We have developed a novel tissue banking protocol using mechanical thrombectomy to capture thrombus along with arterial blood proximal and distal to it. The protocol provides high-quality specimens, facilitating analysis of the initial molecular response to ischemic stroke in the human condition for the first time. This approach will permit reverse translation to animal models for treatment development.

Background: Older-age individuals are at the highest risk for disability from a traumatic brain injury (TBI). Astrocytes are the most numerous glia in the brain, necessary for brain function, yet there is little known about unique responses of astrocytes in the aged-brain following TBI. Methods: Our approach examined astrocytes in young adult, 4-month-old, versus aged, 18-month-old mice, at 1, 3, and 7 days post-TBI. We selected these time points to span the critical period in the transition from acute injury to presumably irreversible tissue damage and disability. Two approaches were used to define the astrocyte contribution to TBI by age interaction: (1) tissue histology and morphological phenotyping, and (2) transcriptomics on enriched astrocytes from the injured brain. Results: Aging was found to have a profound effect on the TBI-induced loss of astrocyte function needed for maintaining water transport and edema-namely, aquaporin-4. The aged brain also demonstrated a progressive exacerbation of astrogliosis as a function of time after injury. Moreover, clasmatodendrosis, an underrecognized astrogliopathy, was found to be significantly increased in the aged brain, but not in the young brain. As a function of TBI, we observed a transitory refraction in the number of these astrocytes, which rebounded by 7 days postinjury in the aged brain. Transcriptomic data demonstrated disproportionate changes in genes attributed to reactive astrocytes, inflammatory response, complement pathway, and synaptic support in aged mice following TBI compared to young mice. Additionally, our data highlight that TBI did not evoke a clear alignment with the previously defined "A1/A2" dichotomy of reactive astrogliosis. Conclusions: Overall, our findings point toward a progressive phenotype of aged astrocytes following TBI that we hypothesize to be maladaptive, shedding new insights into potentially modifiable astrocyte-specific mechanisms that may underlie increased fragility of the aged brain to trauma.

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AbstractBackgroundOlder-age individuals are at the highest risk for disability from a traumatic brain injury (TBI). Astrocytes are the most numerous glia in the brain, necessary for brain function, yet there is little known about unique responses of astrocytes in the aged-brain following TBI.MethodsOur approach examined astrocytes in young adult, 4-month-old, versus aged, 18-month-old mice, at 1, 3, and 7 days post-TBI. We selected these time points to span the critical period in the transition from acute injury to presumably irreversible tissue damage and disability. Two approaches were used to define the astrocyte contribution to TBI by age interaction: 1) tissue histology and morphological phenotyping, and 2) transcriptomics on enriched astrocytes from the injured brain.ResultsAging was found to have a profound effect on the TBI-induced loss of homeostatic astrocyte function needed for maintaining water transport and edema – namely, aquaporin-4. The loss of homoeostatic responses was coupled with a progressive exacerbation of astrogliosis in the aged brain as a function of time after injury. Moreover, clasmatodendrosis, an underrecognized astrogliopathy, was found to be significantly increased in the aged brain, but not in the young brain. As a function of TBI, we observed a transitory refraction in the number of these astrocytes, which rebounded by 7 days post-injury in the aged brain. The transcriptomics found disproportionate changes in genes attributed to reactive astrocytes, inflammatory response, complement pathway, and synaptic support in aged mice following TBI compared to young mice. Additionally, our data highlight that TBI did not evoke a clear alignment with previously defined “A1/A2” dichotomy of reactive astrogliosis.ConclusionsOverall, our findings point toward a progressive phenotype of aged astrocytes following TBI that we hypothesize to be maladaptive, shedding new insights into potentially modifiable astrocyte-specific mechanisms that may underlie increased fragility of the aged brain to trauma.

SummaryThe E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response – two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNAseq highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression, a disrupted TCA cycle, and are inherently pro-glycolytic, while spatial transcriptomics and MALDI mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism.

Delayed cerebral vasospasm is thought to be caused by factors released from a subarachnoid blood clot. Because vasospasm occurs several days after hemorrhage, we hypothesized that clotted blood releases vasoactive factors as it ages. Targeted proteomics identified histidine-rich glycoprotein (HRG) as a potentially vasoactive factor released within the first 72 hours of clot formation. In vitro studies revealed that HRG caused moderate (B30%) dilation of cannulated cerebral arterioles and proliferation of cerebrovascular endothelial cells. We conclude that HRG released from clotted blood, while unlikely to contribute to cerebral vasospasm, might provide important vasodilatory or angiogenic stimuli after hemorrhagic stroke.

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Apolipoprotein E4 (APOE4) is the strongest risk allele associated with the development of late onset Alzheimers disease (AD). Across the CNS, astrocytes are the predominant expressor of ApoE while also being critical mediators of neuroinflammation and cerebral metabolism. APOE4 has been consistently linked with dysfunctional inflammation and metabolic processes, yet insights into the molecular constituents driving these responses remain unclear. Utilizing complementary approaches across humanized APOE mice and isogenic human iPSC astrocytes, we demonstrate that ApoE4 alters the astrocyte immunometabolic response to pro-inflammatory stimuli. Our findings demonstrate that ApoE4-expressing astrocytes acquire distinct transcriptional repertoires at single-cell and spatially-resolved domains, which are driven in-part by preferential utilization of the cRel transcription factor. Further, inhibiting cRel translocation in ApoE4 astrocytes abrogates inflammatory-induced glycolytic shift and in tandem mitigates production of multiple pro-inflammatory cytokines. Altogether, our findings elucidate novel cellular underpinnings by which ApoE4 drives maladaptive immunometabolic responses of astrocytes.