Washout of heme-containing proteins dramatically improves tetrazolium-based infarct staining
Washout of heme-containing proteins dramatically improves tetrazolium-based infarct staining
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DOI：
10.1016/j.vascn.2006.06.005
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发表时间：
2007-03-01
影响因子：
1.9
通讯作者：
Toombs, Christopher F.
中科院分区：
文献类型：
--
作者：
Pitts, Kelly R.;Stiko, Ann;Toombs, Christopher F.
简介：缺血-再灌注损伤后确定梗死面积的方法包括氯化三苯基四唑（TTC）大体染色和灌注有色染料以划分非缺血区。切除的组织（INF）通常会呈现斑驳的棕褐色至褐色，使得INF和TTC阳性组织之间的边界难以辨别。我们实验室以前的工作表明，TTC染色后，长时间清洗厚切片会使该边界急剧变尖。方法：成年大鼠结扎左前降支（LAD），缺血30 min，再灌注/恢复24 h。然后收获心脏，厚切片，并用TTC染色。将染色的切片在PBS中在4 ° C下储存长达3周。结果如下：在收获后直接固定的梗死心脏的薄切片上的组织学显示INK内广泛出血，然而，当心脏在PBS中储存3周时，这种出血被冲洗掉。在第1、2和3周采集的PBS样品的SDS-PAGE显示随着时间的推移出现低分子量条带。肽序列分析显示存在几种蛋白质，包括含血红素蛋白（HCP）血红蛋白、细胞色素c和肌红蛋白。从厚切片到PBS的HCP损失对应于每个切片内先前斑驳的INF的漂白。这些样品的HPLC分析证实HCP的损失有助于INF变白。此外，对有或无HCP的心脏切片得出的梗死面积值的分析显示，当值来自无HCP的切片时，测量误差显著降低。讨论内容：这些数据表明，心脏组织中的HCP有助于INF的不均匀和变色外观，并且这些蛋白质的洗脱产生更容易与相邻的非梗死组织区分的INF。这种方法大大减少了与梗塞测量相关的误差，并改善了对药物治疗和其他旨在影响缺血再灌注损伤的干预措施的影响的分析。(c)2006年爱思唯尔公司All rights reserved.
Introduction: Methods to determine infarct size following ischemia-reperfusion injury include gross staining with triphenyltetrazolium chloride (TTC) and perfusion of colored dyes to demarcate the non-ischemic zone. Infarcted tissue (INF) can typically appear a mottled tan to brownish color, making a border between INF and TTC-positive tissue difficult to discern. Previous work in our lab indicated that following TTC staining, prolonged washing of thick sections dramatically sharpened this boundary. Methods: Adult rats underwent 30 min ischemia via LAD ligation and reperfusion/recovery over 24 h. Hearts were then harvested, thick-sectioned, and stained with TTC. Stained sections were stored in PBS at 4 degrees C for up to 3 weeks. Results: Histology on thin sections from infarcted hearts fixed directly after harvest revealed extensive hemorrhage within the INK However, this hemorrhage is washed out when hearts are stored in PBS for 3 weeks. SDS-PAGE of PBS samples taken at 1, 2, and 3 weeks showed a low molecular weight band appearing over time. Peptide sequencing revealed the presence of several proteins including the heme-containing proteins (HCPs) hemoglobin, cytochrome c, and myoglobin. The loss of HCPs from thick sections to PBS corresponded with the blanching of the previously mottled INF within each section. HPLC analysis of these samples confirmed the loss of HCPs contributes to INF whitening. Further, analysis of infarct size values derived from heart slices with or without HCPs showed a significant decrease in measurement error when values were derived from slices without HCPs. Discussion: These data suggest that HCPs in the heart tissue contribute to the non-uniform and discolored appearance of the INF, and that washout of these proteins produces an INF more easily distinguished from neighboring non-infarcted tissue. This method greatly reduces the error associated with infarct measurements and improves the analysis of the effects of drug treatments and other interventions designed to impact ischemia reperfusion injury. (c) 2006 Elsevier Inc. All rights reserved.