CN113397757A - 用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法 - Google Patents
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Abstract
本发明特别是公开了一种用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,先建立急性缺血性脑卒中小鼠模型,然后进行行为学试验和冰冻切片,行为学试验包括圆筒试验和网格爬行试验,使用以上2种行为学试验测定小鼠各阶段的运动功能,以评估Sham组,IS组和LQ给药组的运动功能相关指标;冰冻切片可用于尼氏染色和TUNEL细胞凋亡试剂盒染色,观察小鼠病灶区尼氏染色的图像和TUNEL细胞凋亡免疫染色的图像。采用上述结构,提供了一种能验证拉喹莫德具有促进急性缺血性脑卒中后的恢复作用的方法。
Description
技术领域
本发明涉及用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法。
背景技术
脑卒中,俗称中风,是由脑部血管受损导致的脑组织损伤。具有死亡率高、发病率高、致残率高点的特点,对人们的生命和健康造成严重威胁,是神经功能障碍和死亡的重要原因。脑卒中按照病因可分为出血性脑卒中和缺血性脑卒中,其中,缺血性脑卒中在临床上最为常见,约占我国脑卒中的60%-80%。
缺血性脑卒中指因脑的供血动脉(颈动脉和椎动脉)狭窄或闭塞,引起脑部的血液供应不足,使得局部脑组织发生缺血、缺氧而坏死或软化,进而导致神经功能障碍的疾病。神经元比邻近的星形胶质细胞更容易受到缺血性损伤,星形胶质细胞是复杂的高分化细胞,以连续的方式分布在中枢神经系统中,并对健康中枢神经系统的正常功能做出许多重要贡献,包括调节血流、向神经元提供能量代谢物、参与突触功能和可塑性以及维持细胞外离子、液体和递质平衡。此外,星形胶质细胞通过通常称为反应性星形胶质细胞增生的过程对所有形式的中枢神经系统损伤(如感染、创伤、缺血和神经退行性疾病)做出反应,这涉及其分子表达和形态的变化,在严重情况下,还涉及疤痕形成,在卒中后的晚期恢复阶段,胶质瘢痕可能阻碍轴突再生,并随后降低功能结果。尽管治疗缺血性脑卒中的药物越来越多,但是除了阿司匹林和在极少数急性缺血性脑卒中患者中进行溶栓治疗外,尚未证实任何其他药物干预对大多数急性缺血性脑卒中患者有效。
发明内容
为了克服现有技术的不足,本发明提供了一种能验证拉喹莫德具有促进急性缺血性脑卒中后的恢复作用的方法。
为了实现上述目的,本发明采用的技术方案是:一种用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,具体方法如下:
(1)先建立急性缺血性脑卒中小鼠模型
1)选用成年野生型雄性ICR小鼠,周龄为6-8周,重量约为25±2g,在温度25±2℃、湿度70±5%和光照12h:黑暗12h循环情况下,于无病原体饲养中心培养小鼠;
2)采用光化学栓塞法制作小鼠脑缺血模型,共设Sham组、IS3d组、IS5d组、IS 3d+LQ-25mg/kg组、IS 3d+LQ-50mg/kg组、IS5d+LQ-25mg/kg组、IS 5d+LQ-50mg/kg组7组;
3)按随机数表法,将70只周龄为8周的ICR小鼠进行分组,除Sham组外,其余各组小鼠腹腔注射浓度为10mg/ml的玫瑰红100mg/kg,5分钟后,腹腔再注射浓度为5%的水合氯醛200mg/kg,麻醉后,对小鼠进行缺血手术,小鼠脑立体定位于S1FL区;
4)缺血手术结束后30分钟,向IS3d+LQ-25mg/kg组、IS 3d+LQ-50mg/kg组、IS5d+LQ-25mg/kg组以及IS 5d+LQ-50mg/kg组小鼠灌胃Laquinimod-L12699;
5)术后密切观察各组小鼠生命体征,造模完毕后将小鼠置于37℃热板复苏,待苏醒后将其置于鼠笼饲养;
(2)进行行为学试验和冰冻切片
