本文選題：彌漫性軸索損傷　切入點：小膠質(zhì)細(xì)胞　出處：《河北醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的:彌漫性軸索損傷(diffuse axonal injury, DAI)是指在頭部遭受直線加速性或(和)旋轉(zhuǎn)性暴力打擊時,腦組織由于加速運動而產(chǎn)生剪切、伸展和壓縮應(yīng)力,導(dǎo)致腦白質(zhì)廣泛發(fā)生以神經(jīng)軸索損傷為特征的一系列病理生理變化。意識障礙是其典型的表現(xiàn),臨床診斷較困難,預(yù)后多差。近年來研究發(fā)現(xiàn),彌漫性軸索損傷造成外傷后的腦功能障礙不僅是由于最初的機械外力對組織的剪切、積壓等造成的,更是由于損傷后數(shù)小時甚至數(shù)天中發(fā)生的復(fù)雜的“二次打擊”造成的。以往的研究主要集中在損傷后神經(jīng)元的變化,近年有研究發(fā)現(xiàn)外傷性腦損傷(traumic brain injury, TBI)后中樞神經(jīng)系統(tǒng)中的星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞增生,且隨時間的變化而變化。 小膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)的免疫效應(yīng)細(xì)胞,在中樞神經(jīng)系統(tǒng)的免疫應(yīng)答中發(fā)揮關(guān)鍵性作用。小膠質(zhì)細(xì)胞也是一種可塑性細(xì)胞,不僅能釋放神經(jīng)生長因子,也能釋放神經(jīng)毒因子,其保護(hù)作用和損傷神經(jīng)元的效應(yīng)依賴于周圍環(huán)境及活化因子的變化。有實驗表明,小膠質(zhì)細(xì)胞是第一個針對損傷發(fā)生應(yīng)答的細(xì)胞。在短暫性前腦缺血模型中,發(fā)現(xiàn)小膠質(zhì)細(xì)胞的反應(yīng)早在20分鐘內(nèi)即發(fā)生。外傷性腦損傷后有IL-6、IL-10、IL-1、IFN-γ等炎性細(xì)胞因子表達(dá)增高,且其血漿含量可作為反映傷情嚴(yán)重程度的可靠指標(biāo)。IL-6在創(chuàng)傷性炎癥反應(yīng)的發(fā)生發(fā)展過程中起重要的作用,屬于致傷因子,具有啟動和促進(jìn)炎癥反應(yīng)的作用,與損傷的嚴(yán)重程度密切相關(guān)。然而,小膠質(zhì)細(xì)胞是否在DAI后被激活,是否與促炎因子IL-6一起參與了二次打擊的過程,從而導(dǎo)致其自身及神經(jīng)元的進(jìn)一步損傷目前尚不清楚。 本實驗采用Marmarou A等人于1994年首次報道的自由落體撞擊方法復(fù)制大鼠DAI模型,觀察小膠質(zhì)細(xì)胞是否在DAI后被激活,促炎因子IL-6的表達(dá)情況,進(jìn)一步探討DAI后的神經(jīng)組織中神經(jīng)元和小膠質(zhì)細(xì)胞的相互作用,及損傷后炎癥反應(yīng)和細(xì)胞損傷的關(guān)系,為進(jìn)一步闡明DAI的病理生理學(xué)機制奠定基礎(chǔ)。 方法:健康雄性Sprague-Dawley(SD)大鼠60只,體重300克左右,隨機分為正常對照組、假手術(shù)組和打擊后不同時間組(0h、1h、3h、6h、12h、24h、48h、72h、5d和7d)。正常對照組大鼠不作處理;假手術(shù)組大鼠給予手術(shù)及打擊前處理,但不予真正的打擊;打擊組大鼠經(jīng)過手術(shù)后,給予打擊,打擊后按不同時間處死。觀察打擊后大鼠的行為學(xué)表現(xiàn),麻醉灌注后取全腦,制作石蠟切片,采用HE染色、銀染、焦油紫尼氏小體染色和Weil’s鐵蘇木素髓鞘染色的方法觀察大鼠腦組織的病理改變。將經(jīng)以上方法驗證符合標(biāo)準(zhǔn)的大鼠列入打擊組,用免疫組織化學(xué)的方法測定腦組織中各個腦區(qū)小膠質(zhì)細(xì)胞激活標(biāo)志物CD11b及IL-6在損傷后不同時間點的表達(dá)情況。 