i.t. SO3 or SNX-111 150ng, 300ng, 600ng, 900ng or 1200ng(in 20μL normal saline) respectively. Thermal hyperalgesia and mechanical allodynia were tested by measuring paw withdrawal latency(PWL) in response to thermal stimulation andby von-Frey filaments. Nociceptive thresholds were expressed as x ±s. Data were compared by t tests (comparisons to base-line values obtained just before injection). Results The CCI procedure leads to the development of spontaneous pain, heat hyperalgesia and both thermal and tactile allodynia in the hind foot innervated by the injured nerve. Intrathecal bolus injections of 150 ng to 1200 ng SO3 produced significant, dose-dependent antinociceptive effects on both thermal hyperalgesia and mechanical allodynia; and the effects were similar to those produced by i.t. SNX-111. Conclusion The results show that selective N-type VSCC blockers are potent and efficacious antinociceptive agents when they are administered by the spinal route, and they are effective in a rat model of neuropathic pain.Experiment III The analgesic effect of continuous intrathecal infusion ofω-conopeptide SO3 on an animal model of neuropathic pain Objective To evaluate the effect of continuous intrathecal infusion of ω -conopeptide SO3 on pain related behaviors and whether tolerance developed with chronic infusion in a rat model of chronic constriction injury (CCI). Methods 24 SD rats of CCI model were randomly divided into four groups of 6 animals each. 14 days after CCI, the rats were implanted with chronic lumbar i.t. catheters link to osmotic pump . In the control group the animals were continuous i.t. normal saline 0.25 μL/hr for 28 days. In other groups the animals were continuous i.t. SO3 15ng/h, 30ng/h or 60ng/h for 28 days respectively. Thermal hyperalgesia and mechanical allodynia were tested before and 3,7,14,21,28,30 days after the pumpbeing implanted. Nociceptive thresholds were expressed as x+s. Data were compared by / tests and one-way ANOVA. Results All doses of SO3 produced a significant reduction of the response to the thermal stimulation, ice-cold water and von-Frey filaments in a dose-dependent manner at 3 to 28 days of infusion compared to saline infusion. The effect of SO3 was reversible, as shown by a return to nociceptive responses similar to saline-infused rats 2 days after the minipumps had been disconnected after a 28-day infusion period. Conclusion These data indicate that chronic infusion of ω -conopeptide SO3 produce a powerful antinociception, with minimal development of tolerance. Part II Mechanisms of the analgesic effect of intrathecal administration of ω -conopeptide SO3Experiment I Effect of intrathecal SO3 on the changes in intracellular calcium ion concentration of the spinal dorsal root ganglion (DRG) neuronsin a rat model of chronic constriction injury(CCI)Objective To evaluate the effect of intrathecal administration of SO3 on the changes in intracellular calcium ion concentration of the DRG neurons in a rat model of CCI. Methods 40 male SD rats weighing 230-270g were randomlydivided into five groups of 8 animals each. Rats in the N group served as unoperated controls; in group C the rats were placed four loose ligatures around the right sciatic nerve; in group CN the rats were placed four loose ligatures around the right sciatic nerve and i.t. normal Saline 20 fi L; in group CS1 SO3 600ng was administered 14 days after operation; in group CS7 SO3 (30ng/h) was chronically administered for 7 days from the 7th day post-operation. The animals were decapitated on time. The DRGs (L4-L6) were homogenized into single cells and intracellular calcium ion concentration ([Ca2+]i) was measured by flow cytometry. Results In CCI rats, significant increases were observed in [Ca2+]i on the contralateral side and ipsilateral side of DRGs on day 14 after operation. I.t. NS made no difference in [Ca2+]i to those in group C. The treatment with i.t. SO3 600ng suppressed the increases in [Ca2+]i of bilateral side of DRGs. Chronically administration of SO3 suppressed the increases in [Ca2+]i of bilateral side of DRGs further. Conclusion The antinociceptive effect provided by intrathecal SO3 might be associated with N-type calcium channel, which suppressing the increasees in [Ca2+]i in the DRGs.Experiment II Effect of intrathecal SO3 on the expression of CGRP,substance P, Fos and Jun protein in the spinal cord of CCI rats Objective To investigate the effect of intrathecal administration of SO3 on the expression of CGRP, substance P, Fos and Jun protein in the spinal cord of CCI rats. Methods 72 male SD rats weighing 230-270g were randomly divided into nine groups of 8 animals each. After i.t. NS or SO3, the animals were perfused intracardially with 4% paraformaldehyde and the L4-L6 spinal cord were removed immediately. The changes of CGRP, substance P, Fos and Jun protein expression in the spinal cord were detected by immunohistochemical techniques. Results The expression of CGRP, substance P, Fos and Jun protein in the spinal cord was minimal in sham operation group; CCI induced significant increase in the expression of CGRP, substance P, Fos and Jun protein in both the ipsilateral and the contralateral spinal cords. Acutely or chronically i.t. NS had no effect on these expression. Both acutely or chronically i.t. SO3 can significantly inhibited...