Studies Of Effect And Mechanism Of Regulating GABA_A Receptors Of Dorsal Root Ganglion In Treating Neuropathic Pain

Part1 Therapeutical effect of GABAA receptor regulation of DRG in rats with neuropathic painObjective To investigate the role of the GABAA receptors in the injured and uninjured DRG of the SNL rats in the mechanism of the neuropathic pain, in order to discover the new target to treat the neuropathic pain.Methods 100 female SD rats were randomly divided into the six groups. The rats in the A (n=10) and B (n=10) group were topically injected with 50μl NS in the L5 and L4 DRG after the L5 spinal nerve ligation. C group (n=30) were randomly divided into C1 (n=10), C2 (n=10) and C3 (n=10) groups. The rats in the C1,C2 and C3 group were respectively topically injected with muscimol(20μg), bicuculline(0.15μg) and muscimol(20μg)+bicuculline(0.15μg) in the L5 DRG after the L5 spinal nerve ligation. D group (n=10) were topically injected with muscimol (20μg) in the L5 DRG in the post-operative 5 day. E group (n=30) were randomly divided into E1 (n=10), E2 (n=10) and E3 (n=10) groups. The rats in the E1,E2 and E3 group were respectively topically injected with muscimol(20μg), bicuculline(0.15μg) and muscimol(20μg)+bicuculline(0.15μg) in the L4 DRG after the L5 spinal nerve ligation. F group (n=10) were topically injected with muscimol (20μg) in L4 DRG in the post-operative 5 day. The mechanical and thermal pain thresholds on the injured paw were measured through behavioral test respectively on the pre-operative 1 day and post-operative 1-10 days. Results The mechanical threshold in the A and B group decreased in the post-operative 1-10 days (P<0.05). The thermal pain threshold decreased too in the post-operative 3day, and sustained to postoperative 10 day (P<0.05). There was no significant difference between A and B group (P>0.05). Compared with A group, the mechanical and thermal thresholds of the C1 group were significantly increased in the post-operative days (P<0.05). Compared with the pre-operative thresholds in the C1 group, there was no significant difference (P>0.05). In the C2 group, the thermal pain thresholds were significantly decreased in the post-operative 1 day, and sustained to the post-operative 10 days (P<0.05). Compared with A group, the thermal and mechanical thresholds in the C2 group significantly decreased in the postoperative days (P<0.05). There was no significant difference between the pain threshold in C3 and A group (P>0.05). In D group, the thermal and mechanical thresholds increased in the post-injection 1 day (P<0.05), and reached to peak in the post-injection 2 day (P<0.05), but decreased since post-injection 3 day. There was no significant difference with A group in post-injection 4 day (P>0.05). Compared with B group, the mechanical threshold in El group significantly increased after operation, and sustained to post-operative 10 days (P<0.05). But compared with the pre-operative thresholds, the post-operative mechanical thresholds significantly decreased (P<0.05). There was no significantly difference between the thermal thresholds in the E1 and B group (P>0.05). In the E2 group, the thermal thresholds significantly decreased in the post-operative 1 day (P<0.05). Compared with B group, the thermal and mechanical thresholds significantly decreased in the post-operative days (P<0.05). In E3 group, the pain thresholds were no significant difference with B group (P>0.05). In F group, the thermal and mechanical thresholds increased in the post-injection 1 day, and reached to peak in the post-injection 2 day (P<0.05), but decreased since post-injection 3 day. There was no significant difference with B group in post-injection 4 day (P>0.05).Conclusion Agonism to the GABAA receptor in the injured DRG can prevent the NP after SNL, but can only transiently alleviate the symptom of NP when NP has been existing. Agonism to the GABAA receptor in the adjacent uninjured DRG can prevent the mechanical hyperalgesia after SNL, but only transiently alleviates the thermal allodynia.Part2 Alteration of GABA and GABAA receptor of rats'DRG neurons in neuropathic painObjective To investigate the expression of the GABA and the GABAA receptor on the injured and uninjured DRG in the rats with neuropathic pain.Methods 30 SD rats were randomly divided into two groups. SNL group (n=20) suffered from the L5 spinal nerve ligation. C group (n=10) suffered from the same procedure as the SNL group except for L5 spinal ligation. The pain thresholds on the ligated paw were measured through behavioral test respectively on pre-operative day and post-operative 3 day in these two