~(60)Co-γ射线照射对小鼠SVZ神经干细胞增殖的影响

目的:探讨60Co-γ射线照射对成体小鼠侧脑室室管膜下区(SVZ)神经干细胞增殖的影响。方法:28只昆明小鼠随机分成4组,每组7只,分别以0 Gy、5 Gy(小剂量)、15 Gy(中剂量)、30 Gy(大剂量)剂量进行照射,于照射后1周观察侧脑室室管膜下区(SVZ)的5-溴脱氧核苷尿嘧啶(BrdU)阳性细胞形态和数目。结果:照射后1周,各照射组SVZ的BrdU阳性细胞形态与对照组无差别,呈圆形、椭圆形以及梭形等;15 Gy、30 Gy照射组SVZ的BrdU阳性细胞数明显降低(P<0.01),5 Gy照射组SVZ的BrdU阳性细胞数无差别(P>0.05)。结论:大中剂量60Co-γ照射会抑制SVZ神经干细胞的增殖分裂能力。     

褪黑素通过上调紧密连接蛋白减轻蛛网膜下腔出血大鼠脑水肿

目的:研究褪黑素对蛛网膜下腔出血(SAH)大鼠脑水肿的影响及分子机制。方法:雄性SD大鼠分为假手术组(sham)、SAH组(SAH)和褪黑素预处理组(SAH+Mel)。利用注射自体血液的方法制备SAH大鼠模型,SAH+Mel组大鼠在模型制备前1周通过皮下注射给予褪黑素预处理。利用湿干比方法检测大鼠脑含水量,利用伊文思蓝渗透实验检测血脑屏障的完整性,利用Western Blot检测大鼠皮层中闭合小环蛋白-1(ZO-1)、咬合蛋白(occludin)和闭合蛋白-5(claudin-5)的表达。结果:和假手术组大鼠相比,SAH术后48 h大鼠脑含水量增加明显(P<0.05),脑组织中伊文思蓝含量增加(P<0.05),皮层中ZO-1、occludin和claudin-5的表达下降。然而,经褪黑素预处理后,SAH大鼠脑含水量下降(P<0.05),脑组织中伊文思蓝含量减少(P<0.05),ZO-1、occludin和claudin-5的表达上调,(P<0.05)。结论:褪黑素通过上调紧密连接蛋白表达减轻SAH大鼠脑水肿。     

全身照射小鼠血清中不同化学形态铜元素含量变化研究

目的采用火焰吸收原子分光光度法(FAAS)检测全身照射小鼠血清中不同化学形态铜元素含量,探究辐照后小鼠血清铜元素含量变化机制及其作为生物剂量计的可行性。方法昆明种雄性小白鼠78只,鼠龄5周,体质量20~22 g,清洁级。按随机数字表法,根据照射剂量0、4.0、6.0、8.0 Gy分为对照组与照射组,再根据照射时间1、4、10、24 h共分为13组,每组6只。照射组1~4亚组给予4.0 Gy剂量照射,照射时间为1.5 min;5~8亚组给予6.0 Gy剂量照射,照射时间为2 min 15 s;9~12亚组给予8.0 Gy剂量照射,照射时间为3 min;对照组不给予照射。从小鼠眼眶取血后分离血清,用乙醇蛋白沉淀法分离血清中不同化学形态铜元素,火焰原子吸收光谱法测量血清铜质量浓度。采用单因素方差分析法对所得数据进行处理。结果不同剂量照射小鼠游离态血清铜质量浓度24 h内变化无统计学意义(P0.05);照射后24 h内小鼠结合态血清铜质量浓度变化有统计学意义(P0.05),其中照射剂量为4.0 Gy、6.0 Gy时变化趋势为先上升后下降,峰值分别位于4 h、10 h处;照射剂量为8.0 Gy时变化趋势为单调上升。照射后24 h小鼠结合态血清铜质量浓度、血清铜总质量浓度与辐照剂量呈良好线性关系,直线方程为Y=0.047 3X+0.365 4、Y=0.048 6X+0.530 9(R2=0.972 6、0.982 6)。结论照射小鼠体内血清铜元素含量发生变化,主要体现在血清中参与细胞保护与修复等生理活动的结合态铜元素含量变化;生物体在调控结合态铜元素含量变化来进行细胞保护修复的同时并未产生细胞毒性;照射后24 h小鼠结合态血清铜质量浓度、血清铜总质量浓度具备发展成为新型生物剂量潜力。     