行为学试验包括圆筒试验和网格爬行试验,使用以上2种行为学试验测定小鼠各阶段的运动功能,以评估Sham组,IS组和LQ给药组的运动功能相关指标;
冰冻切片可用于尼氏染色和TUNEL细胞凋亡试剂盒染色,观察小鼠病灶区尼氏染色的图像和TUNEL细胞凋亡免疫染色的图像。
作为本发明的进一步设置,所述圆筒试验用于评估小鼠上肢协调性的情况,观察小鼠在直立状态下使用上肢的偏好,可以通过评估损伤侧肢体的使用频率和意愿反应小鼠的恢复情况,具体步骤如下:
1)试验在安静的环境下进行,选取小鼠最活跃的时间段晚上7:00-10:00进行;
2)小鼠被放置在圆筒状的透明有机玻璃里一共3min,全程录像,圆筒后面设置玻璃以获取小鼠背对摄像机的情形;
3)每次小鼠直立状态下双上肢接触圆筒壁然后又放回地面记为一组。
作为本发明的进一步设置,所述网格爬行试验用来评估小鼠运动的协调性,具体步骤如下:
1)试验在安静的环境下进行,选取小鼠最活跃的时间段晚上7:00-10:00进行;
2)小鼠被放置在圆柱形的铁丝网内,其直径和高为27.5cm x 22cm,铁丝网的网格为边长是1cm的正方形,铁丝网被固定在实验室工作台上,一共1min,全程录像;
3)拍摄小鼠所有的运动,记录小鼠的步行错误。
作为本发明的进一步设置,所述冰冻切片的具体步骤如下:
1)用5%水合氯醛分别麻醉各组小鼠,先用1×PBS溶液心脏灌注,再用4%PFA溶液灌注,将灌注后的脑组织浸入4%PFA中24h;
2)组织脱水:将组织换入30%的蔗糖溶液,待组织沉下即完成脱水;
3)取出脑组织用OCT包埋后于-80℃冰箱放置10min;
4)使用冰冻切片机切片,每张组织厚度为30um,得到的冰冻切片可直接进行下一步染色或于组织冻存液保存。
作为本发明的进一步设置,所述尼氏染色的具体步骤如下:
1)取出切片在室温放置,待表面没有水分后放在55℃烘箱烘15-30min,使组织贴紧载玻片;
2)将切片放入装有尼氏染色液的染缸中,放置时间根据染色情况而定,若染色效果不好可以放入37℃烘箱加热;
3)尼氏染色液上色完成后放入1×PBS中快速洗一次;
4)放入95%酒精中洗脱1-3min;
5)放入无水乙醇中洗脱5min×2次,然后放入二甲苯中5min×2次;
6)滴适量的中性树胶到盖玻片上,取出二甲苯中的切片快速沿着边缘将盖玻片贴上,用指甲油粘住边角,晾干后拍照或者常温保存。
作为本发明的进一步设置,所述TUNEL细胞凋亡试剂盒染色的具体步骤如下:
1)取出切片浸入1×PBS溶液10min×3次,以洗去切片组织上的OCT;
2)用4%PFA溶液固定20min,1×PBS溶液洗10min 3次;
3)用ddH2O将50×柠檬酸钠抗原修复液稀释成1×柠檬酸钠抗原修复液,并在恒温水浴锅预热至96℃,放入组织切片抗原修复6min-10min;
4)切片抗原修复后冷却至室温,用3%双氧水或甲醇封闭10min,用0.1%Triton打孔液冰上2min;
5)加一抗并在4℃的环境中孵育过夜,一抗用5%的BSA溶液配制,一抗的稀释浓度:NeuN浓度为1:800,细胞核标记物DAPI浓度为1:1000,二抗的稀释浓度均为1:1000;
6)去除一抗,用1×PBS洗一抗10min×3次,加入对应种属的荧光二抗以及按试剂盒配置的TUNEL试剂,放与37℃恒温箱孵育90min,1×PBS洗二抗10min×3次
7)在盖玻片上加适量的抗荧光淬灭剂,封片,之后用指甲油粘住边角,晾干后拍照。
上述技术方案的有益效果为,拉喹莫德(Laquinimod,LQ)是一种具有高口服生物利用率的新合成化合物,它目前正在临床开发用于治疗多发性硬化症,可降低多发性硬化症的复发率、脑萎缩和残疾进展。动物研究表明其引起Th1(辅助T细胞1,其产生促炎细胞因子)抗炎属性向Th2(辅助T细胞2,其产生抗炎细胞因子)转化,对周围的炎症有很好的治疗效果,能显著降低刺激星形胶质细胞的促炎因子,但不降低小胶质细胞的促炎因子。也有研究表明,LQ通过在体内和体外干扰星形胶质细胞NF-kB的激活来减少星形胶质细胞的炎症反应,减弱了血肿周围小胶质细胞/巨噬细胞和星形胶质细胞的活化。通过对急性缺血性脑卒中小鼠模型给予LQ灌胃,浓度分别为25mg/kg和50mg/kg;通过行为学试验发现给予LQ组小鼠较对照组小鼠运动和感觉能力有明显改善,但LQ-25mg/kg组小鼠行为学检测与LQ-50mg/kg组小鼠无明显差异;通过尼氏染色发现LQ-25mg/kg组小鼠较对照组小鼠病灶面积有明显变小;通过TUNEL细胞凋亡试剂盒染色发现LQ-25mg/kg组小鼠较对照组小鼠凋亡细胞数明显减少。
