Simultaneous flow cytometric characterization of multiple cell types and metabolic states in the rat brain after repeated mild traumatic brain injury

Abstract

BackgroundCellular responses at the sub-acute phase of mild traumatic brain injury (mTBI), and their contribution to ongoing damage, are unclear, complex and require simultaneous assessment of multiple cells to elucidate. New methodAn 11-colour flow cytometry method for analysing brain cells was evaluated in a weight-drop rat model of repeated mTBI. Animals received sham, one, two or three mTBI delivered at 24 h intervals (n = 6/group). Cerebrum homogenates were prepared 11 days after first mTBI, in two cohorts of n = 3/group to enable same-day staining of fresh tissue. Percentages of neurons, astrocytes, microglia, mature oligodendrocytes and NeuN + CC1+ cells, neutrophils, macrophages and non-myeloid leukocytes, and their immunoreactivity for cell damage indicators (inducible nitric oxide synthase; iNOS, proliferating cell nuclear antigen; PCNA, 8-Oxo-2′-deoxyguanosine; 8OHDG and 4-hydroxynonenal; HNE), were assessed. ResultsMedian fluorescence intensity (MFI) of iNOS in activated microglia increased following two, but not one or three, mTBI (p = 0.04). However, there were differences between processing cohorts in terms of percentages and MFI of some PCNA+, iNOS+, 8OHDG + and HNE + cell populations. Comparison with existing methodsPrevious applications of flow cytometry for rat brain analysis were typically limited to three or four markers. This method uses 11 markers to identify nine cell populations and evaluate their immunoreactivity to four metabolic indicators of cell damage. ConclusionsFlow cytometry can be useful for discerning injury-related changes in multiple rat brain cells. However, markers sensitive to subtle changes in experimental conditions must be identified in pilot experiments and subsequently analysed in the same tissue-processing cohort.