Ifenprodil减轻大鼠蛛网膜下腔出血后脑水肿及皮质凋亡

目的:探讨N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)受体GluN2B亚基(GluN2B-NMDA受体)的负向变构剂艾芬地尔(ifenprodil)对大鼠蛛网膜下腔出血(subarachnoid hemorrhage, SAH)后神经功能缺损、血脑屏障通透性、脑水肿及皮质细胞凋亡的影响。方法:选取成年健康雄性SD大鼠90只,分为假手术组(n=18)、SAH+安慰剂组(n=36)和SAH+ifenprodil组(n=36),通过血管内穿刺法建立SAH模型。在建模后2、24和48 h,腹腔注射安慰剂或ifenprodil。在建模后72 h,Garcia评分标准评价神经缺损,脑干湿重法检测脑水肿,伊文思蓝(Evans blue)法检测血脑屏障通透性,TUNEL染色检测皮质细胞凋亡,Western blot法检测凋亡信号分子的表达。结果:与假手术组比较,SAH+安慰剂组的神经功能评分降低,脑组织含水率增加,伊文思蓝有显著溢出,TUNEL阳性细胞数显著增多、Bcl-2的表达减弱,Bax及activated caspase-9/3的表达增强(P0.05);与SAH+安慰剂组相比,SAH+ifenprodil组神经功能评分升高,脑组织含水率降低,伊文思蓝溢出量降低,TUNEL阳性细胞减少,Bcl-2的表达增加,Bax及activated caspase-9/3表达减少(P0.05)。结论:ifenprodil能够减轻SAH后神经功能缺损和脑水肿,降低血脑屏障的通透性,减少皮质区细胞凋亡。     

液化石油气中毒对小鼠齿状回及室管膜下区神经发生的影响

目的:探讨液化石油气急性中毒对小鼠海马齿状回及侧脑室室管膜下区神经发生的影响。方法:选择健康成年昆明小鼠随机分三组,分别为对照组,液化石油气(LPG)高、低剂量中毒组。使用密闭中毒箱行染毒实验。用Morris水迷宫训练测试其空间学习与记忆能力;腹腔注射5-溴脱氧尿苷(BrdU)后通过免疫组织化学方法来显示BrdU阳性细胞。结果:两个中毒组动物的学习记忆能力均弱于对照组(P<0.01),齿状回和室管膜下区内BrdU阳性细胞数目也低于对照组(P<0.01)。结论:液化石油气急性中毒通过抑制齿状回和室管膜下区的神经发生,影响小鼠的学习记忆能力和脑损伤后修复。     

