机械性脑白质损伤后皮层神经元凋亡的证据及其分布特征

目的　探讨细胞凋亡在机械性脑白质损伤后皮层神经细胞病理变化中的作用及其分布特征。方法　雄性健康SD大鼠 3 6只随机分为假手术组 (1 2只 )和模型组 (2 4只 ) ,用模型组动物制作大脑皮层下横切模型。于致伤后不同时间处死动物 ,用TUNEL原位末端标记和常规病理组织学方法观察损伤区及相应皮层神经细胞变化的性质和规律。结果　致伤后横切处出血 ,损伤组织内神经元出现坏死改变 ,胶质细胞增多。大脑皮层Ⅱ Ⅴ层神经细胞多出现萎缩改变。伤后 1 2d时 ,相应皮层内即出现散在早期凋亡神经元 ,随后逐渐增多 ,7d后又趋减少 ,伤后 1 9d皮层内仍可检出凋亡神经元。在损伤区凋亡神经元少见 ,可见部分胶质细胞凋亡。结论　脑白质损害时 ,损伤相应部位皮层神经元主要表现为凋亡。在损伤区 ,神经元和胶质细胞主要表现为坏死。皮层内凋亡神经元主要分布于第Ⅱ Ⅴ层 ,其时间分布呈一单峰状曲线 ,凋亡峰出现在伤后第 7d前后 ,至少持续 1 9d。     

神经细胞凋亡在脊髓缺血再灌注损伤延迟性瘫痪中的作用

目的研究细胞凋亡在脊髓缺血再灌注损伤发生延迟性瘫痪中的作用。方法将48只新西兰白兔随机分为2组:对照组(sham)和缺血再灌注组(IR)。参照并改进Zivin方法建立兔脊髓腰骶段缺血再灌注延迟性瘫痪模型,比较各组动物不同时间点后肢运动功能及病理形态学变化;采用原位末端脱氧核糖核酸酶转移介导的脱氧尿三磷酸(dUTP)标记法(TUNEL法)检测神经细胞凋亡水平。结果HE染色显示,再灌注8 h组神经细胞形态基本正常,结构清楚,灰质中有少量空泡,但神经元细胞结构完好;再灌注24 h组:灰质中前角神经元细胞破坏严重,空泡形成,无明显炎症细胞浸润;再灌注72 h组:灰质前角中大量空泡形成,尚残存数个结构清楚的运动神经元,有明显炎症细胞浸润;再灌注168 h组:灰质中运动神经元消失,残存数个固缩坏死神经元。TUNEL法染色显示,sham组及再灌注8 h后,仅见非特异性染色。再灌注24 h后出现大量阳性细胞,至再灌注72 h阳性细胞数量达到最高峰,主要分布在前角运动神经元。再灌注1周后,灰质结构破坏严重,仅有少量神经元幸存,但其阳性细胞平均积分吸光度值仍较对照组高。结论脊髓缺血再灌注后发生延迟性瘫痪时,神经元死亡的方式主要是细胞凋亡。     

Apoptosis of motor neurons in the spinal cord after ischemia reperfusion injury delayed paraplegia in rabbits

Objective To clarify the pathologic change of the motor neuron on spinal cord ischemia reperfusion injury delayed paraplegia. Methods The infrarenal aorta of White New Zealand rabbits （n=24） was occluded for 26 minutes using two bulldog clamps. Rabbits were killed after 8, 24, 72, or 168 hours （n=6 per group）, respectively. The clamps was placed but never clamped in sham-operated rabbits （n=24）. The lumbar segment of the spinal cord （L5 to L7） was used for morphological studies, including hematoxylin and eosin staining, the expression of bcl-2 and bax proteins in spinal cord was detected with immunohistochemistry. The apoptotic neurons in spinal cord were measured with terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end-labeling of DNA fragments （TUNEL） staining. Results Delayed paraplegia occurred in all rabbits of ischemia reperfusion group at 16-24 hours, but not in sham groups. Motor neurons were selectively lost at 7 days after transient ischemia. After ischemia, the positive expression of bcl-2 protein were in the sham controls but decreased significantly as compared with that of the IR group （P〈0.01）, especially in 72 hours reperfusion. The positive expression of bax protein were also in the sham controls, but increased in the IR group, especially in 72 hours reperfusion; In addition, TUNEL study demonstrated that no cells were positively labeled until 24 hours after ischemia, but nuclei of some motor neurons were positively labeled at peak after ischemia reperfusion at 72 hours. Oenclusion Spinal cord ischemia in rabbits induces morphological and biochemical changes suggestive of apoptosis. These data raise the possibility that apoptosis contributes to neuronal cell death after spinal cord ischemia reperfnsion.     

