Abstract：
BACKGROUND:Experimental stroke studies use multiple techniques to evaluate histopathological damage. Unfortunately, sensitivity and reproducibility of these techniques are poorly characterized despite pivotal influence on results. METHOD:The present study compared several quantification methods to differentiate between two severities of global cerebral ischemia and reperfusion. Male Sprague-Dawley rats were randomized to moderate (10min) or severe (14min) ischemia by bilateral carotid occlusion (BCAO) with hemorrhagic hypotension. Neuronal cell count was determined in hippocampus at bregma -3.14mm and -3.8mm on day 3 and 28 post insult by counting neurons in the whole CA1 or in one to three defined regions of interest (ROI) placed in NeuN and Fluoro-Jade B stained sections. RESULTS:In healthy rats hippocampal neurons were arranged uniformly, while distribution became inhomogeneous after ischemia. The number of NeuN and Fluoro-Jade B positive cells was dependent on localization. Differences between ischemia severities became more prominent at 28 days compared to 3 days. Fluoro-Jade B positive cell count increased at 28 days, staining rather injured not dying neurons. COMPARISON WITH EXISTING METHODS:Placement of counting windows has a major influence on extent of differences between degree of neuronal injury and variations within groups. CONCLUSIONS:The investigated quantification methods result in inconsistent information on the degree of damage. To obtain consistent and reliable results observation period should be extended beyond 3 days. Due to inhomogeneous distribution of viable neurons in CA1 after ischemia neuronal counting should not be performed in a single ROI window, but should be performed in multiple ROIs or the whole CA1 band.
journal_name
J Neurosci Methodsjournal_title
Journal of neuroscience methodsauthors
Wang J,Jahn-Eimermacher A,Brückner M,Werner C,Engelhard K,Thal SCdoi
10.1016/j.jneumeth.2014.11.001subject
Has Abstractpub_date
2015-01-30 00:00:00pages
67-76eissn
0165-0270issn
1872-678Xpii
S0165-0270(14)00393-8journal_volume
240pub_type
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