X射线全身照射对小鼠免疫功能的影响

目的:通过X-射线体外全身照射BALB/c小鼠建立小鼠辐射损伤模型,检测放射线对小鼠脾组织、单核巨噬细胞及T、B淋巴细胞损伤的剂量范围,探索放射线剂量与组织损伤程度之间的量效关系。方法:采用9种不同剂量的X-射线体外全身照射BALB/c小鼠,于照射后1d、60d、120d取腹腔细胞做大吞噬实验;分离脾细胞,MTT法检测T细胞增殖活性,同时取脾脏组织切片、HE染色,检测组织损伤程度;照射后120d取小鼠脾细胞用流式细胞仪检测T、B细胞数量百分比。结果:不同剂量X-射线照射的小鼠其大吞噬细胞的吞噬指数及T细胞的增殖指数之间均存在差异,且与放射剂量呈负相关性。FCM检测结果显示0.1Gy及以上剂量组与正常对照组相比较,小鼠T、B淋巴细胞数量减少(P<0.05)。0.25Gy和0.5Gy组T、B淋巴细胞数量明显高于除0.05Gy以外的其余各照射组,但是仍低于正常组(P<0.05)。脾组织切片显示,照射后1d 0.1Gy以上照射组脾脏炎性细胞浸润;60d后,4.0Gy和8.0Gy组脾脏明显萎缩、巨噬细胞增生、瘤样变,其余组损伤不明显;120d后8.0Gy组脾脏萎缩、红白髓分界不清,白髓损伤尤为严重,其余组不明显。结论:0.1Gy以上X-射线体外全身照射可引起小鼠免疫功能及组织损伤,且损伤程度与放射性强度呈剂量依赖性。     

人脐带间充质干细胞对大鼠创伤性脑损伤后血脑屏障的保护

背景:人脐带间充质干细胞在创伤性脑损伤后血脑屏障修复过程中发挥着至关重要的作用。目的:探讨人脐带间充质干细胞移植对创伤性脑损伤大鼠血脑屏障的保护作用及可能机制。方法:①将60只SD大鼠随机分为假手术组、脑损伤模型组、人脐带间充质干细胞组及抑制剂Sunitinib组,每组15只。除假手术组外,其他3组采用大鼠可控性皮质撞击仪制作创伤性脑损伤模型;②造模后0.5,24,48 h,假手术组、脑损伤模型组大鼠经尾静脉注射1 mL生理盐水,人脐带间充质干细胞组、抑制剂Sunitinib组大鼠经尾静脉注射2×10^9 L^-1人脐带间充质干细胞1 mL,抑制剂Sunitinib组大鼠从造模前1 d开始至处死每天按80 mg/kg的剂量口服PDGFR-β通路抑制剂Sunitinib;③造模后3 d,利用干湿比重法计算各组大鼠脑组织含水量,伊文思蓝法检测血脑屏障的通透性,免疫荧光染色观察GFAP和vWF的表达,Western blot检测血脑屏障相关蛋白及PDGFR-β蛋白的表达。结果与结论:①与假手术组比较,脑损伤模型组的脑含水量、伊文思蓝含量、vWF和GFAP表达明显升高(P<0.05),ZO-1、Oclaudin-5、PDGFR-β蛋白表达明显降低(P<0.05);②与脑损伤模型组比较,人脐带间充质干细胞组的脑含水量、伊文思蓝含量、vWF和GFAP表达明显降低(P<0.05),ZO-1、Oclaudin-5、PDGFR-β蛋白表达明显升高(P<0.05);使用PDGFR-β抑制剂后人脐带间充质干细胞的治疗作用被明显抑制;③结果表明人脐带间充质干细胞可以降低创伤性脑损伤大鼠血脑屏障的通透性,发挥神经保护作用,其作用机制可能是其提高了损伤区PDGFR-β蛋白的表达。     

CART肽抑制缺血再灌注小鼠脑组织水通道蛋白4表达并减轻脑水肿

目的:研究可卡因及苯丙胺调节转录物(CART)肽对小鼠脑缺血再灌注急性期脑水肿的作用,并探讨其作用机制。方法:清洁级雄性ICR小鼠随机分为假手术(sham)组、大脑中动脉栓塞(MCAO)组和CART组,复制小鼠MCAO模型以模拟局灶性脑缺血再灌注损伤病理过程,TTC染色法计算脑梗死体积及脑水肿体积,干湿重法测定脑组织中水含量,通过检测伊文思蓝含量判断血脑屏障(BBB)的完整程度,免疫荧光和Western blot观察脑组织水通道蛋白4(AQP-4)的表达变化。结果:小鼠MCAO后24 h,CART肽处理组小鼠脑梗死体积和脑水肿体积均明显小于MCAO组(P0.01),脑组织水含量亦明显低于MCAO组(P0.01)。伊文思蓝测定表明,CART肽能够有效维持血脑屏障的完整性。免疫荧光和Western blot结果显示,MCAO小鼠脑组织中AQP-4表达明显升高,而CART处理明显抑制脑缺血再灌注诱导的AQP-4表达(P0.05)。结论:CART肽可减轻脑缺血再灌注损伤小鼠急性期脑水肿,维持血脑屏障完整性,其作用机制可能与抑制脑组织AQP-4表达水平密切相关。     

