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.

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.

BackgroundVascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia. High levels of plasma homocysteine or hyperhomocysteinemia has been characterized as a risk factor for VCID however, the mechanism underlying the connection between hyperhomocysteinemia and development of VCID pathology remains elusive. I hypothesize that hyperhomocysteinemia initiates a pro‐inflammatory cascade that increases the activity of MMP9 causing both perivascular astrocytes to dissociate from their vessels and initiating the degradation of endothelial cell tight junction proteins, leading to blood brain barrier dysfunction and the progression toward VCID pathology.MethodFor in vivo studies, C57BL6 WT and MMP9KO mice were placed on a control diet or a diet deficient in folate, vitamins B6 and B12 and enriched in methionine to induce hyperhomocysteinemia for 4, 8, 12, and 16 weeks. For in vitro, experiments, primary astrocytes from WT and MMP9KO mice were treated with a 50μM homocysteine stimulus or an inflammatory stimulus (TNF‐a, IL‐1b and C1q) for 48 hours and the conditioned media was collected. This homocysteine or inflammatory astrocyte conditioned media was then used to treat WT endothelial cells. Immunohistochemistry and gene expression analysis were used to determine neuroinflammatory changes while histology was used to identify changes in astrocytic end‐feet proteins, tight junction proteins, vascular density and microhaemorrhages. Both gel and in situ zymography were used to assess proteinase activity of MMP9 and related gelatinases. Western blots were used to investigate substrates of MMP9 including claudin, occludin and β‐dystroglycan. Behaviour was assessed using rotarod, novel object recognition and spontaneous alternation testing. Transendothelial electrical resistance and sodium fluorescein assays were used to measure the integrity of endothelial cell tight junctions in vitro.ResultStudies are underway to examine changes in cognition, microhaemorrhages, neuroinflammation, astrocyte end‐foot integrity, tight junction protein integrity and activity of MMP9.ConclusionCollectively, our findings suggest that astrocytic MMP9 may play an integral role in the mechanism associating homocysteine induced neuroinflammation with vascular pathogenesis leading to VCID, highlighting this pathway as an important subject for future study.

BackgroundVascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia. Hyperhomocysteinemia has been identified as a risk factor for VCID though the mechanism behind this connection has yet to be determined. Hyperhomocysteinemia increases inflammation inducing the upregulation of matrix metalloproteinase 9 (MMP9) activity. MMP9 has been shown to degrade several substrates along the neurovascular unit including claudin 5, collagen 4, occludin and β‐dystroglycan. I hypothesize that increased MMP9 activity following the induction of hyperhomocysteinemia leads to the cleavage of important anchoring proteins along the neurovascular unit weakening the integrity of the blood brain barrier which contributes to the progression of VCID pathology.MethodC57BL6 WT and MMP9 knockout mice were placed on a control diet or a diet deficient in vitamins B6, B12 and folate and enriched in methionine to induce hyperhomocysteinemia for 4, 8, 12 and 16 weeks. Western blot analysis was performed to investigate the substrates of MMP9 including claudin, occludin, and β‐dystroglycan.ResultStudies are underway to examine if MMP9 activation contributes to blood brain barrier dysfunction directly by degrading tight junction proteins leading to a weakened endothelial cell barrier and/or indirectly by cleaving proteins responsible for maintaining the connection between astrocytic end feet and the endothelial basement membrane.ConclusionOur findings suggest that increased levels of MMP9 activity may impact several aspects of the neurovascular unit to contribute to VCID pathology. Further investigations focused on inhibition of MMP9 activation could provide the field with an important target for mitigating progression of this disease.