【作者】 冉然；

【导师】 姚尚龙；

【作者基本信息】 华中科技大学 ， 麻醉学， 2011， 博士

【摘要】 第一部分背根神经节GABAA受体调控对大鼠慢性神经痛的治疗作用目的探讨损伤和邻近未损伤大鼠背根神经节GABAA受在L5SNL大鼠神经痛发生发展中的作用,为神经痛的治疗寻找新的靶点。方法选用成年雌性SD大鼠100只,随机分为六组,A组和B组为SNL组,各10只,于L5脊神经结扎后分别于L5和L4DRG局部注射生理盐水50μl, C组为损伤DRG治疗组,30只,分为C1、C2和C3亚组,每组10只,分别于L5脊神经结扎术后损伤L5背根神经节局部注射muscimol(20μg),bicuculline(0.15μg)和muscimol(20μg)+bicuculline(0.15μg),D组10只,于L5脊神经结扎术后5天L5背根神经节局部注射rnuscimol20μg,E组为未损伤DRG治疗组,30只,分为E1、E2和E3亚组,每组10只,分别于L5脊神经结扎术后未损伤L4背根神经节局部注射muscimol(20μg),bicuculline(0.15μg)和：muscimol(20μg)+bicuculline(0.15μg),F组10只,于L5脊神经结扎术后5天L4背根神经节局部注射muscimol20μg,从术前一天至术后10天内,每日行为学检测各组大鼠术侧后爪热痛阈和机械痛阈。结果A组和B组大鼠L5脊神经结扎术后1天机械痛阈开始下降,持续至术后10天,热痛阈于术后3天开始下降,持续至术后10天,两组间痛阈无明显差异(P>0.05),同A组相比,C1组动物术后各时间点机械痛阈和热痛阈明显升高(P<0.05),并且同术前相比没有明显差异(P>0.05),C2组于术后1天就出现明显热痛阈下降,持续至术后10天(P<0.05),同A组相比,术后各时间点热痛阈和机械痛阈明显下降(P<0.05),C3组术后同A组比较没有明显差异(P>0.05),D组动物注射后1天,机械痛阈和热痛阈开始上升(P<0.05),于注射后2天达到高峰,注射后3天机械痛阈和热痛阈开始下降,于注射后4天同A组比较差异无统计学意义(P>0.05)。同B组动物相比,E1组术后机械痛阈明显升高(P<0.05),持续至术后10天,但同术前相比仍然较低(P<0.05),热痛阈于术后同B组相比无明显差异(P>0.05),E2组从术后1天开始热痛阈出现明显下降,同B组相比,热痛阈和机械痛阈在术后各个时间点上明显下降(P<0.05),E3组动物术后同B组比较没有明显差异(P>0.05),F组动物注射后1天,同B组相比机械痛阈和热痛阈都有所上升(P<0.05),注射后2天达到高峰,注射后3天下降,于注射后第4天同B组比较没有明显差异(P>0.05)。结论SNL术后受损DRG局部GABAA受体调控可以预防痛觉过敏的发生,但对已经发生的痛觉过敏只起到暂时的缓解作用。未损伤DRG局部GABAA受体对神经痛机械痛敏的预防有一定的作用,但对神经痛热痛觉过敏只有短暂的治疗效果。第二部分神经痛大鼠背根神经元GABA含量及GABAA受体的改变目的探讨神经痛大鼠损伤和未损伤DRG上GABA及其受体GABAA的表达,从而为DRG局部注射GABAA受体激动剂治疗神经痛提供理论依据。方法选取30只SD大鼠随机分为两组,SNL组大鼠20只,行L5脊神经结扎诱导神经痛,对照组大鼠10只,行假手术,不结扎L5脊神经,分别于术前和术后3天行行为学测手术侧大鼠后爪痛阈,于手术后第5天处死大鼠,取出同侧L4及L5DRG,运用免疫组化SABC法检测大鼠GABA及其受体GABAAα1亚基的表达,并分类统计各组中具有免疫阳性细胞所占比例。结果SNL大鼠术后出现明显神经痛症状,对照组大鼠无明显神经痛症状,与对照组相比,SNL组L5DRG中具有GABA免疫阳性的神经元比例出现明显下降(P<0.05),主要为大和中DRG神经元中具有GABA免疫阳性的神经元所占比例出现下降(P<0.05),小DRG神经元没有明显改变(P>0.05); L4DRG中GABA免疫阳性神经元的比例也没有发现明显改变(P>0.05)。L5DRG中GABAA受体免疫阳性神经元的比例没有发现明显改变(P>0.05)，但大和中DRG中GABAA免疫阳性神经元比例明显下降(P<0.05)。L4DRG中具有GABAA受体免疫阳性的神经元比例出现明显下降(P<0.05),主要为大和中DRG神经元中具有GABA免疫阳性的神经元所占比例出现下降(P<0.05),小DRG神经元没有明显改变(P>0.05)。结论受损和未受损DRG上GABA能神经元减少,具有GABAA受体的神经元数量下降,导致GABA能传递减弱,可能是神经痛发病机制。第三部分损伤和未损伤背根神经节GABA激活电流的改变以及对神经元电生理学特征的影响目的运用膜片钳技术研究损伤和未损伤DRG神经元GABA激活电流的改变及其对神经元的影响。方法成年SD大鼠30只,随机分为两组, SNL组20只,行L5脊神经结扎,对照组10只,行假手术,不结扎脊神经,术前及术后3天通过行为学测定来判断造模成功后立即处死大鼠,急性分离对照组L4-6及SNL组L4和L5背根神经元,建立膜片钳全细胞电压钳记录模式,在电流钳模式下给予阶跃刺激诱发动作电位,记录后通过细胞外加药排管给予100μmol/lGABA诱发GABA电流，记录神经元上电流幅度,再次转入电流钳下记录动作电位,对比用药前后静息电位及动作电位的改变。结果SNL术后4天,大鼠患肢机械痛阈和热痛阈出现明显下降(P<0.05),对照组大部分DRG神经元对100μmol/lGABA产生一快速失活的内向电流,这些神经元主要集中在大DRG神经元和中DRG神经元上,同对照组相比,SNL组L4和L5DRG神经元中GABA电流发生率明显降低,主要为大和中DRG神经元的发生率出现下降(P<0.05),小DRG神经元发生率没有明显变化(P>0.05), SNL组L4和L5大和中DRG神经元GABA电流幅度同对照组相比均明显降低(P<0.05),小DRG神经元GABA电流没有明显差异(P>0.05)。在具有GABA电流的神经元中,100μmol/lGABA可以诱发神经元去极化,静息电位绝对值、动作电位幅度及诱发动作电位的基强度同用药前相比出现明显下降(P<0.05),对阈电位没有明显影响(P>0.05),对比SNL组和对照组用药前后DRG神经元各参数的变化幅度发现,SNL组L5大和中DRG神经元静息电位下降幅度、动作电位下降幅度以及基强度下降幅度同对照组相比明显较低(P<0.05),小DRG神经元没有明显改变(P>0.05)。L4大DRG神经元超射值和基强度的下降幅度出现降低(P<0.05)中DRG神经元静息电位,基强度和超射值的变化幅度明显降低(P<0.05)。另外,SNL组L4和L5DRG中以及对照组中都有部分神经元出现自发放电,具有自发放电的神经元集中在小DRG神经元中,自发放电的神经元没有GABA电流出现。结论神经损伤后损伤和邻近未损伤大和中DRG神经元上GABA介导的抑制信号的减弱,引起神经元致敏,可能是神经痛的发病机制之一。更多还原

【Abstract】 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更多还原