重组链激酶预防蛛网膜下腔出血后脑血管痉挛的实验研究

重组链激酶预防蛛网膜下腔出血后脑血管痉挛的实验研究作者：王志刚，冀勇，丁璇    作者单位：250033济南，山东大学第二医院神经外科    【摘要】  目的 研究重组链激酶（rSK）对蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)的预防作用。方法 采用枕大池二次注血法制成大白兔SAH模型，24 h后经侧脑室穿刺置管,治疗1组注入1.25 mg rSK，闭管6 h后开放引流，6 h后再次注射、闭管、引流，如此反复3次；治疗2组一次性注入rSK 3.75 mg；SAH组注入生理盐水1 ml，方法同治疗1组；给药后6 h、12 h和1 d、3 d、5 d、7 d经各引流管引流的脑脊液（CSF）用于检测氧合血红蛋白（OxyHb）含量；制模前及制模后7 d分别进行基底动脉（BA）造影，计算口径比，并进行组织学检查。结果 （1）CSF中OxyHb含量SAH组逐渐上升，治疗1组、2组逐渐下降，注药后1～7 d治疗1组、2组OxyHb含量较SAH组明显下降（P<0.05～0.01）；第7 d治疗1组OxyHb含量显著低于治疗2组（P<0.05）。(2)SAH组BA管径明显缩小，呈重度痉挛；治疗1组、2组管径无明显变化。（3）组织学检查发现SAH组脑底表面有含铁血黄素沉着，血管周围有血凝块，BA内膜皱缩；治疗1组脑底无含铁血黄素沉着及血凝块，BA未见病理改变;治疗2组脑底有少量含铁血黄素沉着及血凝块，BA内膜轻度皱缩。结论 侧脑室短期、少量反复注射rSK并CSF引流可预防SAH后CVS的发生。    【关键词】  重组链激酶；蛛网膜下腔出血；脑血管痉挛　　Experimental study of preventive effect of recombinant streptokinase on cerebral vasospasm after subarachnoid hemorrhage  WANG Zhigang， JI  Yong， DING  Xuan .　Department of Neurosurgery, the Second Hospital of Shandong University, Jinan 250033, China    Abstract：Objective  To study the preventive effect of recombinant streptokinase (rSK) on cerebral vasospasm (CVS) after  subarachnoid hemorrhage(SAH).Methods  Rabbit models of SAH  were made by double blood injection into cisterna magna. A tube was inserted into lateral ventricle after 24 h. In group treating one, 1.25 mg rSK were injected into lateral ventricle, and the tube was opened for drainage after closed 6 h, and then 6 h later, injection, closed pipe and drainage were recycled for another 2  times. In the group SAH , 1 ml normal saline (NS) was injected into lateral ventricle, and then the way was  the same as group treating one. In group treating  two, 3.75 mg rSK were injected for once. 6 h, 12 h ,1 d, 3 d, 5 d and 7 d later, 1 ml CSF was drained from the lateral ventricle in groups SAH , treating  one and treating two ,respectively. The level of OxyHb in CSF was detected, angiograms of basilar artery(BA) were performed before and after 7 d of SAH,calculation the rate of calibre and histological