缓激肽对大鼠缺血区血脑屏障的影响

目的研究缓激肽对局部脑缺血区超微结构、血脑屏障的通透性及继发性脑水肿的影响。方法制备大鼠大脑中动脉缺血模型,在大脑中动脉缺血2hr再灌注1hr末,颈内动脉灌注小剂量缓激肽(10μg/kg/min),应用电镜观察血脑屏障超微结构的改变;测定脑组织伊文思蓝含量来判断血脑屏障的通透性;应用干湿法测定脑含水量的变化。结果缓激肽灌注大鼠脑毛细血管紧密连接开放,对照组大鼠紧密连接完整;与对照组相比,缓激肽灌注组缺血侧脑组织伊文思蓝含量明显高于缺血对照组(P<0.01),但再灌注24hr后脑含水量并没有增加(P>0.05)。结论小剂量缓激肽通过开放紧密连接来增加缺血区血脑屏障的通透性,但并不会增加继发性脑水肿的程度。     

低频超声开放大鼠血脑屏障的研究

目的探讨1MHz低频超声在不同功率、不同照射时间对大鼠脑细胞及血脑屏障的影响,寻求开放血脑屏障的最佳条件。方法119只Wistar大鼠颅顶右前方开窗后超声照射,采用伊文思蓝(EB)法检测血脑屏障通透性的变化;应用透射电镜观察内皮细胞紧密连接的变化。结果1MHz、12mW照射20s或30s,照射后3hrs,血脑屏障通透性均显著高于对照组(P<0.05),电镜下微血管内皮紧密连接呈开放状态,照射时间30s时,可见部分神经元核膜变形;照射后12hrs,与对照组相比血脑屏障通透性无明显差异(P<0.05),且部分神经元核膜的变形无明显恢复。1MHz、8mW照射的各组与对照组比较血脑屏障通透性无明显差异(P<0.05)。结论1MHz、12mW、照射时间20s可能是可逆性地开放血脑屏障的适宜条件。     

脊髓刺激术对神经病理性痛模型大鼠痛行为和脊髓背角内小胶质细胞激活的影响

目的:探讨脊髓刺激术(spinal cord stimulation,SCS)对神经病理性痛(neuropathic pain,NP)模型大鼠痛行为及脊髓背角内小胶质细胞激活的影响。方法:成年大鼠20只,随机分为4组:(1)正常对照组(control组);(2)SCS组:正常大鼠给予SCS刺激;(3)脊神经结扎(spinal nerve ligation,SNL)假刺激组(SNL+shamSCS组):SNL且植入SCS装置,但不刺激;(4)SNL+SCS组:SNL且给予SCS刺激。术前连续3 d、术后第5 d检测各组大鼠足底机械痛敏阈值(mechanical withdrawal threshold,MWT)。SCS组和SNL+SCS组术后第2-5 d给予SCS刺激,每d持续8 h;且在每次给予SCS 8 h刺激前进行90 min行为学测试,即SCS刺激30 min,以及刺激结束后的60 min内(共90 min),每15 min测量一次MWT。在第5 d给予SCS 8 h刺激结束后处死动物,利用免疫组织化学染色结合平均光密度(average optical density,AOD)分析的方法检测各组大鼠腰5节段脊髓背角内小胶质细胞特异性标志物OX-42的表达情况。结果:(1)行为学结果显示:术后第5 d,SNL+shamSCS组和SNL+SCS组大鼠手术侧后爪的MWT由术前26.00±0.0 g分别降至5.50±0.96 g和6.40±0.40 g(P<0.05);SNL+SCS组给予SCS刺激30 min后大鼠手术侧后爪的MWT明显有所提高,达16.20±2.60 g,与刺激前(6.40±0.40 g)相比有显著性差异(P<0.05);但停止SCS刺激60 min后,大鼠的MWT明显有所下降,与刺激前几乎没有明显差别。(2)免疫组化染色结果显示:术后第5 d,SNL+SCS组脊髓背角内OX-42的表达明显弱于SNL+shamSCS组,但二者都强于control组和SCS组;AOD结果也证实:SNL+SCS组大鼠脊髓背角内OX-42的AOD(1.29±0.28)明显低于SNL+shamSCS组(2.66±0.38),但仍高于control组(0.14±0.21)和SCS组(0.24±0.08)。结论:SCS对SNL模型大鼠的神经病理性痛有较好的镇痛效果;该作用可能与SCS刺激显著抑制脊髓背角内小胶质细胞的激活密切相关。     