Apoptosis of lumbar spinal cord neurons in cauda equina syndrome rats

Objective To explore the law of apoptosis of lumbar spinal cord neurons in cauda equina syndrome (CES). Methods Cauda equina of rats was compressed by a piece of silica gel stick. From day 1 to day 28, the lumbar spinal cord specimens were harvested and assessed by Nissl's staining and TUNEL staining. Results Compression of cauda equina caused lesion and apoptosis of neurons in lumbar spinal cord, and the extent of apoptosis reached the peak on 7th day after compression. Conclusion Apoptosis of neurons in lumbar spinal cord might be one of the reasons why patients with CES get poor prognosis.     

Effects of erigeron breviscapus （Vant.） Hand-Mazz pretreatment on pathology and oxyradical level following spinal cord ischemia-reperfusion injury in rabbits

Objective To investigate the effects of erigeron breviscapus (Vant.) Hand-Mazz (erigeron breviscapus) pretreatment on pathology and oxyradical level in the spinal cord after ischemia-reperfusion (I/R) injury in rabbits. Methods A total of 40 New Zealand white rabbits were randomly divided into three groups: sham-operation group with 10 rabbits treated with only abdominal aorta exposure without occlusion, control group with 15 rabbits that underwent ischemia for 50 minutes and treated with matched saline, and experimental group with 15 rabbits that underwent ischemia for 50 minutes and treated with erigeron breviscapus (9mg/kg) injection before ischemia. Malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in the spinal cord were examined at 6 and 24 hours after I/R, respectively. The morphological changes and the number of the spinal cord anterior horn motor neurons were observed and counted under the light microscope and electron microscope, respectively. Results The level of MDA was markedly decreased and SOD activity was increased in the experimental group compared with those in the control group (P<0.01). Compared with that in the control group, the number of motor neurons in the experimental group significantly increased at 24h after I/R (P<0.01) and the morphous of the motor neurons improved. Conclusion Erigeron breviscapus can reduce oxyradical production and the apoptosis of nerve cells, and protect nerve tissue structure and function after spinal cord I/R.     

大鼠急性脊髓损伤中caspase-1的表达及意义

目的研究半胱氨酸天冬氨酸特异性蛋白酶-1(cysteinyl aspartate specific protease-1,caspase-1)在大鼠急性脊髓损伤后的表达及意义。方法取健康成熟SD大鼠36只,随机分为对照组和损伤组,各18只,每组再分8 h、3 d7、d共3个时间点,每个时间点6只。对照组仅做单纯T8、T9椎板切除术,损伤组则按Nystrom法建立大鼠急性脊髓损伤动物模型。HE染色观察脊髓组织病理学变化,免疫组化测定各时间点caspase-1的表达变化。结果对照组大鼠脊髓组织中少量细胞caspase-1表达阳性。脊髓损伤后8 h时,损伤组caspase-1表达高于对照组,3 d时表达最高,7 d时略有降低,但仍高于对照组。脊髓损伤组各时间点caspase-1表达阳性细胞数较对照组明显升高(P<0.01)。结论大鼠脊髓损伤后caspase-1的表达迅速增强,可能是导致脊髓继发性损伤的因素之一。     

