Introduction: Small vessel ischemic strokes account for 25% of strokes in the US. They often occur silently, increasing the prevalence 5-10 fold and are progressive with new strokes occurring adjacent to prior strokes. In this common form of stroke, there is a local injury damaging axons and white matter, and a distant injury damaging the neurons with axons affected by the stroke, leading to cortical thinning in the connected cortex. This selective neuronal loss contributes to minor stroke related cognitive dysfunction and disability yet the molecular response of neurons with stroke-injured axons remains challenging to study.
Hypothesis: White matter stroke injures cortical projection neurons and triggers a unique molecular program that contributes to selective neuronal loss.
Methods: To determine the neuronal effects of a white matter stroke, we produced a subcortical white matter stroke below the forelimb motor cortex in adult male C57/Bl6 mice resulting in a focal white matter lesion. Retrograde neuronal tracing identifies individual neurons damaged by the white matter stroke. Layer 5 cortical neurons were isolated by magnetic microbead separation of non-neuronal cells, followed by fluorescent-activated cell sorting (FACS) isolation of retrogradely-labeled cells.
Results: Stereologic measurement of the neurons with stroke-injured axons co-labeled with the Layer 5 neuronal marker CTIP-2 reveals that focal white matter stroke selectively identifies between 15-25% of the Layer 5 cortical neurons in both sensory and motor cortex with spanning the cortical regions of interest, compared to only ∼3% in sham injured animals. Using FACS isolation, we compared the transcriptional profile of white matter stroke injured cortical projection neurons to uninjured Layer 5 neurons at one week after stroke. An average of 6,297 cells were collected per isolation, RNA isolated and analyzed by qPCR and RNA-seq.
Conclusions: Bioinformatic analysis of differentially expressed genes indicates that white matter stroke activates both degenerative and regenerative pathways in stroke-induced axonally-injured neurons. These data can be harnessed to prevent selective neuronal loss after white matter stroke and induce neural repair after stroke.