【作者】 赵燕邦；

【导师】 屠冠军；

【作者基本信息】 中国医科大学 ， 外科学， 2010， 博士

【摘要】 脊髓损伤(Spinal cord injury,SCI)后的脊髓水肿是脊髓继发损伤的关键病理生理过程之一,对预后影响较大。近年发现作为“神经甾体”的黄体酮(progesterone, PG)对中枢神经系统具有保护作用,在脑外伤及中风动物模型中能显著减轻脑水肿,提高神经功能恢复程度。人们对其治疗机理产生兴趣。目前,已普遍接受AQP4对中枢神经水肿起到主要的调节作用。AQP4主要在中枢神经系统星型胶质细胞表达。国内外对黄体酮减轻脊髓水肿机理的研究未见报道。目的1.观察大鼠急性脊髓损伤后及给与黄体酮后脊髓组织含水量的变化,验证黄体酮能否减轻脊髓水肿。2.观察大鼠急性脊髓损伤后AQP4和GFAP分布及表达量的变化。3.进一步观察给予黄体酮后AQP4和GFAP表达的变化,探讨黄体酮能否对AQP4和GFAP的表达产生影响。方法造模和取材：成年雄性Sprague-Dawley(SD)大鼠144只,体重250-300g。随机分为三组。分别为：1、假手术+药用大豆油(溶媒剂)组(Sham+vehicle, SV组)；2、脊髓损伤+药用大豆油组(Lesion+vehicle, LV组)；3、脊髓损伤+黄体酮组(Lesion+progesterone, LP组)。每组内按时间点不同又分为0h,6h,12h,24h,48h和72h六个亚组,每组9只。每个亚组选3只大鼠进行灌注,固定。采用钳夹法制作脊髓损伤模型。以T6棘突为中心,取后正中纵切口,长约3cm,暴露T5-T7的棘突,咬除T6棘突及全椎板,暴露脊髓硬膜约0.5cm长。调节自制脊髓夹的限宽为1mm。将脊髓夹的两个夹片分别插入脊髓硬膜与两侧椎弓根之间的间隙内。迅速夹两夹片至限宽,夹持时间约1分钟。观察到大鼠尾及双下肢出现痉挛、摆动时,证明大鼠脊髓损伤,迅速撤去脊髓夹。假手术组仅暴露硬膜。LP组大鼠于术后0.5h腹腔内注射黄体酮,24h、48h皮下注射黄体酮,16mg／kg。SV组,LV组大鼠于术后0.5h腹腔内注射药用大豆油,24h、48h皮下注射药用大豆油(溶媒剂),16mg／kg。利刀切取T4-5节段脊髓组织,测干湿重。利刀切取T7-8节段脊髓组织,仔细剥去硬膜,放入EP管内,迅速放入-70℃冷冻冰箱保存。4%多聚甲醛左心室灌注。利刀切取T4-8节段脊髓组织将取出的大鼠脊髓放到事先准备好的4%多聚甲醛中固定,过夜,然后移至30%蔗糖中,4℃冰箱中进行充分脱水(3—7天),沉底。检测方法1：测干湿重比。2.HE染色观察光镜下脊髓损伤区水肿带的变化。3.免疫荧光双标法观察AQP4和GFAP表达分布的变化。4.Western blot法检测AQP4和GFAP表达量的变化。5.统计分析。结果1.脊髓组织含水量SV组脊髓含水量最低,LP组较高,LV组最高。各组间比较有统计学差异。2.HE染色光镜下观察损伤组病灶区可分为血肿区、血肿周围区及邻近的正常组织区。出血区可见大量的红细胞,血肿周围有少量肿大的神经细胞,散在的炎性淋巴细胞。血肿内可见到坏死的和变性的神经细胞散在于大量的红细胞中,血肿周围可见水肿带；72h水肿最明显,血肿周围开始出现泡沫细胞,并可见周围组织疏松,形成周围明显的水肿带。3.脊髓免疫荧光标记假手术组及未损伤区的脊髓免疫荧光标记显示GFAP和AQP4在脊髓灰质,白质中均匀一致表达。AQP4在脊髓表面的软脊膜,脊髓实质内构成血脑屏障的小血管周围表达较多。AQP4在星型胶质细胞参与形成血脑屏障的足突上表达较多。与其他文献报道一致。损伤后脊髓的GFAP和AQP4的双荧光共同标记显示二者的表达强度不一致,分布区域不一致。损伤后的GFAP荧光标记(绿光)显示LV组和LP组在短暂减弱后均明显增强,LP组增强的程度比LV组明显。损伤区与水肿区的移行部位增强表达明显。说明星型胶质细胞在损伤周围区明显增多。损伤后的AQP4荧光标记(红光)显示LV组有增强趋势,LP组有轻度的下降趋势,损伤区与水肿区的移行部位未见表达。说明损伤周围区新增多的星型胶质细胞膜上没有AQP4表达。4. Western blot测定脊髓损伤对照组(LV组)AQP4在损伤后12、24、48、72小时与假手术对照组比较均明显表达增强(P<0.01)。脊髓损伤治疗组(LP组)AQP4在损伤后12、24、48、72小时表达与假手术对照组比较有下降趋势(P<0.05)；与脊髓损伤对照组(LV组)比较损伤后6、12、24、48、72小时下降明显(P<0.01)。与假手术组(SM组)比较,脊髓损伤对照组(LV组)和脊髓损伤治疗组(LP组)GFAP在损伤后6、12、24、48、72小时均显著表达下降(P<0.01),12小时下降到最低,24小时后恢复升高。；与脊髓损伤对照组(LV组)比较,脊髓损伤治疗组(LP组)在6、12、24小时表达无明显差异(P>0.05)：在48、72小时GFAP的表达明显增加(P<0.05)。结论1.黄体酮能够降低急性脊髓损伤后脊髓组织的含水量,减轻脊髓水肿。2.在正常脊髓上,AQP4与GFAP的表达分布均匀一致。AQP4在星型胶质细胞上表达,表达部位集中在参与形成血脑屏障的足突。在损伤区脊髓上,AQP4与GFAP的表达分布不一致,损伤区周围没有AQP4表达。3.黄体酮促进脊髓损伤区周围大量反应性星形胶质细胞聚集。4.黄体酮降低了脊髓损伤周围区AQP4的表达水平。更多还原

