大鼠弥漫性轴索损伤后小胶质细胞的活化与IL-6的表达

发布时间：2018-03-24 12:45

本文选题：弥漫性轴索损伤　切入点：小胶质细胞　出处：《河北医科大学》2008年硕士论文

【摘要】： 目的:弥漫性轴索损伤(diffuse axonal injury, DAI)是指在头部遭受直线加速性或(和)旋转性暴力打击时,脑组织由于加速运动而产生剪切、伸展和压缩应力,导致脑白质广泛发生以神经轴索损伤为特征的一系列病理生理变化。意识障碍是其典型的表现,临床诊断较困难,预后多差。近年来研究发现,弥漫性轴索损伤造成外伤后的脑功能障碍不仅是由于最初的机械外力对组织的剪切、积压等造成的,更是由于损伤后数小时甚至数天中发生的复杂的“二次打击”造成的。以往的研究主要集中在损伤后神经元的变化,近年有研究发现外伤性脑损伤(traumic brain injury, TBI)后中枢神经系统中的星形胶质细胞和小胶质细胞增生,且随时间的变化而变化。小胶质细胞是中枢神经系统的免疫效应细胞,在中枢神经系统的免疫应答中发挥关键性作用。小胶质细胞也是一种可塑性细胞,不仅能释放神经生长因子,也能释放神经毒因子,其保护作用和损伤神经元的效应依赖于周围环境及活化因子的变化。有实验表明,小胶质细胞是第一个针对损伤发生应答的细胞。在短暂性前脑缺血模型中,发现小胶质细胞的反应早在20分钟内即发生。外伤性脑损伤后有IL-6、IL-10、IL-1、IFN-γ等炎性细胞因子表达增高,且其血浆含量可作为反映伤情严重程度的可靠指标。IL-6在创伤性炎症反应的发生发展过程中起重要的作用,属于致伤因子,具有启动和促进炎症反应的作用,与损伤的严重程度密切相关。然而,小胶质细胞是否在DAI后被激活,是否与促炎因子IL-6一起参与了二次打击的过程,从而导致其自身及神经元的进一步损伤目前尚不清楚。本实验采用Marmarou A等人于1994年首次报道的自由落体撞击方法复制大鼠DAI模型,观察小胶质细胞是否在DAI后被激活,促炎因子IL-6的表达情况,进一步探讨DAI后的神经组织中神经元和小胶质细胞的相互作用,及损伤后炎症反应和细胞损伤的关系,为进一步阐明DAI的病理生理学机制奠定基础。方法:健康雄性Sprague-Dawley(SD)大鼠60只,体重300克左右,随机分为正常对照组、假手术组和打击后不同时间组(0h、1h、3h、6h、12h、24h、48h、72h、5d和7d)。正常对照组大鼠不作处理;假手术组大鼠给予手术及打击前处理,但不予真正的打击;打击组大鼠经过手术后,给予打击,打击后按不同时间处死。观察打击后大鼠的行为学表现,麻醉灌注后取全脑,制作石蜡切片,采用HE染色、银染、焦油紫尼氏小体染色和Weil’s铁苏木素髓鞘染色的方法观察大鼠脑组织的病理改变。将经以上方法验证符合标准的大鼠列入打击组,用免疫组织化学的方法测定脑组织中各个脑区小胶质细胞激活标志物CD11b及IL-6在损伤后不同时间点的表达情况。计量资料数据用均数±标准差(Mean±SD)表示,用SPSS统计分析软件进行统计学分析,各组均数的比较行单因素方差分析(ANOVA),用最小显著差法(LSD)作两两比较,P0.05为有显著性差异。结果:1组织病理学变化: HE染色形态学变化:正常对照组与假手术对照组的大鼠脑组织结构清晰,均匀致密;打击组大鼠大脑皮层、脑干等部位可见局灶性点状出血,大鼠打击后6h开始出现脑组织充血水肿,24h达高峰,48h以后肿胀有所减轻。 Bielschowsky’s镀银染色轴索的形态学变化:正常对照组与假手术对照组大鼠脑组织结构清晰,神经元轴突直且长,表面光滑;打击组大鼠打击后12h可见胼胝体、脑干和小脑等部位神经轴索扭曲、肿胀,呈串珠状、波浪状改变,周围间质水肿,上述表现于24h及48h更加明显,5d明显好转。 Weil’s髓鞘染色髓鞘的形态学变化:正常对照组与假手术对照组大鼠脑组织结构清晰,均匀致密,髓鞘横切面呈环状,纵切面呈鱼刺状;打击组大鼠打击后6h可见大鼠脑干等部位损伤轴索的周围髓鞘间隙明显增宽,髓鞘显著疏松、水肿及崩解出现空白区,上述改变在12h、24h和48h更加明显,5d明显好转。 Cresyl violet染色神经元尼氏小体的形态学变化:正常对照组与假手术对照组大鼠脑组织结构清晰,脑干、皮质和小脑等部位神经元内可见呈蓝紫色,细小颗粒状,均匀致密,环绕在细胞核的周围的尼氏小体;打击组大鼠打击12h后可见脑干等部位损伤轴索的周围神经元淡染出现空白区,尼氏小体明显减少,且呈粗大颗粒状,上述改变在24h和48h显著,5d明显好转。 2 CD11b阳性小胶质细胞在不同时间点的活化:正常对照组与假手术对照组大鼠脑组织CD11b阳性小胶质细胞淡染,呈棕黄色、分枝状,胞体小,具有伸向各个方向的细小突起;打击组大鼠打击后3h可见皮层、小脑、脑干等脑区的小胶质细胞CD11b表达升高,主要分布于损伤轴索及小血管的周围,且与对照组有明显差异(P0.05),其表达量12h时增高达到峰值,活化的小胶质细胞数量明显增加,突起变短、变粗,胞体增大、变圆,CD11b表达于24h后开始下降,72h后明显低于12h组,但与对照组仍然有明显的差异(P0.05)。 3 DAI后IL-6在不同时间点的表达:正常对照组与假手术对照组大鼠的脑组织IL-6有表达,其量很低;打击组大鼠打击后6h可见皮层、小脑、脑干等损伤部位脑区神经细胞有IL-6表达的升高,与对照组有明显差异(P0.05),12h至24h表达量增高达到峰值,48h其表达开始下降,7d时IL-6的表达较24h有非常明显的降低,但较对照组仍然有明显的差异(P0.05),阳性细胞主要是神经胶质细胞,神经元也有一定量IL-6的表达。结论:采用Marmarou A的自由落体撞击方法复制了大鼠弥漫性轴索损伤模型;DAI后脑组织中CD11b阳性小胶质细胞活化,12h达到峰值;DAI后大鼠脑组织中IL-6的表达升高,12h达到峰值,阳性细胞主要是神经胶质细胞,神经元也有一定量IL-6的表达。