全脑缺血再灌注损伤后JUN蛋白在大鼠海马的表达及热休克预处理对其表达的影响

目的研究全脑缺血再灌注损伤后JUN蛋白在大鼠海马的表达及热休克预处理对其表达的影响。方法以四血管法建立全脑缺血再灌注模型。将大鼠置于42℃中15 min行热休克预处理,全脑缺血6 min后行2 h、6 h、12 h、24 h3、d、5 d再灌注后处死,取海马脑组织行HE染色、JUN蛋白免疫组化染色,TUNEL法检测凋亡细胞。结果缺血再灌注后2 h JUN蛋白在CA1区开始表达,6 h达到高峰,5 d时仍有表达;CA3区JUN蛋白的表达弱于CA1区(P<0.05);同时CA1区细胞损伤明显。热休克预处理组JUN蛋白表达在CA1和CA3区相应时点减弱(P<0.05),细胞损伤轻微,凋亡细胞减少(P<0.05)。结论全脑缺血再灌注损伤后JUN蛋白过度表达参与了神经元损伤过程,热休克预处理通过下调JUN蛋白过度表达具有脑保护作用。     

The development of an OxyHb animal model in mice and the study on OxyHb-induced apoptosis of mouse brain cells in vivo

Objective On the basis of developing a new animal model for oxyhemoglobin (OxyHb) injection into subarachnoid space in mice, this research was to explore the temporal dependence and spatial distribution of OxyHb- induced apoptosis in the mouse brain cells in vivo and the mechanism of neurocyte injury induced by OxyHb. Methods The animal model for OxyHb injection into subarachnoid space in mice was developed. Mice were divided randomly into the experimental group (n=40) and the control group (n= 35). The control group received saline injection (50 μL ) and the experimental group received OxyHb injection (50 μL ), both into the subarachnoid space. The mice of the two groups were subdivided according to different postoperative time (3 h, 6 h, 12 h, 24 h and 48 h). The apoptosis or necrosis of cells was distinguished with microscopy (HE staining), transmission electron microscopy and TUNEL method. Results The distribution of apoptosis was mainly in the ipsilateral neocortex and bilateral hippocampal gyrus. The apoptotic mouse brain cells showed morphological changes in the experimental group by HE staining and transmission electron microscopy. The count of TUNEL-positive cells showed substantial increase in the experimental group, and there was a significant difference between the control and experimental groups, and the number of OxyHb- induced apoptotic cells decreased with time. Conclusion OxyHb in subarachnoid space in mice can induce apoptasis, but not necrosis of mouse brain cells in viro. The apoptotic brain cells show the pattern of temporal dependence and spatial distribution. It is suggested that the early treatment should be the method of first choice for treating the hemorrhagic brain injury.     

EFFECTS OF NERVE GROWTH FACTOR ON ENDOTHELIN AFTER SPINAL CORD INJURY IN RATS

Ｗｉｔｈｔｈｅｄｅｖｅｌｏｐｍｅｎｔｏｆｍｅｄｉｃｉｎｅ ,ｓｐｉｎａｌｃｏｒｄｉｎｊｕｒｙ (ＳＣＩ)ｈａｖｅｂｅｅｎｔｈｅｍｏｓｔｄａｎｇｅｒｏｕｓｆａｃｔｏｒｆｏｒｔｈｅｈｅａｌｔｈ .ＢｕｔｎｅｒｖｅｇｒｏｗｔｈｆａｃｔｏｒｍａｙｂｅｔｈｅｈｏｐｅｏｆｔｒｅａｔｍｅｎｔｆｏｒＳＣＩ .Ｗｅｏｂｓｅｒｖｅｄｔｈｅｃｈａｎｇｅｓｏｆｅｎ ｄｏｔｈｅｌｉｎ(ＥＴ)ｓｅｒｕｍｌｅｖｅｌａｆｔｅｒｔｈｅｉｎｊｕｒｙｏｆｓｐｉｎａｌｃｏｒｄ ,ｔｈｅｒｅｓｕｌｔｓｗｅｒｅｒｅｐｏｒｔｅｄｈｅｒｅｔｏｉｎｖｅｓｔｉ…     