groups. On the post-operative 5 day, the L4 and L5 DRG in these two groups were dissociated. The expression of GABA and GABAA receptor on these DRGs were measured through immunohistochemistry (SABC). The ratio of the cell with the positive immune-reaction was recorded.Results Rats in SNL group showed the obvious symptoms of NP after operation. Compared with C group, the proportion of GABA positive neurons in SNL group significantly decreased in the L5 DRG (P<0.05). Most of the DRG neurons with the decreased GABA expression were large and medium DRG neurons (P<0.05), but not the small neurons. There was no significant difference in the expression of GABA in L4 DRG between SNL group and C group (P>0.05). There was no significant difference in the expression of GABAA receptor in L5 DRG between SNL group and C group (P>0.05). The proportion of neurons with GABAA receptor expression significantly decreased in L5 large and medium DRG neurons (P<0.05). Compared with C group, the expression of GABAA receptor in the L4 DRG significantly decreased in SNL group (P<0.05). Most of the DRG neurons with the decreased GABAA receptor expression were large and medium DRG neurons (P<0.05), but not the small neurons (P>0.05).Conclusion Down-regulation of GABA and GABAA receptor expression in the injured and uninjured DRG may weaken the GABAergic transition, which may be the mechanism of the neuropathic pain. The changes on the adjacent uninjured DRG neurons may be the important role in the mechanical hyperalgesia, but not in the thermal allodynia.Part3 The alteration of GABA active currents in injured and intact DRG neurons in neuropathic pain and the effect of these alterations on the electrophysiology characteristics of neuronsObjective To investigate the changes of GABA currents in the injured and adjacent uninjured DRG neurons in the rats with neuropathic pain and the role of these changes in the neuropathic pain with whole-cell patch clamp.Methods 30 SD rats were randomly divided into two groups. SNL group (n=20) suffered from the L5 spinal nerve ligation. C group (n=10) suffered from the sham operation without the L5 spinal nerve ligation. The neuropathic pain was identified through behavioral test respectively on pre-operative day and post-operative 3 day. Then, the L4-6 DRG neurons in C group and the L4 or L5 DRG neurons in SNL group were acutely isolated after decapitation, and the whole cell patch clamp were established in these acute isolated neurons. In the currents clamp mode, the action potential was activated and recorded under the step currents stimulation. Then, under the voltage clamp mode, the GABA currents were activated and recorded with the 100μmol/1 GABA extracellularly applied from an array of fused silica glass tubes. The action potential was recorded again in this condition under the currents clamp. The changes of resting potential and action potential were recorded.Result The mechanical and thermal pain threshold significantly decreased in post-operative 4 day (P<0.05). Most of the DRG neurons had the fast inactive inward current with the 100μmol/1 GABA. Compared with C group, the incidence of GABA currents in the L4 and L5 DRG neurons in SNL group significantly decreased (P<0.05), and most of the neurons with decreased GABA currents were the medium and large DRG neurons. There were no significant changes in small neurons in SNL group (P>0.05).100μmol/1 GABA induced the depolarization of neurons, and the resting potential and the amplitude and rheobase of action potential significantly decreased (P<0.05). There was no significant change in the threshold potential after GABA application (P>0.05). Compared with C group, the amplitudes of alteration induced by GABA were significantly decreased in resting potential, amplitude and rheobase of action potential in the L5 large and medium DRG neurons (P<0.05), but not in the L5 small DRG neurons (P>0.05). The amplitudes of alteration induced by GABA were decreased in the amplitude and rheobase of action potential in L4 large DRG neurons (P<0.05), but not in the resting potential (P>0.05). The amplitudes of alteration of resting potential, amplitude and rheobase of action potential induced by GABA were significantly decreased in L4 medium DRG neurons (P<0.05). The spontaneous discharge was found in some neurons in L4 and L5 DRG. Furthermore, the neurons with the spontaneous discharge were small DRG neurons, and without GABA currents.Conclusion The weak of inhibiting signal mediated by GABAA receptor in large and medium neurons in injured and intact uninjured DRG induce the neurons sensitization, which may be the mechanism of NP...