examinations were also performed. Results  (1)The level of OxyHb in CSF of group SAH was increased gradually while decreased in groups treating one and treating two. Compared with group SAH, the levels of  OxyHb were significant lower in groups treating one and treating two at 1～7 d after administration（P<0.05～0.01）; and  there was obviously difference between groups treating one and treating two at 7 d after administration（P<0.05）.(2)CVS of BA were found in group SAH and no change occurred for groups treating one and treating two.(3)The histology showed that in group SAH there was hemosiderin pigmentation in surface of pavimentum cerebri , blood clot was around blood vessel, the endomembrane of BA was shrinkage. However, in  group treating one, BA was no change and no blood clot around blood vessel. In group treating two, there was a little pigmentation in surface of pavimentum cerebrilighter and the endomembrane of BA was  shrinkage gently , no blood clot was around blood vessel.  Conclusion  Repeated injections with lower dosage of rSk into lateral ventricle and CSF drainage is a good method for  prevention from CVS after SAH.    Key words：recombinant streptokinase；subarachnoid hemorrhage；cerebral vasospasm　　脑血管痉挛（CVS）是自发性蛛网膜下腔出血（SAH）后常见的并发症，也是致残和死亡的主要原因之一。本实验采用重组链激酶（rSK）侧脑室注射并行脑脊液（CSF）外引流, 观察不同给药方式对SAH后CVS的预防作用。1  材料与方法　　1.1  动物及分组  新西兰纯种健康清洁级大白兔24只（山东大学第二医院实验动物中心），雌雄不限，月龄4～6个月，质量（2.5±0.4）kg。随机分为正常对照组、SAH组、治疗1组、治疗2组，每组6只。　　1.2  方法　　1.2.1  SAH模型制作  应用30%乌来糖3 ml/kg静脉麻醉，将SAH组、治疗1组及2组的动物采用枕大池二次注血建立兔SAH模型。动脉血采自兔耳中央动脉, 首次注入0.5 ml/kg，48 h后再次注入0.3 ml/kg；再次注血24 h后为SAH第1 d。　　1.2.2  药物干预方法  （1）治疗1组：制模后24 h从侧脑室置管并注入rSK 1.25 mg，闭管6 h后开放引流，6 h后再次注药、闭管、引流，如此反复共3次，rSK总量3.75 mg；（2）治疗2组：制模24 h后从侧脑室置管并一次性注入rSK 3.75 mg；（3）SAH组：制模24 h后从侧脑室置管并注入生理盐水1 ml，余操作方法同治疗1组；（4）正常对照组：无手术及药物干预。　　1.2.3  CSF中氧合血红蛋白（OxyHb）检测  各组给药后6 h、12 h和1 d、3 d、5 d、7 d经引流管各留取CSF 1 ml，以2700 r/min离心3 min，分光光度计测定CSF A540、A560、A576值；OxyHb=(1.4747A576-0.682A560-0.5329A540)×10-4mol/L。　　