【Abstract】 The edema followed spinal cord injury(SCI) is a major factor effecting the function recovery. Recently, many studies demonstrate that progesterone(PG) and some of its metabolites can significantly reduce cerebral edema and enhance functional recovery from TBI and stroke in several animal modele. Now,it has been accepted that AQP4,which mainly express at astrocyte in CNS, has a dominating role in regulating the edema in CNS. The mechanisms of progesterone modulating spinal edema have not been detected.Objective1. To observe the changes of spinal cord water content after injury and therapied with PG.2. To observe the change of AQP4 and GFAP in distribute and quantity after spinal cord injury.3. To observe the change of AQP4 and GFAP in distribute and quantity in injuried spinal cord after therapied with PG. Investigate the posibility that PG modulate the epression of AQP4 and GFAP in spinal cord.Materials and MethodsA total of 144 adult male Sprague-Dawley (SD) rats served as subjects, Weight range:250-300g. the rats SCI model was made based on previous documents, with some modification. The cords at T6 was injured by bilateral compression for 1 min using forceps that were 1mm wide with a 1mm spacer. Rapidly remove the forceps,when find the tails and double low limbs began to swing or spasm, which evidence the spinal cord has been injuried.Based on previous studies determining optimal dose response, all progesterone-treated animals received 16mg/kg progesterone in Soybean Oil (vehicle).The animals received the first dose of either Progesterone or an equal volume of Soybeal Oil at 0.5h after the injury,intraperitoneal injection,subsequent injections were given at 24h and 48h after injury,hypodermic injection.The experimental animals were divided randomly into three groups:Sham+ vehicle (SV), Lesion+vehicle (LV), Lesion+progesterone (LP). Every group wase divided into 6 subgroups, according different time points of tissue specimens, i.e. Oh, 6h,12h,24h,48 h,72h.There are 9 animals in each subgroup.3 rats of them, spinal cords were removed from T4-T8 vertebral body levels after perfused with 4% paraformaldehydein 0.1 M phosphate buffer via the heart. postfixed for overnight at 4℃, cryoprotected in 30% sucrose in phosphate buffer 3-7 days, and embedded in OCT compound, prepare for Double immunofluorescence staining.6 rats of them, spinal cords were removed from T4-T5 vertebral body levels, weighed, heated at 80℃for 48 h and re-weighed. Percent water content was calculated as [wet weight-dry weight)/wet weight×100]. Spinal cords were removed from T7-T8 vertebral body levels, freezed rapidly in-70℃, prepare for Western blotting.Result1. Spinal cord water content:the water content in SV group is lowest, LP group is higher(P<0.05), LV group is the highest(P<0.01).2. Results with HE staining:the lesion areas in LV group and LP group contains hematoma