脊髓刺激术对神经病理性痛模型大鼠脊髓背角内NR2B受体和星形胶质细胞激活的影响

目的:探讨脊髓刺激术(spinal cord stimulation,SCS)对L5脊神经结扎(spinal nerve ligation,SNL)诱导的神经病理性痛(neuropathic pain,NP)大鼠脊髓背角内NMDA受体亚单位NR2B的表达和星形胶质细胞激活的影响。方法:成年雄性SD大鼠48只,随机分为4组:正常组(不做任何处理);SCS组(植入SCS装置并给予SCS刺激);SNL+sham SCS组(给予SNL手术并植入SCS装置,但不进行刺激);SNL+SCS组(SNL手术并给予SCS刺激)。SCS刺激是在SNL术后第6～10 d进行(8 h/d),第10 d刺激结束后处死动物。运用行为学方法检测慢性痛状态下大鼠后肢对机械性刺激的反应阈值;采用免疫组织化学染色和Western blot方法分别检测脊髓背角内NR2B和星形胶质细胞的标志物GFAP的表达变化。结果:(1)SNL术后大鼠手术侧后足机械性痛敏显著增加,第6～10 d给予SCS刺激后,可观察到大鼠的痛行为学表现有明显缓解;(2)免疫组化结果显示:与SNL+sham SCS组相比,SNL+SCS组大鼠脊髓背角内NR2B和GFAP免疫阳性细胞的数量显著减少;(3)Western blot结果显示:给予SCS刺激后,SNL大鼠腰膨大段脊髓背角内NR2B的表达量显著下调,同时GFAP的表达量也明显有所降低。结论:给予SCS刺激可以有效地缓解SNL模型大鼠的神经病理性痛的行为学表现;该作用可能与SCS刺激抑制脊髓背角内NR2B的表达和星形胶质细胞的激活密切相关。     

