Use of a spectrophotometric hemoglobin assay to objectively quantify intracerebral hemorrhage in mice
Use of a spectrophotometric hemoglobin assay to objectively quantify intracerebral hemorrhage in mice
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DOI：
10.1161/01.str.28.11.2296
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发表时间：
1997-11-01
期刊：
影响因子：
8.3
通讯作者：
Pinsky, DJ
中科院分区：
文献类型：
--
作者：
Choudhri, TF;Hoh, BL;Pinsky, DJ
背景与目的
开发新型抗凝或溶栓策略治疗缺血性脑卒中备受关注。然而，目前准确评估这些药物出血可能性的方法有限。本研究旨在开发并验证一种准确量化小鼠模型中脑出血（ICH）程度的方法。
方法
在小鼠模型中，通过立体定向脑实质内单独输注胶原酶B（6×10⁻⁶ U；n = 5）或先输注胶原酶B然后静脉注射重组组织型纤溶酶原激活剂（rt - PA）（0.1 mg/kg；n = 6）来诱导脑出血。对照组包括立体定向输注生理盐水的假手术组（n = 5）或未处理的动物组（n = 5）。脑出血（1）通过基于冠状切片上最大出血直径的量表进行分级，（2）通过分光光度法测定，该方法测量匀浆小鼠脑化学还原提取物中的氰化高铁血红蛋白。使用已知量的血红蛋白或自体血液添加到另一组匀浆大脑中对该分光光度法进行了验证。利用该方法，对大脑中动脉局灶性闭塞/再灌注后接受闭塞后高剂量静脉注射rt - PA（10 mg/kg；n = 11）的小鼠以及发生脑卒中但用生理盐水处理的对照小鼠的出血程度进行了量化。
结果
添加到新鲜全脑组织匀浆中的已知量的血红蛋白或自体血液在添加量与氰化高铁血红蛋白吸收峰处的光密度（OD）之间呈线性关系（分别为r = 1.00和0.98）。当进行体内研究以量化实验诱导的脑出血时，接受脑内输注胶原酶B的动物的光密度显著高于输注生理盐水的对照组（增加2.1倍；P = 0.05）。在大脑中动脉闭塞和再灌注的脑卒中模型中，再灌注后给予rt - PA与接受生理盐水的动物相比，光密度增加了1.8倍（P
Background and Purpose There is great interest in developing novel anticoagulant or thrombolytic strategies to treat ischemic stroke. However, at present there are limited means to accurately assess the hemorrhagic potential of these agents. The present studies were designed to develop and validate a method to accurately quantify the degree of intracerebral hemorrhage (ICH) in murine models.Methods In a murine model, ICH was induced by stereotaxic intraparenchymal infusion of collagenase B alone (6x10(-6) U; n=5) or collagenase B followed by intravenous recombinant tissue plasminogen activator (rt-PA) (0.1 mg/kg; n=6). Controls consisted of either sham surgery with stereotaxic infusion of saline (n=5) or untreated animals (n=5). ICH was (1) graded by a scale based on maximal hemorrhage diameter on coronal sections and (2) quantified by a spectrophotometric assay measuring cyanomethemoglobin in chemically reduced extracts of homogenized murine brain. This spectrophotometric assay was validated with the use of known quantities of hemoglobin or autologous blood added to a separate cohort of homogenized brains. With this assay, the degree of hemorrhage after focal middle cerebral artery occlusion/reperfusion was quantified in mice treated with postocclusion high-dose intravenous rt-PA (10 mg/kg; n=11) and control mice subjected to stroke but treated with physiological saline solution (n=9).Results Known quantities of hemoglobin or autologous blood added to fresh whole brain tissue homogenates showed a linear relationship between the amount added and optical density (OD) at the absorbance peak of cyanomethemoglobin (r=1.00 and .98, respectively). When in vivo studies were performed to quantify experimentally induced ICH, animals receiving intracerebral infusion of collagenase B had significantly higher ODs than saline-infused controls (2.1-fold increase; P=.05). In a middle cerebral artery occlusion and reperfusion model of stroke, administration of rt-PA after reperfusion increased the OD by 1.8-fold compared with animals that received physiological saline solution (P