zone, peri-hematoma zone and normal tissue zone. There are a lot of eryrocytes in hematoma zone, necrotic and degenerated nerve cell; surrounding the hematoma are a small quantity swelling nerve cell, inflammatory lymphocyte. The edema zone surround the hematoma, whith noticeable at 72h with foam cell emerging. The edema necrosis and inflammatory reaction in LP group are all relieved compared with LV group.3. Double immunofluorescence staining:AQP4 was expressed in gray and white matter, co-localization of GFAP labeled astrocytes (green) with AQP4 labeling (red) in the white matter and grey matter of the normal spinal cord. AQP4 was abundantly expressed around blood vessels, in the glia limitans externa facing CSF in subdural spaces fluid (CSF).,thus supporting the hypothesis that AQP4 mediates water transport between the spinal cord parenchyma and blood or CSF.After SCI, there were some GFAP positive cells in the injured regions of the spinal cord that did not express AQP4. This figure shows apparently in injured groups at 72h. It might be that these GFAP-positive cells, which did not express AQP4 after SCI, are astrocytes that migrated into the lesion site.4. AQP4 and GFAP expression in electrophoresis and Western blotting:Compared with SV groups, the AQP4 expression levels in LV groups are increased significantly at 12h,24h,48h and 72h after the injury (P<0.01); the LP groups are moderately decreased at 12h,24h,48h and 72h (P<0.05). Compared with LV groups, the AQP4 expression levels in LP groups are decreased significantly at 12h, 24h,48h and 72h after the injury (P<0.01).Compared with SV groups, the GFAP expression levels in LV and LP groups are all decreased significantly at 6h,12h,24h,48h and 72h after the injury (P<0.01), lowest at 12h, restore to step-up at 24h. Compared with LV groups, the GFAP expression levels in the LP groups are no obviously diference (P>0.05) at 6h,12h and 24h after injury; but significantly increased at 48h and 72h (P<0.05).Conclusion1. The progesterone can reduce the water content in injuried spinal cord in acute course, extenuate spinal cord edema.2. In normal spinal cords, AQP4 and GFAP are co-localization in normal spinal cords. AQP4 was expressed in astrocytes, concentrate on the foot process involved in the construction of the BBB.In injuried spinal cords, the distribution of AQP4 and GFAP are discrepancy. There are no AQP4 expression in peri-lesion areas.3. Progesterone encourage reactive astrocyte accumulated around the lesion of spinal cord。4. Progesterone reduced the AQP4 expression levels in spinal cord damage zone.更多还原