Biology subjects

Buckwalter, M. S.

Publications and source records attributed to Buckwalter, M. S..

10 recordsLinked to original sources

bioRxiv2026

Blood-brain barrier dysfunction predicts cognitive trajectory after ischemic stroke

Ischemic stroke doubles the risk of dementia.1-4 Stroke severity and location affect cognition early,5,6 but late dementia risk is not related to infarct characteristics, nor is it reduced by preventing additional strokes,3,6,7 and its mechanism is unknown. We identified a plasma proteomic signature of chronic stroke that was consistent with blood-brain barrier (BBB) dysfunction, including a 58% decrease in plasma levels of platelet-derived growth factor B and downregulation of its pathway compared to healthy controls. During 2 years of follow-up, the stroke-specific proteome was accentuated in stroke survivors who subsequently declined in the processing speed/executive function cognitive domain. To test BBB function, we performed dynamic contrast-enhanced MRI 6-9 months after stroke in an additional cohort and found 1.7-fold higher whole brain BBB leakage compared to controls. Finally, we compared autopsy tissue from people with infarcts and dementia at death to those with infarcts and no dementia. Those who died with dementia had dramatic loss of vascular mural cell coverage compared to those without dementia (median 0.7% vs. 27%). Thus, our proteomic, functional, and structural data implicate chronic BBB dysfunction in cognitive decline late after stroke, revealing potential proteomic and imaging biomarkers and, importantly, a novel target for intervention.

neuroscience↗

bioRxiv2026

ABERRANT HIPPOCAMPAL NEUROGENESIS IS A CONSERVED RESPONSE TO STROKE IN MICE: A MULTI-CENTER MULTIMODEL STUDY

BackgroundAdult hippocampal neurogenesis is markedly altered after cerebral ischemia. Although stroke increases the production of newborn neurons, many of these cells display aberrant morphological and positional features that impair their functional integration and contribute to long-term cognitive decline. Given the clinical heterogeneity of ischemic stroke and the persistent translational failures of preclinical approaches relying on single-model studies it remains unknown whether post-stroke neurogenic alterations are conserved across different experimental paradigms. This study aimed to define common and model-specific features of hippocampal neurogenesis across complementary focal ischemia models. MethodsWe performed a multi-center, multimodel analysis within the STROKE-IMPaCT consortium using permanent and transient middle cerebral artery occlusion (MCAO) paradigms (MCAO via ligation or cauterization under normoxic (dMCAO) or hypoxic conditions (dMCAO+Hypoxia); and filament-based tMCAO across six international sites. Brains from adult C57BL/6J mice were collected 3 days, 7 days, or 2 months after ischemia, sham, or naive conditions. Hippocampal cell proliferation (Ki67) and neuroblast density (DCX) were quantified, and the morphological maturation of newborn neurons was evaluated using high-resolution analyses of dendritic architecture and somatodendritic polarity. All analyses were performed blind to experimental group. ResultsAcross all stroke models, ischemia induced a robust bilateral increase in hippocampal cell proliferation, most pronounced at 3 days and still elevated at 7 days, with levels returning to baseline by 2 months. Neuroblast density was similarly increased at 7 days, particularly in the ipsilateral hippocampus, but normalized by 2 months. Despite recovery in cell number, long-term morphological analysis revealed a consistent reduction in apical dendrite length and a higher proportion of neurons exhibiting aberrant features including ectopic localization, multipolar or inverted polarity, and abnormal lateral growth across all models. These abnormalities were observed both when pooling data across sites and when analyzing each model or center individually. ConclusionsIschemia induces an early, transient increase in hippocampal neurogenesis across diverse stroke paradigms, but the newborn neurons generated after stroke consistently display maladaptive morphological features. These cross-model, cross-site abnormalities indicate that aberrant hippocampal neurogenesis represents a robust hallmark of post-stroke pathology within the investigated species, independent of ischemia type or surgical approach, despite known differences in the spatial distribution of primary injury across experimental stroke models. Our findings support the concept that maladaptive neurogenesis may contribute to chronic post-stroke cognitive impairment and underscore the need to consider the quality not only the quantity of newborn neurons when developing therapeutic strategies.

animal behavior and cognition↗

bioRxiv2025

Blockade of VCAM1 or VLA4 preserves cerebrovasculature and prevents cognitive decline late after stroke