THE EXPRESSION OF BCL-2, BAX AND CASPASE-3 IN NEURON OF THE SPINAL CORD ANTERIOR HORN AFTER CAUDA EQUINA ACUTE COMPRESSION

Objective To explore the influence of the acute cauda equina compression on the iumbosacral spinal cord; To clarify the pathologic change of the motor neuron after acute cauda equina compression. Methods 27 canis familiaris were randomly divided into 9 groups （3 in each） ： one for normal group, one for control group, and seven for compression groups. The control group and compressed groups was given operation and the sac made of silica gel was placed under the lamina of L5-6. Water was injected into the sac until their posterior legs paralysis in compressed groups, the animals had been compressed for 4, 8, 12, 24, 48, 72, 168 hours. The control group were not injected water. Cells apoptosis was investigated with the technology of TdT-mediated biotin dUTP nick end-labeling （TUNEL） staining. The Bcl-2 Bax and Caspase-3 protein was investigated by immunohistochemical method. Results TUNEL staining cells in anterior horn presented after compressed 8-12 hours, and at 72 hours the number of positive cells got to maximum, it decreased subsequently after 168 hours. The protein of Bax, Bcl-2 expressed a little in normal motor neuron. The caspase-3 protein didn＇t express in normal ceil. They all reached the peak at 72 hours after compression. Conclusion The apoptosis of motor neuron occurred earlier after eauda equina acute compression. Bax protein restrained Bcl-2 protein then active caspase-3 and conduced apoptosis of motor neuron.     

急性脊髓损伤中内皮素与钙离子变化的关系

目的　通过观察急性脊髓损伤 (Acutespinalinjury ,ASI)后脊髓组织的细胞内钙含量和血浆内皮素 (Endothelin ,ET)含量的变化 ,探讨ET与Ca2 +在ASI发病机制的关系。方法　选用Wistar雄性大鼠 2 8只 ,应用改良Allen s方法 ,制成脊髓损伤模型 ,造成T1 2 脊椎节段脊髓 5g×1 0cm中度损伤的ASI后 ,分别应用原子吸收光谱分析法和放射免疫法测定ASI后 1、4、8、2 4、72和 1 68h脊髓组织中钙离子含量和血浆中ET浓度。结果　损伤节段组织细胞内钙离子含量及血浆中ET浓度均随病程而改变 ,于 8～ 72h达高峰 ,与正常值有显著性差异。结论　ASI后脊髓组织中细胞内Ca2 +含量及血浆中ET浓度迅速升高 ,提示ET受体阻滞剂与钙离子拮抗剂联合使用为治疗ASI提供了新的途径。     

C57/BL6小鼠海马结构发生、发育和衰老过程中的细胞凋亡

目的探讨C57/BL6小鼠海马结构发生发育及衰老过程中细胞凋亡的变化规律。方法采用Hoechst 33258染色检测不同阶段C57/BL6小鼠海马结构中的细胞凋亡;免疫荧光和Western blotting技术检测不同阶段C57/BL6小鼠海马结构中Caspase-3的表达。结果 Hoechst 33258染色结果显示:胚龄(embryonic day,E)12d～18d凋亡细胞渐增多;生后(postnatal day,P)1～7d渐减少;P14d～2月主要于齿状回、海马沟和海马槽的白质区可见少量凋亡细胞;3月～18月仅在齿状回亚颗粒细胞层见零星分布。阿蒙角(Ammons horn,CA)及齿状回(dentate gyrus,DG)多形层及分子层细胞凋亡率P1～P14d渐下降。CA及DG主细胞层细胞凋亡率P1d最高,以后逐渐降低。Caspase-3免疫荧光结果示:E12～E16d阳性表达细胞渐增多;E18～P3d继续增多,主要分布于CA锥体层及DG颗粒层;P5～P14d分布渐减少;P21d～18月各区仅见少量阳性细胞表达。Western Blotting结果示E18～P3dCaspase-3表达增加;P5～P21d表达降低;P21d后趋于稳定。结论生后1d为小鼠海马结构凋亡高峰;Caspase-3为重要凋亡因子。     