[Abstract]:Objective: diffuse axonal injury (diffuse axonal, injury, DAI) refers to the linear acceleration of the head suffered sexual violence or (and) rotary blow when the brain produces shear due to accelerated motion, stretching and compression stress, resulting in brain white matter occurs widely in a series of pathophysiological changes in axonal injury characterized. Disturbance of consciousness is its typical manifestation, clinical diagnosis is difficult, the prognosis is poor. In recent years, the study found that diffuse axonal injury caused by traumatic brain dysfunction is due not only to the original mechanical force to the organization pressure caused by shear, product, is due to the number of hours after injury and the number of in the days of occurring complex "two hit" caused. Previous studies focused on changes in neuronal injury after traumatic brain injury in recent years, a study found that (traumic brain injury, TBI) after the central nervous system in astrocytes The stromal cells and microglia proliferated and changed with time.
Microglia is the immune effector cells of the central nervous system and play a key role in the immune response in the central nervous system. Microglia is a kind of cell plasticity, can not only release nerve growth factor, can also release neurotoxic factors, the protective effect and injury of neurons depends on the surrounding environment and change activation factor. Experiments have shown that microglia is the first one for the injury response of cells. After transient forebrain ischemia model, found that microglia reaction occurs early in 20 minutes. The traumatic brain injury after IL-6, IL-10, IL-1, IFN- and other inflammatory cytokines expression of gamma increased, and their plasma concentrations can be used as a reliable marker of.IL-6 severity play an important role in the occurrence and development of traumatic inflammation, which belongs to the injury factor, has initiated and promoted anti inflammation The effect is closely related to the severity of injury. However, whether microglia is activated after DAI and whether it is involved in the two attack with the proinflammatory factor IL-6, leading to further damage to its own neurons and neurons is not clear.
