【作者】 李晓慧；

【导师】 金学隆； 王世忠；

【作者基本信息】 天津医科大学 ， 生理学， 2008， 硕士

【摘要】 目的白细胞在血液中的流变行为是其在体内发挥功能的重要病理生理机制。本研究拟从细胞流变学的角度出发,利用动物在体实验的方法,阐明脑出血急性期白细胞的变形性、趋化性和粘附性的变化规律,对该问题的深入阐明不仅可对脑出血时白细胞的功能有崭新的认识,而且对于采取积极有效的干预措施,改善脑出血患者的预后有十分重要的临床意义。方法1大鼠自体血内囊出血动物模型的建立:应用48只健康成年雄性Wistar大鼠(清洁级),用10%水合氯醛腹腔注射麻醉后,俯卧位固定在立体定向仪上。颅顶备皮,医用酒精消毒手术区皮肤,正中矢状切口约1.5cm,剥离皮下组织及骨膜,暴露“十字缝”和“人字缝”。根据包新民等著“大鼠脑立体定位图谱”确定右侧内囊后肢位置:前囟后3.3mm,矢状缝右侧3.2mm,垂直进针7.1mm。确定位置后用牙科钻(钻头直径1mm)钻破颅骨,采取二次注血、二次停针法,缓慢注入在大鼠断尾处采取的自体血液0.1ml,建立内囊出血动物模型,假手术只做刺入动作,不注射血液,作为手术对照组。大鼠清醒后采用Berderson评分法进行行为学评价,评分在手术后3h、6h、12h、24h进行。2大鼠内囊出血后脑组织形态学的观察:不同实验组的大鼠断头处死,小心剥离颅骨,取出完整脑组织,固定在4%多聚甲醛24h。将固定好的脑组织取出放在冰盘上以针孔为中心连续冠状切片(片厚5um)。切片常规脱蜡,HE染色,在光镜下观察病灶处血肿及组织学改变。3微循环观察:运用微循环显微镜、计算机图像分析系统对大鼠肠系膜后静脉内白细胞的流变行为(趋边、粘附及变形)进行直观研究。通过图像分析系统上回放录像带,选择无分支、长度100μm、直径10～30μm的微血管进行数据分析。4统计学方法:应用SPSS 15.0医学统计软件包,各组数据结果用(?)±s表示,组间比较均采取方差分析中的SNK法,P＜0.05为差异有显著性。结果1实验组不同时期脑组织病理形态在光镜下均可见脑实质内出血灶,圆形或椭圆形,大小不一,低倍视野内遍布红细胞,出血灶边缘脑组织疏松水肿(见图2-5)。ICH后3h,血肿周围即出现部分神经细胞、胶质细胞肿胀,细胞间隙出现大小不等的空泡,少数神经细胞坏死、消失,白细胞浸润。上述病理变化随着血肿形成的时间延长而逐渐严重。2从脑出血后3h到12h,白细胞沿壁滚动速度明显减慢。白细胞滚动速度从(14.94±4.23)um/s降低到(3.03±0.47)um/s,各组之间比较均有统计学意义(P＜0.05)。3从脑出血后3h到12h,白细胞粘附数、滚动数显著增多。白细胞粘附数从(2.96±0.33)个/100um增加到(15.29±1.96)个/100um,滚动数从(8.83±0.96)个/min增加到(36.71±1.33)个/min,各组之间比较均有统计学意义(P＜0.05)。4从脑出血后3h到12h,总体白细胞-内皮细胞接触时间(TLECT)显著延长。TLECT从(2.89±0.24)s增加到(17.24±2.99)s,各组之间比较均有统计学意义(P＜0.05)。结论1通过改良后的脑立体定向术可以准确复制大鼠自体血内囊出血动物模型。2脑出血急性期血肿压迫产生的占位效应、血肿成分及其降解产物的化学毒性作用、血肿腔内炎症反应和炎症因子等因素都可能造成微循环障碍,引起血肿周围组织的继发性损伤。3脑出血急性期,机体常处于高度应激状态,由于植物神经中枢受损,神经-体液调节功能紊乱而发生应激性溃疡等其它器官的应激性病变。脑出血的刺激对白细胞的生理功能有显著影响,体现在变形性降低、趋化性增强、膜流动性改变以及白细胞数目的增加。微循环障碍是应激性溃疡等疾病发生的重要病理生理机制之一,白细胞的流变行为可直接影响微循环的灌注,同时,微循环障碍也影响了白细胞的流动状态,这种相互作用,在脑出血急性期,随着血肿时间的延长而逐渐增强。白细胞流变行为的异常不仅是ICH后继发性脑损伤的病理机制之一,还参与了应激性溃疡等其它器官应激的病理生理过程。因此,研究急性脑出血后外周血中白细胞流变行为的改变对早期预防和治疗脑出血并发症有重要的临床指导意义。更多还原