下面结合附图对本发明作进一步描述。
附图说明
附图1为本发明具体实施例圆筒试验的检测图;
附图2为本发明具体实施例网格爬行试验的检测图;
附图3为本发明具体实施例各组小鼠病灶区尼氏染色的图像;
附图4为本发明具体实施例各组小鼠冰冻切片TUNEL细胞凋亡免疫染色的图像。
具体实施方式
本发明的具体实施例如图1-4所示,一种用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,具体方法如下:
(1)先建立急性缺血性脑卒中小鼠模型
本研究选用成年野生型雄性ICR小鼠(6-8周龄),重量约为25±2g。在控制条件(温度25±2℃,湿度70±5%)和12:12h光照:黑暗循环情况下,于无病原体饲养中心培养动物。采用光化学栓塞法制作小鼠脑缺血模型,共设Sham组、IS3d组、IS5d组、IS 3d+LQ-25mg/kg组、IS 3d+LQ-50mg/kg组、IS 5d+LQ-25mg/kg组、IS5d+LQ-50mg/kg组7组。按随机数表法,将70只ICR小鼠(雄性、8周)进行分组。除Sham组外,其余各组小鼠腹腔注射浓度为10mg/ml的玫瑰红100mg/kg,5分钟后,腹腔再注射浓度为5%的水合氯醛200mg/kg,麻醉后,用剃毛机将手术部位及周围剃毛,碘伏溶液消毒手术部位3次。小鼠脑立体定位于S1FL区,缺血手术结束后30分钟,向IS3d+LQ-25mg/kg组、IS 3d+LQ-50mg/kg组、IS5d+LQ-25mg/kg组以及IS5d+LQ-50mg/kg组小鼠灌胃Laquinimod-L12699(25mg/kg,50mg/kg,Aladdin,中国)。术后密切观察各组小鼠生命体征。造模完毕后将小鼠置于37℃热板复苏,待苏醒后将其置于鼠笼饲养。
观察和检测方法:
1.行为学试验:
使用以下2种行为试验测定小鼠的运动功能,以评估Sham组,IS组和LQ给药组的运动功能相关指标:
1-1圆筒试验:
这项试验用来评估小鼠上肢协调性的情况,观察小鼠在直立状态下使用上肢的偏好,可以通过评估损伤侧肢体的使用频率和意愿反应小鼠的恢复情况。具体步骤如下:
1)试验在安静的环境下进行,选取小鼠最活跃的时间段晚上7:00-10:00进行;
2)小鼠被放置在透明有机玻璃的圆筒状里(13.6cm x 19.9cm:D x H)一共3min,全程录像,圆筒后面设置玻璃以获取小鼠背对摄像机的情形。
3)每次小鼠直立状态下双上肢接触圆筒壁然后又放回地面记为一组。
1-2网格爬行试验:
这项试验用来评估小鼠运动的协调性。具体步骤如下:
1)试验在安静的环境下进行,选取小鼠最活跃的时间段晚上7:00-10:00进行;
2)小鼠被放置在圆柱形的铁丝网内(27.5cm x 22cm:D x H),铁丝网的网格为1cm的正方形,即铁丝网上单个的镂空孔为1cm的正方形,铁丝网被固定在实验室工作台上约50cm处,小鼠在铁丝网的底面上爬行,一共1min,全程录像;
3)拍摄小鼠所有的运动,记录小鼠的步行错误。
2.冰冻切片:
1)5%水合氯醛分别麻醉各组小鼠。先用1×PBS溶液心脏灌注,再用4%PFA溶液灌注,将灌注后的脑组织浸入4%PFA中24h;
2)组织脱水:将组织换入30%的蔗糖溶液,待组织沉下即完成脱水;
3)取出脑组织用OCT包埋后于-80℃冰箱放置10min;
4)使用冰冻切片机切片,每张组织厚度为30um,得到的冰冻切片可直接进行下一步染色或于组织冻存液保存。
3.冰冻切片尼氏染色:
1)取出切片在室温放置,待表面没有水分后放在55℃烘箱烘15-30min,使组织贴紧载玻片;
2)将切片放入装有尼氏染色液的染缸中,放置时间根据染色情况而定,若染色效果不好可以放入37℃烘箱加热;
3)尼氏染色液上色完成后放入1×PBS中快速洗一次;
4)放入95%酒精中洗脱1-3min(时间可根据脱色情况而定);
5)放入无水乙醇中洗脱5min×2次;然后放入二甲苯中5min×2次;
6)滴适量的中性树胶到盖玻片上,取出二甲苯中的切片快速沿着边缘将盖玻片贴上,注意防止气泡产生,用指甲油粘住边角,防止盖玻片滑脱,晾干后即可拍照或者常温保存。
4.冰冻切片TUNEL细胞凋亡试剂盒染色:
1)取出切片浸入1×PBS溶液10min×3次,以洗去切片组织上的OCT;
2)4%PFA溶液固定20min,1×PBS溶液洗10min 3次
3)用ddH2O将50×柠檬酸钠抗原修复液稀释成1×柠檬酸钠抗原修复液,并在恒温水浴锅预热至96℃,放入组织切片抗原修复6min-10min;
4)切片抗原修复后冷却至室温,3%双氧水(甲醇)封闭10min,0.1%Triton打孔液冰上2min;
5)加一抗并在4℃的环境中孵育过夜,一抗用5%的BSA溶液配制,一抗的稀释浓度:NeuN浓度为1:800,细胞核标记物DAPI浓度为1:1000;二抗的稀释浓度均为1:1000);
6)去除一抗,用1×PBS洗一抗10min×3次,加入对应种属的荧光二抗以及按试剂盒配置的TUNEL试剂,放与37℃恒温箱孵育90min,1×PBS洗二抗10min×3次
7)在盖玻片上加适量的抗荧光淬灭剂,封片,之后用指甲油粘住边角,防止盖玻片滑脱,晾干后拍照。
实验结果可证明拉喹莫德能改善缺血性脑损伤后小鼠的运动功能,如附图1、2所示,图示为各组小鼠手术前1天、手术后1天、3天和5天的行为学检测(圆筒试验和网格爬行试验)。*p<0.05,**p<0.01,#p<0.05,##p<0.01,n=10。
拉喹莫德能促进缺血性脑损伤后小鼠病灶面积的恢复,如附图3所示,图示为各组小鼠病灶区尼氏染色的图像,比例尺=500um。
拉喹莫德能抑制缺血性脑损伤病灶及周围区域神经元凋亡,如附图4所示,图示为各组小鼠冰冻切片TUNEL细胞凋亡免疫染色的图像,比例尺=100um。
本发明不局限于上述具体实施方式,本领域一般技术人员根据本发明公开的内容,可以采用其他多种具体实施方式实施本发明的,或者凡是采用本发明的设计结构和思路,做简单变化或更改的,都落入本发明的保护范围。
Claims (6)
1.一种用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,其特征在于:具体方法如下:
(1)先建立急性缺血性脑卒中小鼠模型
1)选用成年野生型雄性ICR小鼠,周龄为6-8周,重量约为25±2g,在温度25±2℃、湿度70±5%和光照12h:黑暗12h循环情况下,于无病原体饲养中心培养小鼠;
2)采用光化学栓塞法制作小鼠脑缺血模型,共设Sham组、IS3d组、IS5d组、IS 3d+LQ-25mg/kg组、IS 3d+LQ-50mg/kg组、IS 5d+LQ-25mg/kg组、IS 5d+LQ-50mg/kg组7组;
3)按随机数表法,将70只周龄为8周的ICR小鼠进行分组,除Sham组外,其余各组小鼠腹腔注射浓度为10mg/ml的玫瑰红100mg/kg,5分钟后,腹腔再注射浓度为5%的水合氯醛200mg/kg,麻醉后,对小鼠进行缺血手术,小鼠脑立体定位于S1FL区;
4)缺血手术结束后30分钟,向IS3d+LQ-25mg/kg组、IS 3d+LQ-50mg/kg组、IS5d+LQ-25mg/kg组以及IS 5d+LQ-50mg/kg组小鼠灌胃Laquinimod-L12699;
5)术后密切观察各组小鼠生命体征,造模完毕后将小鼠置于37℃热板复苏,待苏醒后将其置于鼠笼饲养;
(2)进行行为学试验和冰冻切片
行为学试验包括圆筒试验和网格爬行试验,使用以上2种行为学试验测定小鼠各阶段的运动功能,以评估Sham组,IS组和LQ给药组的运动功能相关指标;