亚胺环己酮与甲基强的松龙治疗大鼠脊髓损伤的初步研究

目的　为脊髓损伤的临床药物治疗提供新的理论依据。方法　将 48只大鼠用 Allen法制成脊髓损伤模型 ,分别予以亚胺环己酮、甲基强的松龙、二药联合应用及不予治疗。观察动物不同时期神经功能评分 ,斜板试验及体表诱发电位。结果　从伤后 3周起 ,治疗组各种指标与对照组有显著差异 ,而各治疗组间无差异。结论　早期应用亚胺环己酮与甲基强的松龙治疗脊髓损伤均有效 ,但二者合用不能提高疗效     

EXPRESSION OF NESTIN AND GLIAL FIBRILLARY ACIDIC PROTEIN IN DIFFERENT PERIOD AFTER SPINAL INJURY IN ADULT RATS

Objective To study the expression of Nestin and glial fibrillary acidic protein (GFAP) in different period after spinal injury in adult rats. Methods Animal moels were created by artery clamp. Expression of Nestin and GFAP in different period ( 1,3,5days; 1-8 weeks) and different area( injury locus and its surrounding tissue ) after spinal injury were observed pathologicaly using immunofluorescence and LeicaQ5001W imaging processing system.Results There was expression of Nestin and GFAP in injured area 1 day after injury. The expression increased not only in in injured area but its sourrounding 3--7 days later and gradually got to peak value. As the time went on, expression of Nestin and GFAP dereased. Conclusion Spinal injury can induce the expression of Nestin. Nerve stem cell has response to spinal injury. There is positive correlation between expression of Nestin and hyperplasia of reactivity astrocyte. Nerve stem cell maybe invovled in the repair of central nervous system (CNS).     

脑弥漫性轴索损伤实验装置的研制及动物模型的建立

目的 自制一种脑弥漫性轴索损伤(DAI)装置并成功建立DAI动物模型.方法 自制头颅瞬间旋转损伤装置,使大鼠头颅在瞬间(<3ms)旋转90°造成剪切损伤.观察损伤后大鼠的生命体征及行为学改变;于伤后2、6、12、24、36、72h及10d分别处死损伤组动物,制备脑石蜡切片,行镀银及HE染色,光镜下观察神经轴索变化.结果 伤后大鼠均即刻出现原发昏迷,其中2只于损伤后20min内死亡,余持续时间1-30min不等;伤后大鼠呼吸节律紊乱,瞳孔对光反射减弱或消失,醒后均有程度不等的反应性下降,肢体活动迟缓;肉眼可见广泛蛛网膜下腔出血或脑室出血;光镜下可见皮髓交界区、胼胝体区、脑干、小脑白质等部位的神经轴索有不同程度的肿胀、断裂、轴索球形成,后期有小胶质细胞增生,局部呈巢样聚集.结论 本装置能造成大鼠脑DAI,且具有简便、可控、确切的特点,适合进行中、小型动物DAI模型的实验研究.     

细胞外ATP对大鼠脊髓损伤后运动功能恢复的影响

目的研究细胞外ATP对大鼠脊髓损伤后运动功能恢复的影响。方法健康成年Wistar大鼠20只,雌雄不限,体重280-320 g,平均300 g,制作成脊髓打击伤动物模型,并随机分为两组:A组(ATP组)和B组(对照组),每组10只。伤后1、3、7、14、28 d用改良的Tarlov评分、斜板试验观察大鼠运动功能的恢复情况。结果大鼠脊髓损伤后A组改良的Tarlov评分、斜板试验优于对照组,在14 d和28 d,改良的Tarlov评分、斜板试验A组明显优于B组(P<0.05)。结论细胞外ATP能促进大鼠脊髓损伤后运动功能的恢复。     

