光化学法诱导建立兔脑梗死模型的效果及特征(英文)

Establishment of cerebral infarction model in rabbits induced by photochemical injury

背景:脑梗死动物模型是研究人的脑梗死疾病的前提和条件。传统的模型制作方式有两种,一种是采用开颅结扎或电凝血管方法阻断供血动脉;另一种是采用导管法引用栓子或水凝胶微球栓塞供血动脉。但这两种模型制作不易,结果不稳定,应用受到限制。光化学法诱导建立脑梗死动物模型是一种新方法。目的:应用光化学诱导法建立兔局灶性脑梗死模型。设计:单一样本实验。可行性及其特征,探索人类脑梗死疾病的新的实验研究手段。单位:南通大学实验动物中心。材料:实验于2003-05/11在南通大学实验动物中心(二级实验室)完成。随机选取1012月龄日本大耳兔63只,雌33只,雄30只,体质量1.7～3.3kg。方法:兔麻醉后,沿颅骨的中央与眼后角垂直交叉处纵行切开皮肤约2cm,暴露颅骨,刮离骨膜,在矢状缝左(或右)侧外0.5cm与冠状缝后0.5cm处,用钻头直径0.5cm的颅钻钻透颅骨,造成颅骨圆形洞窗,随即由耳缘静脉按1mL/kg一次性缓慢注入35g/L四氯四碘荧光素钠盐。约3min后用冷光源(波长540nm,功率140lx)对准颅骨洞窗,连续照射8min后,缝合皮肤切口。术后24h,进行神经缺失功能5分制评分(0分:无神经损伤症状;1分:对侧(左侧)后下肢肌张力降低,回缩反射减弱;2分:对侧肢体瘫痪明显,后肢外展;3分:行走明显向外侧拖行,躯体倾向对侧;4分:不能自发行走和意识丧失)。术后48h处死兔,测量梗死灶面积、体积,并制作病理切片观察损伤程度。主要观察指标:兔肢体功能状态,脑梗死灶面积和体积,病理损伤程度。结果:59只兔成功建立起梗死灶,模型成功率为98%。①神经功能缺失评分:56只兔为1或2分,有3只兔达到3分。②梗死灶平均面积为(0.465±0.012)cm2,平均体积(长×宽×高)为(0.268±0.009)cm3。③切片观察损伤程度:梗死灶呈典型的损伤、渗出和炎症反应病理过程。轻度22只(37%)。中度32只(54%)。重度5只(9%)。结论:①应用光化学法诱导兔脑梗死模型制作简单、快速、重复性好,短时间内即可制作较多的供各种研究目的使用的模型。②模型动物存活时间长,死亡率低,有利于慢性脑血管病的研究。③此方法避免了对脑血管和脑组织的机械损伤。④可根据需要选定皮质区域制作梗死灶,梗死灶大小恒定并可控制其大小和深度,为皮层定位研究提供方法。⑤该模型可造成血小板聚集及血管内皮细胞损伤,为抗血小板聚集药物及脑缺血保护疗法的研究提供可能性。⑥但此模型也有其局限性犤9犦:由于血栓阻塞发生于终末动脉,所以不利于侧支循环及再灌注损伤的研究;该模型更近似于人类微血管病变,而不能说明人类其他缺血性卒中的发生机制。

BACKGROUND： Cerebral infarctional animal model provide basis for studying human cerebral infarction（CI）. There are two traditional CI models, one is reproduced by craniotomy or electro-coagulation by which supplying artery are blocked, another is achieved by embolus or water gelatin micro-thrombosis. But both are difficult to perform and results were instable, which limit the application. Photochemical injury is a novel way to reproduce CI model on experimental animals.OBJECTIVE： To explore a new method of experimental research of local cerebral infarction model which is induced by photochemical injury in rabbits DESIGN： Single sample study SETTING： Experimental Animal Center of Nantong University. MATERIALS： This study was conducted at the Experimental Animal Center of Nantong University from May to December 2003 （secondary laboratory）. Totally 63 Japanese flap-eared rabbits, with birth age of 10-12 month, 33 females and 30 males, with body mass of 1.7-3.3 kg, were randomly selected. METHODS： After anaesthetized, rabbits were cut at the skin for 2 cm long at the crossing of skull center and posterior canthus, skull was exposed and periosteum was separated, then a round skull window with diameter of 0.5 cm was drilled at 0.5 cm left or （right） to sagittal suture and 0.5 cm posterior to coronal suture, after that, 35 g/L rose Bengal was slowly injected from ear-edge vein in dosage of I mL/kg by once. About 3 minutes later, cold light source （wave length of 540 nm, power of 140 lx） was used to cast light directly onto the skull window for consecutively 8 minutes, then incision was sutured. At postoperative 24 hours, neurological defects were scored in five grades [0 score represent no neural impairments; 1 score： the left posterior limhs displayed decreased muscular tension and attenuated contraction reflex; 2 scores： the left posterior limbs were paralyzed, displaying ohvious ahduction; 3 scores： rabbit displayed obvious adductive drag with body leant to the opposite side; 4 scores： unable to walk and unconsciousness], rabbits were put to death at postoperative 48 hours, infarctional area and volume were determined and pathological changes was also observed. MAIN OUTCOME MEASURES： Limb movement, infarctional area and ＇volume and pathological changes. RESULTS： Cl mode was successfully established on 59 rabbits, the successful model reproducible rate was 98%. ① Scores for neural functional defects： 56 rabbits scored for 1 or 2, and 3 rabbits scored for 3. ② The mean infarctional area was （0.465±0.012） cm^2, and the mean volume （length×width×height） was （0.268±0.009） cm^3. ③ Histopathological changes： Infarctional focus displayed typical pathological changes such as impairment, effusion and inflammation. Gentle impairment could be observed in 22 rabbits （37%）, medium in 32 rabbits （54%） and severer in 5 rabbits （9%）. CONCLUSION：① Photochemical methods used for reproducing cerebral infarction model has multiple advantages, such as easy performance, quick and good repeatability, it can be used to reproduce experimental models for various objectives in short time. ② Experimental model animal can survive for a long time with low mortality, benefiting for researches on chronic cerebrovascular diseases.③ Mechanic injury of cerebral vessels and brain tissues could be avoided. ④ The cortical region of Cl, as well as infarctional size and depth are under control, meeting the need of researches on cortical location. ⑤ Platelet aggregation and endothelium injury could be observed in photochemical injury, which provide basis for study on the effect of anti-platelet aggregation medicine and protective therapy for CI.⑥ But there was still some limitations： Since thrombosis was induced at the terminal artery, unfit for the study of lateral circulation and reperfusion; however it was found more similar to human microvascular diseases, thereby incapable of explaining the pathogenesis of other ischemic strokes.