冰冻切片可用于尼氏染色和TUNEL细胞凋亡试剂盒染色,观察小鼠病灶区尼氏染色的图像和TUNEL细胞凋亡免疫染色的图像。
2.根据权利要求1所述的用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,其特征在于:所述圆筒试验用于评估小鼠上肢协调性的情况,观察小鼠在直立状态下使用上肢的偏好,可以通过评估损伤侧肢体的使用频率和意愿反应小鼠的恢复情况,具体步骤如下:
1)试验在安静的环境下进行,选取小鼠最活跃的时间段晚上7:00-10:00进行;
2)小鼠被放置在圆筒状的透明有机玻璃里一共3min,全程录像,圆筒后面设置玻璃以获取小鼠背对摄像机的情形;
3)每次小鼠直立状态下双上肢接触圆筒壁然后又放回地面记为一组。
3.根据权利要求1所述的用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,其特征在于:所述网格爬行试验用来评估小鼠运动的协调性,具体步骤如下:
1)试验在安静的环境下进行,选取小鼠最活跃的时间段晚上7:00-10:00进行;
2)小鼠被放置在圆柱形的铁丝网内,其直径和高为27.5cm x 22cm,铁丝网的网格为边长是1cm的正方形,铁丝网被固定在实验室工作台上,一共1min,全程录像;
3)拍摄小鼠所有的运动,记录小鼠的步行错误。
4.根据权利要求1所述的用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,其特征在于:所述冰冻切片的具体步骤如下:
1)用5%水合氯醛分别麻醉各组小鼠,先用1×PBS溶液心脏灌注,再用4%PFA溶液灌注,将灌注后的脑组织浸入4%PFA中24h;
2)组织脱水:将组织换入30%的蔗糖溶液,待组织沉下即完成脱水;
3)取出脑组织用OCT包埋后于-80℃冰箱放置10min;
4)使用冰冻切片机切片,每张组织厚度为30um,得到的冰冻切片可直接进行下一步染色或于组织冻存液保存。
5.根据权利要求4所述的用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,其特征在于:所述尼氏染色的具体步骤如下:
1)取出切片在室温放置,待表面没有水分后放在55℃烘箱烘15-30min,使组织贴紧载玻片;
2)将切片放入装有尼氏染色液的染缸中,放置时间根据染色情况而定,若染色效果不好可以放入37℃烘箱加热;
3)尼氏染色液上色完成后放入1×PBS中快速洗一次;
4)放入95%酒精中洗脱1-3min;
5)放入无水乙醇中洗脱5min×2次,然后放入二甲苯中5min×2次;
6)滴适量的中性树胶到盖玻片上,取出二甲苯中的切片快速沿着边缘将盖玻片贴上,用指甲油粘住边角,晾干后拍照或者常温保存。
6.根据权利要求1所述的用于测试拉喹莫德在治疗缺血性脑卒中的作用的方法,其特征在于:所述TUNEL细胞凋亡试剂盒染色的具体步骤如下:
1)取出切片浸入1×PBS溶液10min×3次,以洗去切片组织上的OCT;
2)用4%PFA溶液固定20min,1×PBS溶液洗10min 3次;
3)用ddH2O将50×柠檬酸钠抗原修复液稀释成1×柠檬酸钠抗原修复液,并在恒温水浴锅预热至96℃,放入组织切片抗原修复6min-10min;
4)切片抗原修复后冷却至室温,用3%双氧水或甲醇封闭10min,用0.1%Triton打孔液冰上2min;
5)加一抗并在4℃的环境中孵育过夜,一抗用5%的BSA溶液配制,一抗的稀释浓度:NeuN浓度为1:800,细胞核标记物DAPI浓度为1:1000,二抗的稀释浓度均为1:1000;
6)去除一抗,用1×PBS洗一抗10min×3次,加入对应种属的荧光二抗以及按试剂盒配置的TUNEL试剂,放与37℃恒温箱孵育90min,1×PBS洗二抗10min×3次
7)在盖玻片上加适量的抗荧光淬灭剂,封片,之后用指甲油粘住边角,晾干后拍照。
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