银杏叶提取物对大鼠脊髓损伤下肢运动功能的影响

目的　观察银杏叶提取物(Egb)治疗大鼠脊髓损伤,评估它对大鼠脊髓损伤下肢运动功能恢复的效果。方法　60只大鼠分成3组,用Allen s改良法建立脊髓损伤的动物模型,通过Egb腹腔注射,用斜板试验法和Bosso, Beattieand Bresnahan(BBB)评分法观察大鼠脊髓损伤下肢运动功能的变化。结果　斜板试验法临界角度药物组和损伤组比较:7 d,P<0.05;14 d,21 d,P<0.01。BBB评分法药物组和损伤组比较:7 d,P<0.05;14 d,21 d, P<0.01。药物组大鼠脊髓损伤后下肢运动功能恢复快,有显著性差异。结论　Egb对大鼠脊髓损伤后双下肢运动功能的恢复有促进作用。     

控制通气对大鼠膈肌组织形态学及肌球蛋白重链的影响

目的探讨控制通气(CMV)对大鼠膈肌组织形态学的影响,以阐明机械通气膈肌功能异常的机制。方法观察18h和24hCMV后大鼠膈肌超微结构及HE染色的结构改变,并检测膈肌肌球蛋白重链(MHC)表型和肌纤维横截面积(CSA)。结果 24hCMV组膈肌纤维损伤密度远高于对照组(P<0.01),肌纤维形状不规则、肌纤维萎缩及结构松散,18hCMV组仅轻度改变;24hCMV组膈肌MHCfast比率升高,MHCslow比率有下降趋势但无统计学差异;MHCfast和MHCslow肌纤维CSA及MHCfast和MHCslow蛋白表达均降低(P均<0.05);18hCMV组两种肌纤维的阳性比率、肌纤维CSA及MHCfast和MHCslow的蛋白表达均无改变。24hCMV组肌纤维的损伤密度与MHCfast阳性比率呈正相关(P<0.05)。结论短期CMV可造成膈肌损伤、肌纤维萎缩,以及慢肌向快肌的移行改变。CMV所造成的肌肉损伤与适应性改变可能相关联。     

31℃、34℃亚低温对缺血大鼠脑细胞凋亡影响的研究

目的　研究不同程度亚低温对缺氧缺血性脑损害 (HIBD)新生大鼠的脑保护作用。方法　用HE染色、电镜、原位末端标记 (Tunel)技术观察不同程度亚低温干预HIBD新生大鼠后不同脑区脑组织凋亡细胞数。结果　HIBD常温组HIBD后 2 4h为凋亡高峰 ,31℃亚低温组HIBD后 2 4h凋亡细胞数目减少 ,比 34℃组作用显著 ,31℃组凋亡高峰延迟出现 ,34℃组无明显的凋亡高峰。结论　HIBD后亚低温干预可明显抑制细胞凋亡 ,保护脑组织 ,温度越低 ,保护作用越明显     

EXPERIMENTAL STUDY ON SPINAL CORD INJURY TREATED WITH THE COMBINATION OF FETAL SPINAL CORD TRANSPLANTATION AND METHYLPREDNISOLONE

ＴｈｅｔｈｅｒａｐｙｏｆＬａｒｇｅＤｏｓａｇｅｏｆＭｅｔｈｙｌｐｒｅｄｉｎ ｓｏｌｏｎｅ(ＭＰ)ｆｏｒＳＣＩｈａｓｄｅｖｅｌｏｐｅｄｃｌｉｎｉｃａｌｌｙｂｅｃａｕｓｅｉｔｃａｎｇｒｅａｔｌｙｉｍｐｏｖｅｔｈｅｎｅｒｖｅｆｕｎｃｔｉｏｎ .Ｉｎｔｈｅｍｅａｎ ｔｉｍｅ,ａｎｉｍａｌｔｅｓｔｓｈａｖｅａｌｒｅａｄｙｆｏｕｎｄｔｈａｔｉｆｆｅｔａｌｓｐｉｎａｌｃｏｒｄｉｓｔｒａｎｓｐｌａｎｔｅｄｔｏｔｈｅｉｎｊｕｒｅｄｓｐｉｎａｌｃｏｒｄａｒｅａ ,ｉｔｃａｎｓｔｉｍｕｌａｔｅｔｈｅａｘｏｎｓｏｆｔｈｅｈｏｓ…