This experiment by Marmarou A et al reported for the first time in 1994 the Feeney method rat model of DAI replication, observe whether microglia were activated after DAI, the expression of proinflammatory cytokines IL-6, to further explore the interaction between neurons and microglia after DAI neural tissues, and after injury, inflammation and cell damage relations, to further clarify the pathophysiological mechanism of DAI and lay the foundation.
Methods: healthy male Sprague-Dawley (SD) 60 rats, weighing about 300 grams, were randomly divided into normal control group, sham operation group and against different time groups (0h, 1H, 3h, 6h, 12h, 24h, 48h, 72h, 5D and 7D). The rats in the normal control group without treatment; the sham operation group rats were treated with surgery and blow pretreatment, but not a real blow; blow rats after the operation, give blow, blow after the death time. According to the different manifestations of rats after the attack, after whole brain perfusion, paraffin sections with HE staining, silver staining method, cresyl violet stain and Weil 's iron hematoxylin staining to observe the pathological changes of myelin in rat brain tissue. The above method is verified in accordance with the standards of the rats included in the combat group, various brain regions in the brain microglia induced was measured by immunohistochemical method. Markers CD11b and IL-6 the injury is not The expression of the same time point.
The data of measurement were expressed by mean + standard deviation (Mean + SD). SPSS statistical analysis software was used for statistical analysis. The average number of each group was compared by one-way ANOVA (ANOVA), and the 22 difference was compared with the least significant difference (LSD). P0.05 showed significant difference.
Results: 1 the changes of histopathology:
The morphological changes of HE staining: normal control group and sham control group rat brain structure is clear, uniform and compact; combat group rat cerebral cortex, brainstem showed focal petechial hemorrhage, rats hit after 6h began to appear in brain tissue edema, peaked at 24h, decreased 48h after swelling.
The morphological changes of the cable axis Bielschowsky 's silver staining: normal control group and sham control group rat brain structure is clear, straight and long axons, smooth surface; combat rats blow 12h visible corpus callosum, brainstem and cerebellum. The axonal swelling, distortion, beaded, wavy change the surrounding interstitial edema, the performance in the 24h and 48h are more obvious, 5D improved significantly.
The morphological changes of myelin Weil s myelin staining: normal control group and sham control group rat brain structure is clear, uniform and dense, transverse section of myelin is annular, longitudinal section of a fishbone shape; peripheral myelin clearance in rats after injury blow against 6h showed rats brainstem axons increased obviously significant myelin loose, edema and disintegration of blank area, the change in 12h, 24h and 48h are more obvious, 5D improved significantly.
The morphological changes of Cresyl neurons in violet staining Nissl bodies: the normal control group and sham control group rat brain structure, brain stem, cerebellum and cortex neurons in the visible blue and purple, finely granular, dense and uniform, circling around the nucleus of Nissl bodies; combat rats against 12h after injury visible brainstem axonal neurons around the stained blank area, Nissl bodies decreased obviously, and a coarse granular, the change significantly in 24h and 48h, 5D was significantly improved.
2 CD11b positive microglia activation in different time points: the normal control group and sham control group rat brain CD11b positive microglia stained, brownish yellow, branched, small cell body, with thin projections extending in all directions; combat group rats 3h after injury can be seen in cerebellar cortex CD11b, microglia and other brain regions of the brainstem expression increased, mainly distributed in the axonal injury around and small blood vessels, and have significant difference with the control group (P0.05), the expression of 12h increased and reached the peak, the number of activated microglia increased significantly, protrusions shorter, thicker, cell body size, turn round, CD11b expression began to decrease after 24h, 72h was significantly lower than the 12h group and the control group, but there are still significant differences (P0.05).
After 3 DAI of IL-6 at different time points of expression: normal control group and sham operation group rats brain tissue IL-6 expression, its volume is very low; against rats 6h after injury of cortex, cerebellum, brainstem injury brain cells have elevated expression of IL-6 was obviously different from the control group (P0.05), 12h and 24h expression was increased and reached the peak, the expression of 48h began to decrease 7d IL-6 expression compared with 24h decreased obviously compared with the control group, but there are still significant differences (P0.05), positive cells were mainly glial cells, neurons also expressed a certain amount of IL-6.
Conclusion: the Feeney method using Marmarou A replication in the rat model of diffuse axonal injury; brain tissue CD11b DAI positive microglia, 12h reached the peak; increased the expression of IL-6 in rat brain tissue after DAI, peaked at 12h, the positive cells were mainly glial cells, neurons also expressed a certain amount of IL-6.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R741;D919

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