大鼠外周神经损伤后脊髓背角HMGB1表达上调参与机械性痛觉超敏

目的:研究HMGB1(high mobility group box-1)在神经病理性痛大鼠脊髓水平的表达变化,探索HMGB1在神经病理性痛发生发展中的作用,为治疗神经病理性痛提供新的理论依据和治疗靶点。方法:(1)雄性SD大鼠(180～220)g 12只,随机均分为三组:NS组:鞘内注射生理盐水;A组:鞘内注射HMGB1 1μg;B组:鞘内注射HMGB1 10μg。盲法用von Frey测定给药前及给药后1 h、1、3、7、14、21、28 d大鼠50%机械缩足阈值(me-chanical withdrawal threshold,MWT);(2)雄性SD大鼠(180～220)g 5只,免疫荧光双标观察HMGB1在脊髓背角的表达定位;(3)雄性SD大鼠(180～220)g 42只,随机均分为对照组(6只),SNL模型组(每时间点6只),West-ern Blot方法观察大鼠脊髓背角HMGB1对照及术后1、3、7、14、21、28 d的表达变化。结果:(1)大鼠脊髓鞘内注射HMGB1后诱发长时程机械性痛敏,A组在鞘内给药后7 d MWT明显下降(P<0.01),B组给药后1 h MWT即显著下降,且持续存在至少28 d;(2)免疫荧光双标显示:HMGB1主要表达于NeuN标记的神经元,而GFAP阳性的星形胶质细胞以及OX42阳性的小胶质细胞几乎不表达HMGB1;(3)Western Blot结果显示,脊髓背角HMGB1在SNL模型术后缓慢增高,7 d时增高最为显著,且持续至少28 d。结论:以上结果表明,外周神经损伤后脊髓水平HMGB1的表达上调可能在神经病理性痛的产生和维持中起着重要作用。     

Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury

The activation of glial cells in the CNS has been suggested to be involved in abnormal pain sensation after peripheral nerve injury. Previous studies demonstrated phosphorylation of p38 mitogen-activated protein kinase (MAPK) in spinal cord glial cells after peripheral nerve injury, and such phosphorylation has been suggested to be involved in the development of neuropathic pain. The aim of this study was to examine the dorsal column nuclei for phosphorylation of p38 MAPK following peripheral nerve injury and to explore a possibility of its contribution to neuropathic pain. Immunohistochemical labeling for phosphorylated p38 (p-p38) MAPK was performed in histological sections of the rat spinal cord and medulla oblongata after the fifth lumbar (L5) spinal nerve ligation (SNL). The number of p-p38 MAPK-immunoreactive (IR) cells was significantly increased in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury at days 3-21 after SNL. Double immunofluorescence labeling with cell-specific markers revealed that p-p38 MAPK-IR cells co-expressed OX-42, suggesting their microglial identity. Increased immunofluorescence labeling for OX-42 indicated that microglial cells were activated by SNL in the L5 dorsal horn and the gracile nucleus ipsilateral to the injury. Continuous infusion of a p38 MAPK inhibitor into the cisterna magna for 14 days beginning on the day of SNL suppressed the development of tactile allodynia, but not thermal hyperalgesia induced by nerve injury. These results demonstrate that SNL activates p38 MAPK pathway in microglia in the gracile nucleus as well as in the spinal cord dorsal horn. Activation of p38 MAPK in medullary microglia may contribute to the pathogenesis of neuropathic pain.     

脊髓小胶质细胞SFKs介导rrTNF诱导的机械痛敏

目的观察Src-家族激酶(SFKs)的激活在外源性TNFα引起的机械痛敏中的作用。方法大鼠坐骨神经周围施加重组大鼠TNFα(rrTNF),免疫组化观察给药后1、7d脊髓背角SFKs表达的变化,同时在给予rrTNF前鞘内注射SFKs抑制剂PP2,行为学测试检测大鼠50%机械刺激撤足阈值的变化。结果坐骨神经周围施加rrTNF可引起SFKs在双侧L5脊髓背角的表达显著上调,且SFKs的表达仅存在于小胶质细胞。提前鞘内注射SFKs抑制剂可防止rrTNF引起大鼠机械痛敏。结论脊髓背角小胶质细胞SFKs信号途径的激活在rrTNF引起的病理性疼痛的产生中起作用。     

