Comparison of different quantification methods to determine hippocampal damage after cerebral ischemia

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.

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.

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.

Placement of counting windows has a major influence on extent of differences between degree of neuronal injury and variations within groups.

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.