【Abstract】 ObjectiveLeukocyte rheology is probably playing an important role in the physiology mechanism of leukocytal function, so my work focused on the effects of rheological properties of leukocytes in rats in terms of cellular Rheology. To interpret the deformability, chemotropism and adhesiveness of Leukocyte, It can help us not only know leukocytal function further, but also can provide a new theory for early diagnosis and clinical treatment of intracerebral hemorrhage.Methods:1 To make an intracerebral internal capsular hemorrhage animal model: 54 male Wistar rats were used in this experiment. They were positioned in a stereo tactic frame and a cranial burr hole (1 mm) was drilled on the right AF0.1-0.2 mm. 100μl autologous blood was sampled and then infused stereotacticlly into the right internal capsular. About sham control rats, they had only a needle insertion. After the infusion, the needle was removed, the burr hole was sealed with bone wax, the wound was sutured, and the animal was placed in a warm box with free access to food and water. Berderson test was done by a single observer without knowledge of neurological condition. Motor behavior was evaluated using testing each rat at 3h, 6h, 12h and 24h after operation.2 Morphological observation of brain : After finishing the Preparation of acute hemorrhage animal model, take the brain tissue and fix it with 10% formaldehyde solution. Through HE strain, observe their dynamic pathological changes under the light microscope.3 Observation of Microcirculation: By use of Microcirculation microscope and computer-image analyses system , pick out the capillary of no branch ,100μm length and 10～30μm diameter, to observe the leukocyte Rheology.4 Statistical analyses: All the data were analyzed by SNK test with SPSS 15.0 statistical software.Results1 The results showed that, the neurological deficit scores were the highest at 12 h and 24h after operation in the Berderson testing method. At all time points, the scores of the Sham-operated rats were lower than that of the ICH rats and significant differences existed between the two groups.2 The lesion of experiment group was severe than that of the sham group which is irregular or ellipsoidal blood clot, different size, rarefaction, edema on the tissue of perihematoma, A few of neurocyte and gliocyte are cellular swelling. Inequality size of vacuole in the cell spaces, and many of Leukocytes infiltrate. These pathological changes are more severity along with the longer time of haematoma formative.3 The Rheological properties of leukocytes in the microcirculation of mesentery in rats were changed obviously in intracerebral hemorrhage group than those of the control group. from 3h to 24h group, the rolling velocity slowed, the number of rolling leucocytes and adherence on endothelium increased, and TLECT obviously extended.Conclusion1 We can copy the model of intracerebral internal capsular hemorrhage by use of stereo tactic method successfully.2 Inflammatory infiltration, neuron necrosis, glial hyperplasia and brain edema in haematoma surrounding of experimental ICH rats may be one of the causes of brain edema and neurobehavioral changes.3 The rheological properties of leukocytes have been changed by intracerebral hemorrhaged stress respond. Microcirculation disturbance is one important pathomechanism of stress ulcer and other disease. The Rheology properties of Leukocytes can impact on microcirculation directly, and the Microcirculation disturbance can impact on leukocytal circulation. The interaction is more and more obviously along with bleeding time. So, study the changes of rheological properties of leukocytes is useful to prevent and treat the complication of brain hemorrhage beforehand.更多还原