氯胺酮鞘内给药对神经病理性痛大鼠脊髓背角小胶质细胞活化、TNF-α和IL-1β产生的影响

目的:探究鞘内注射氯胺酮(ketamine,KTM)对坐骨神经结扎(CCI)神经病理性疼痛大鼠模型行为、脊髓背角肿瘤坏死因子-α(TNF-α)和白介素1β(IL-1β)以及小胶质细胞标记蛋白OX42表达的影响。方法:(1)建立CCI模型大鼠,鞘内注射KTM或MI(米诺四环素)进行干预,测定大鼠机械缩足阈值(MWT)和热缩足潜伏期(TWL)。(2)运用ELISA法检测TNF-α和IL-1β表达水平。(3)使用Western Blot法检测OX42的表达情况。结果:(1)KTM组和MI组大鼠MWT和TWL值较CCI组显著升高;KTM组大鼠MWT和TWL值高于CCI组(P0.05)。(2)KTM组和MI组TNF-α和IL-1β表达水平低于CCI组(P0.05);其中KTM组TNF-α和IL-1β表达水平显著高于MI组(P0.05)。(3)KTM组和MI组OX42表达水平显著低于CCI组;KTM组OX42表达水平高于MI组(P0.05)。结论:鞘内KTM可以抑制脊髓背角小胶质细胞激活,减少TNF-α和IL-1β表达,显著改善坐骨神经结扎引起的神经病理性疼痛。     

脊髓背角MCP-1-JAK2/STAT3信号转导参与大鼠2型糖尿病神经病理性痛的机制研究

目的:探讨脊髓背角MCP-1-JAK2/STAT3信号转导是否参与大鼠2型糖尿病神经病理性疼痛(DNP)的形成与发展。方法:雄性SD大鼠高糖高脂饲养8周,通过腹腔单次注射链脲佐菌素(STZ)制备大鼠2型DNP模型。将2型DNP大鼠随机分为4组,每组16只:DNP组、DNP+MCP-1抑制剂组(DM组)、DNP+JAK2抑制剂AG490组(DA组)和溶剂对照组(SC组)。DA、DM和SC组分别于注射STZ 14 d后蛛网膜下腔置管,3 d后DM、DA和SC组分别给予MCP-1中和抗体10μL(0.1 mg/L)、AG490 10μL(1 mmol/L)和3.5%DMSO 10μL,每天1次,连续14 d。DNP组不做任何处理。另取16只大鼠为对照(control,C)组,普通饲料喂养。蛛网膜下腔给药后第1、3、7和14天时测定机械缩足阈值(MWT)和热缩足反射潜伏期(TWL),各组随机取4只大鼠处死,取L4-6脊髓膨大,采用Western blot法检测p-JAK2和p-STAT3的表达。结果:与C组比较,DNP和SC组第1、3、7和14天时MWT降低,TWL缩短,脊髓背角p-JAK2和p-STAT3表达上调(P0.05);与DNP组比较,DM和DA组第1、3、7和14天时MWT升高和TWL延长,脊髓背角p-JAK2和p-STAT3表达下调(P0.05);DNP组与SC组各指标比较差异无统计学意义。结论:脊髓背角MCP-1-JAK2/STAT3信号转导参与大鼠2型DNP的产生和维持。     

Differential adaptive changes on serotonin and noradrenaline transporters in a rat model of peripheral neuropathic pain

