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.
Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.
Ischemic injury to white matter tracts is increasingly recognized to play a key role in age-related cognitive decline, vascular dementia, and Alzheimer's disease. Knowledge of the effects of ischemic axonal injury on cortical neurons is limited yet critical to identifying molecular pathways that link neurodegeneration and ischemia. Using a mouse model of subcortical white matter ischemic injury coupled with retrograde neuronal tracing, we employed magnetic affinity cell sorting with fluorescence-activated cell sorting to capture layer-specific cortical neurons and performed RNA-sequencing. With this approach, we identified a role for microtubule reorganization within stroke-injured neurons acting through the regulation of tau. We find that subcortical stroke-injured Layer 5 cortical neurons up-regulate the microtubule affinity-regulating kinase, Mark4, in response to axonal injury. Stroke-induced up-regulation of Mark4 is associated with selective remodeling of the apical dendrite after stroke and the phosphorylation of tau in vivo. In a cell-based tau biosensor assay, Mark4 promotes the aggregation of human tau in vitro. Increased expression of Mark4 after ischemic axonal injury in deep layer cortical neurons provides new evidence for synergism between axonal and neurodegenerative pathologies by priming of tau phosphorylation and aggregation.
Introduction: Microvascular stroke and Alzheimer’s disease (AD) account for the majority of dementia diagnoses with 50% of patients having mixed dementia with features of both microvascular stroke in white matter and AD pathology. Clinico-pathologic studies indicate that white matter hyperintensities present on magnetic resonance imaging correlate with the degree of AD pathology in patients supporting pathologic overlap. This significant co-morbidity indicates an interactive neurobiologic relationship yet it is unclear if these two pathologies synergize or simply co-exist. Prior studies to model stroke and AD pathology have struggled to identify clinically relevant paradigms and time courses that could reasonably link the two disorders. Hypothesis: Subcortical white matter stroke synergizes with AD to worsen outcomes. Methods: We have established a clinically relevant model of mixed dementia by introducing subcortical white matter stroke into EFAD transgenic mice that harbor 5 common AD-associated mutations and knock-in human ApoE alleles under control of the murine ApoE promoter. Strokes were introduced either before or after significant amyloid plaque accumulation and the additive effect of stroke determined. Results: In E4FAD mice (with ApoE4 allele) that have already developed amyloid pathology (>6 months of age), white matter strokes are 70% larger in size (p=0.06) but not associated with worsening gliosis or impaired survival of oligodendrocytes in the peri-infarct region. The molecular organization of axons adjacent to stroke is markedly worse in mice with AD pathology. In younger E4FAD mice, the introduction of a white matter stroke prior to the significant accumulation of amyloid pathology results in worsening plaque burden both globally and in overlying cortex. Conclusions: These data suggest that AD and white matter stroke synergize to worsen both the outcome of stroke occurring in the setting of AD. Similarly early stroke synergizes with amyloid generation to accelerate plaque deposition. This clinically relevant model of mixed dementia will be useful in both preclinical drug disease for both AD and stroke therapies as well as crucial to the discovery of neurobiologic pathways that combine to promote stroke and AD pathologies.
Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.