計量資料數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)表示,用SPSS統(tǒng)計分析軟件進(jìn)行統(tǒng)計學(xué)分析,各組均數(shù)的比較行單因素方差分析(ANOVA),用最小顯著差法(LSD)作兩兩比較,P0.05為有顯著性差異。 結(jié)果:1組織病理學(xué)變化: HE染色形態(tài)學(xué)變化:正常對照組與假手術(shù)對照組的大鼠腦組織結(jié)構(gòu)清晰,均勻致密;打擊組大鼠大腦皮層、腦干等部位可見局灶性點狀出血,大鼠打擊后6h開始出現(xiàn)腦組織充血水腫,24h達(dá)高峰,48h以后腫脹有所減輕。 Bielschowsky’s鍍銀染色軸索的形態(tài)學(xué)變化:正常對照組與假手術(shù)對照組大鼠腦組織結(jié)構(gòu)清晰,神經(jīng)元軸突直且長,表面光滑;打擊組大鼠打擊后12h可見胼胝體、腦干和小腦等部位神經(jīng)軸索扭曲、腫脹,呈串珠狀、波浪狀改變,周圍間質(zhì)水腫,上述表現(xiàn)于24h及48h更加明顯,5d明顯好轉(zhuǎn)。 Weil’s髓鞘染色髓鞘的形態(tài)學(xué)變化:正常對照組與假手術(shù)對照組大鼠腦組織結(jié)構(gòu)清晰,均勻致密,髓鞘橫切面呈環(huán)狀,縱切面呈魚刺狀;打擊組大鼠打擊后6h可見大鼠腦干等部位損傷軸索的周圍髓鞘間隙明顯增寬,髓鞘顯著疏松、水腫及崩解出現(xiàn)空白區(qū),上述改變在12h、24h和48h更加明顯,5d明顯好轉(zhuǎn)。 Cresyl violet染色神經(jīng)元尼氏小體的形態(tài)學(xué)變化:正常對照組與假手術(shù)對照組大鼠腦組織結(jié)構(gòu)清晰,腦干、皮質(zhì)和小腦等部位神經(jīng)元內(nèi)可見呈藍(lán)紫色,細(xì)小顆粒狀,均勻致密,環(huán)繞在細(xì)胞核的周圍的尼氏小體;打擊組大鼠打擊12h后可見腦干等部位損傷軸索的周圍神經(jīng)元淡染出現(xiàn)空白區(qū),尼氏小體明顯減少,且呈粗大顆粒狀,上述改變在24h和48h顯著,5d明顯好轉(zhuǎn)。 2 CD11b陽性小膠質(zhì)細(xì)胞在不同時間點的活化:正常對照組與假手術(shù)對照組大鼠腦組織CD11b陽性小膠質(zhì)細(xì)胞淡染,呈棕黃色、分枝狀,胞體小,具有伸向各個方向的細(xì)小突起;打擊組大鼠打擊后3h可見皮層、小腦、腦干等腦區(qū)的小膠質(zhì)細(xì)胞CD11b表達(dá)升高,主要分布于損傷軸索及小血管的周圍,且與對照組有明顯差異(P0.05),其表達(dá)量12h時增高達(dá)到峰值,活化的小膠質(zhì)細(xì)胞數(shù)量明顯增加,突起變短、變粗,胞體增大、變圓,CD11b表達(dá)于24h后開始下降,72h后明顯低于12h組,但與對照組仍然有明顯的差異(P0.05)。 3 DAI后IL-6在不同時間點的表達(dá):正常對照組與假手術(shù)對照組大鼠的腦組織IL-6有表達(dá),其量很低;打擊組大鼠打擊后6h可見皮層、小腦、腦干等損傷部位腦區(qū)神經(jīng)細(xì)胞有IL-6表達(dá)的升高,與對照組有明顯差異(P0.05),12h至24h表達(dá)量增高達(dá)到峰值,48h其表達(dá)開始下降,7d時IL-6的表達(dá)較24h有非常明顯的降低,但較對照組仍然有明顯的差異(P0.05),陽性細(xì)胞主要是神經(jīng)膠質(zhì)細(xì)胞,神經(jīng)元也有一定量IL-6的表達(dá)。 結(jié)論:采用Marmarou A的自由落體撞擊方法復(fù)制了大鼠彌漫性軸索損傷模型;DAI后腦組織中CD11b陽性小膠質(zhì)細(xì)胞活化,12h達(dá)到峰值;DAI后大鼠腦組織中IL-6的表達(dá)升高,12h達(dá)到峰值,陽性細(xì)胞主要是神經(jīng)膠質(zhì)細(xì)胞,神經(jīng)元也有一定量IL-6的表達(dá)。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R741;D919

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