Serotonin and noradrenaline reuptake inhibitors have shown to produce antinociceptive effects in several animal models of neuropathic pain. In the present work, we have analyzed the density of brain and spinal serotonin and noradrenaline transporters (5-HTT and NAT) in a rat model of neuropathic pain, the spinal nerve ligation (SNL). Quantitative autoradiography revealed a significant decrease in the density of 5-HTT ([(3)H]citalopram binding) at the level of the lumbar spinal cord following 2 weeks of neuropathic surgery (lamina V, -40%: 6.01±0.64 nCi/mg tissue in sham-animals vs 3.59±1.56 in SNL-animals; lamina X, -30%: 9.10±2.00 vs 6.40±1.93 and lamina IX, -22%: 12.01±2.41 vs 9.42±1.58). By contrast, NAT density ([(3)H]nisoxetine binding) was significantly increased (lamina I-II, +34%: 2.20±0.45 vs 2.96±0.65; lamina V, +57%: 1.34±0.28 vs 2.11±0.66; and lamina IX, +58%: 2.39±0.71 vs 3.78±1.10). At supraspinal structures, SNL induced adaptive changes only in the density of 5-HTT (septal nuclei, +33%: 10.18±2.03 vs 13.53±1.14; CA3 field of hippocampus, +18%: 6.94±1.01 vs 8.21±0.81; paraventricular thalamic nucleus, +21%: 15.18±1.88 vs 18.35±2.08; lateral hypothalamic area, +40%: 12.68±1.90 vs 17.8±2.55; ventromedial hypothalamic nuclei, +19%: 7.16±0.92 vs 8.55±0.40; and dorsal raphe nucleus, +15%: 35.22±3.88 vs 40.68±3.11). Thus, we demonstrate, in the SNL model of neuropathic pain, the existence of opposite changes in the spinal expression of 5-HTT (down-regulation) and NAT (up-regulation), and the presence of supraspinal adaptive changes (up-regulation) only on 5-HTT density. These findings may help understanding the pathogeny of neuropathic pain and the differential analgesic action of antidepressants targeting 5-HTT and/or NAT transporters.     

Anatomical localization and expression pattern for the NMDA-2D receptor subunit in a rat model of neuropathic pain

The N-methyl-d-aspartate receptor (NMDAR) has been implicated in the etiology of chronic pain. In this regard, this study sought to characterize the localization and expression pattern for the NMDAR-2D subunit in a rat model of neuropathic pain. To this end, one group of rats, 3 weeks post-dorsal root rhizotomy (DRR) and a second group, 3 weeks post-spinal nerve ligation (SNL) and sham surgery, were generated. Dorsal root ganglia (DRG) and/or lumbar spinal cord were excised from DRR, naïve, SNL and sham rats. Both immunohistochemical and real-time PCR analysis confirmed discrete NMDAR-2D subunit expression within the DRG and dorsal horn. However, no overt differences in staining intensity or expression were noted between DRG and spinal cord sections obtained from the different surgical groups. Results also demonstrated that the NMDAR-2D subunit was present within Neu N+ cells in the spinal cord and DRG, but excluded from cells labeled with the astrocytic marker, GFAP, and the microglial maker, OX-42. Lastly, the NMDAR-2D subunit was not co-expressed within neurokinin-1 (NK-1)+ or neurofilament-52 (N-52)+ neurons, but the antibody did co-label a number of isolectin B4+ (IB4) DRG cells. Together, these findings seem to suggest that the NMDAR-2D receptor subunit is present within the cell body region of a population of small diameter sensory afferents and post-synaptically within second order dorsal horn neurons. Although these data suggest that the NMDAR-2D subunit is well poised anatomically to modulate pain neurotransmission, the expression pattern for this subunit is not altered in rats demonstrating the presence of neuropathic-like pain behavior.     

Spinal cord stimulation induces c-Fos expression in the dorsal horn in rats with neuropathic pain after partial sciatic nerve injury

Spinal cord stimulation (SCS) is an established treatment for intractable neuropathic pain, especially CRPS-1. The mechanisms of action of SCS have only been partly elucidated and include suppression of the hyper-excitability of the Wide Dynamic Range neurons and a GABA increase in the dorsal horn. In the present study we demonstrate an increase of c-Fos immunoreactive cells in the dorsal horn after SCS, suggesting early cellular activation that may preclude earlier described electrophysiological and biochemical changes in the dorsal horn after SCS. In a rat model of neuropathic pain, allodynia was induced and quantified using the von Frey test. In 11 rats a SCS device was implanted and spinal cord stimulation performed. Withdrawal threshold were measured every 15 min up to 90 min. A sham group (n = 6) also had a SCS device implanted, but did not receive SCS. After SCS the animals were perfused and histology was performed for quantification of c-Fos immunoreactivity in the dorsal horns. We found a significant increase in c-Fos in the SCS group compared to our sham group and control tissue, indicating late cellular activity in the dorsal horn after SCS.