Basic NeuroscienceComparison of different quantification methods to determine hippocampal damage after cerebral ischemia

- The number of NeuN and Fluoro-Jade B positive cells in CA1 is dependent on localization within CA1.
- Mean cell number does not change significantly over time from day 3 to 28.
- Variations within groups often become smaller resulting in lower SD and higher effect size at 28 days post insult.
- Number of Fluoro-Jade B positive cells does not decrease over time between 3 and 28 days post insult, suggesting that this staining is not labeling dying cells.
- Placement of counting windows has a major influence on extent of differences between moderate and severe ischemic brain damage.

BackgroundExperimental stroke studies use multiple techniques to evaluate histopathological damage. Unfortunately, sensitivity and reproducibility of these techniques are poorly characterized despite pivotal influence on results.MethodThe 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 (10 min) or severe (14 min) ischemia by bilateral carotid occlusion (BCAO) with hemorrhagic hypotension. Neuronal cell count was determined in hippocampus at bregma −3.14 mm and −3.8 mm 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.ResultsIn 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 methodsPlacement of counting windows has a major influence on extent of differences between degree of neuronal injury and variations within groups.ConclusionsThe 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.