1.2.4  DSA检查  分别在制模前及制模后7 d，30%乌来糖3 ml/kg外缘耳静脉麻醉后，置兔仰卧位，常规消毒，于颈前中央一侧旁开2 cm切开皮肤，分离一侧颈总动脉，结扎同侧颈外动脉，颈内动脉置管，行椎动脉造影。分别测量基底动脉（BA）上、中、下段, 取其平均值为动脉口径。痉挛程度=制模后动脉口径/制模前动脉口径×100%。正常：91%～110%，轻度痉挛：81%～90%，中度痉挛：70%～80%, 重度痉挛：<70%。　　1.2.5  BA的取材及病理检查  实验兔分别在SAH第7 d造影后开胸经右心房以pH 7.4 PBS 300 ml灌洗。将颅顶、枕骨及第1～3颈椎棘突和双侧椎板咬除，钝性分离兔脑及上段颈髓，观察脑底表面含铁血黄素沉着及血凝块情况；取附有BA全长的脑干置于10%的福尔马林中，1周后脱水，石蜡包埋，病理切片厚约3 μm，行HE染色，分别在10倍和20倍光学显微镜下观察BA病理变化。　　1.2.6  统计学方法  数据以均数±标准差(±s)表示,采用StudentNewmanKeuls检验进行统计分析。2  结果　　2.1  各组CSF中OxyHb含量的比较  见表1。制模后CSF中OxyHb含量SAH组逐渐升高，治疗1组、2组则逐渐下降。注药后1～7 d治疗1组、2组OxyHb含量较SAH组明显下降（P<0.05～0.01）；第7 d治疗1组的OxyHb含量较治疗2组下降更显著（P<0.05）。　　表1  各组CSF中OxyHb含量的比较（略）　　注：与同期SAH组比较*P<0.05，**P<0.01；与同期治疗2组比较△P<0.05　　2.2  各组BA管径及CVS程度的比较  见表2。治疗1组、2组未出现CVS，SAH组呈重度CVS。　　表2  各组BA管径的比较（略）　　注：与正常对照组比较*P<0.05；与SAH组比较△P<0.05，△△P<0.01　　2.3  各组脑底表面及BA组织学改变  SAH组脑底表面有含铁血黄素沉着，血管周围有血凝块、BA内膜有皱褶；治疗1组脑底表面无含铁血黄素沉着及血凝块，BA内膜未见明显改变；治疗2组脑底表面有少量含铁血黄素沉着及血凝块，BA内膜轻度皱褶。3  讨论　　SAH后诱发CVS的机制尚不完全清楚。大量研究［2，3］表明SAH后蛛网膜下腔中血凝块中的红细胞裂解，释放多种生物活性物质，尤其是OxyHb,在SAH后7～10 d，其释放达高峰，也是临床上CVS的高发期，是导致CVS的主要因素。当OxyHb被转变成正铁血红蛋白时，CVS可以缓解［4］。研究［5］发现，SAH早期（<72 h）手术清除蛛网膜下腔的血凝块，可有效地预防该部位CVS的发生；但SAH往往非常广泛，手术清除作用有限，或手术强行清除血凝块可导致脑组织及重要穿通支血管的损害。　　组织型纤溶酶原激活剂（tPA）可与血凝块中的纤溶酶原结合并将其激活为纤溶酶，降解血凝块中的纤维蛋白，使红细胞从血凝块中释放；动物实验［6］发现早期脑池局部单次或多次应用tPA和尿激酶（UK）溶化蛛网膜下腔的血凝块，使红细胞从CSF中吸收或引流出蛛网膜下腔，可预防CVS的发生。由于纤溶药物的作用存在量效关系，如何使药物迅速到达脑底蛛网膜下腔并维持一定的有效浓度，快速溶化清除血凝块，以及用药方式、途径等均有待进一步研究。Hariton等［7］在猴双侧SAH动物模型中，用不同剂量UK单次注入一侧脑池，6 d后发现只能清除同侧血凝块；在同样的模型中，Susuki等［8］用单侧注入小剂量rtPA连续6 d，也只能清除同侧血凝块；而经侧脑室注入同位素标记的白蛋白，大部分于4～6 h即可到达脑底表面蛛网膜下腔。因此，从侧脑室给药，可使药物随CSF循环自然到达蛛网膜下腔各脑池中，短期反复给药，可使药物维持一定的有效浓度，快速溶化血凝块，并经CSF引流到体外，应是一种较好的给药途径和SAH后预防CVS的方法。　　本实验中选用rSK作为纤溶药物，治疗1组采用短期多次小剂量给药，治疗2组为1次给药，结果显示治疗1组CSF中OxyHb逐渐降低，脑底表面蛛网膜下腔无含铁血黄素沉着及血凝块，BA无痉挛，BA内膜无皱缩，提示SAH后早期经侧脑室短期反复应用rSK，可预防CVS的发生。治疗2组脑底表面蛛网膜下腔出现少量的含铁血黄素沉着及血凝块，BA内膜轻度皱缩，但无明显的BA痉挛，可能因单次给药很快被CSF稀释，因而有其局限性。SAH组脑底蛛网膜下腔有含血黄素沉着，血管周围血凝块聚集，BA出现明显的痉挛，BA内膜增生、皱缩。表明CSF引流虽可将部分OxyHb排出体外，但不能预防SAH后CVS的发生。    因此，采用侧脑室短期重复应用小剂量rSK并CSF引流，具有预防SAH后发生CVS的作用。【参考文献】　［1］Morreale VM, Meissner I. 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