Infarct-induced neurodegeneration occurs chronically after stroke, doubling the risk of dementia. Endothelial vascular cell adhesion molecule 1 (VCAM1) facilitates blood-brain barrier opening and immune cell diapedesis by binding very late antigen 4 (VLA4) on immune cells. We hypothesized that vascular dysfunction persists after stroke and contributes to chronic neuroinflammation and cognitive decline via signaling through the VLA4/VCAM1 axis. We used adult (3-5 month old) and middle-aged (10 month old) C57BL/6J male & female mice and a permanent middle cerebral artery occlusion stroke model. Sham surgery consisted of an identical procedure without occlusion of the artery. We quantified vascular integrity using blood vessel length, pericyte coverage of vasculature, tight junctions, and extravascular fibrinogen leakage by immunostaining. Cognitive testing was performed using both Barnes maze and novel object recognition prior to stroke, and 1 and 6 weeks after stroke, and replicated in both male and female mice. We utilized anti-VCAM1, anti-VLA4 or isotype control antibodies to block VCAM1 or VLA4 function, and then to confirm mechanisms we utilized single cell RNA sequencing on immune and endothelial cells, aptamer-based plasma proteomics, and additional immunostaining for vascular integrity. Mouse brains exhibited signs of persistent vascular dysfunction and loss of blood-brain barrier integrity at 8 weeks after stroke, compared to sham animals. We observed reduced ZO-1 tight junction and pericyte coverage of vasculature, and increased extravascular fibrinogen. Mice with stroke also developed a cognitive deficit in both Barnes maze and novel object by 6 weeks. Treatment with anti-VCAM1 or anti-VLA4 resulted in mice with stroke performing as well as sham mice treated with isotype control antibody on both the Barnes maze and novel object recognition tasks. Anti-VCAM1 and anti-VLA4 both increased expression of blood-brain barrier maintenance genes in brain endothelial cells, while only minimally altering immune cell gene expression. Immune cell infiltration was reduced by anti-VCAM1 but not anti-VLA4 in tissue sections. In contrast to this, both antibodies increased blood vessel length and pericyte vascular coverage. Finally, extravascular fibrinogen was reduced by both antibody treatments in multiple brain regions. Together, our findings establish the VLA4/VCAM1 axis as a promising target to preserve vascular integrity and prevent cognitive decline late after stroke. Our data is consistent with a model where blocking either VCAM1 or VLA4 chronically after stroke promotes new blood vessel growth and maturation and restores the blood-brain barrier to prevent infarct-induced neurodegeneration.

neuroscience↗

bioRxiv2025

Cell-surface proteomic profiling identifies CD72 as a regulator of microglial tiling

Microglial tiling--the phenomenon of consistent cell-to-cell distances and non-overlapping processes--is regarded as a qualitative indicator of homeostasis, but mechanisms of microglial tiling are unknown. We used cell-surface proximity labeling and mass spectrometry to profile the microglial cell-surface proteome in an in vitro model of homeostatic glial physiology and used single-cell RNA sequencing and public databases to identify candidate cell-surface proteins that might modulate tiling. We designed an image-based functional assay which measures six morphological/spatial readouts to screen these proteins for modulation of tiling. CD72, a coreceptor to the B cell receptor that is expressed by microglia, disrupted tiling; we validated its effects in vitro and in situ in organotypic hippocampal brain slices. Phosphoproteomic studies revealed that CD72 modulates pathways associated with cell adhesion, repulsive receptors, microglial activation, and cytoskeletal organization. These results lay the groundwork for further investigation of the functional roles of tiling in homeostasis and disease.

neuroscience↗

bioRxiv2025

Assessing post-stroke cognition in pre-clinical models: lessons and recommendations from a multi-center study

Cognitive decline is a significant long-term consequence of stroke and has no available treatments. To aid in therapy development, we sought to achieve robust detection of cognitive performance after stroke in a multi-site design. Ischemic stroke was induced in adult and middle-aged male C57BL/6J mice utilizing three well-established models: distal middle cerebral artery occlusion (dMCAO), dMCAO with hypoxia and transient MCAO. Cognitive outcomes were assessed via Novel Object Recognition (NOR) and Barnes Maze (BM) tests prior to surgery, and during sub-acute (1-2 weeks) and chronic (8 weeks) phases post-stroke. Histology and immunostaining were used to assess infarct size, tissue damage and neuronal loss, and plasma neurofilament light was quantified. We did not detect a reliable cognitive deficit after stroke using NOR but saw a promising signal from BM (single site tested only). Overall, our study highlights the often-encountered challenges in detecting post-stroke cognitive impairment within the pre-clinical stroke community, as well as a number of complexities in the design and execution of pre-clinical stroke cognition studies, particularly as applied to a multi-site structure. We provide recommendations and suggest important aspects of stroke cognition studies to consider in the future, whether operating as an individual lab or a multi-site group.

animal behavior and cognition↗

bioRxiv2025

Juvenile mice are susceptible to infarct-induced neurodegeneration that causes delayed cognitive decline in a new model of pediatric ischemic stroke

Over half of pediatric stroke survivors have permanent neurologic and cognitive deficits, and some children develop new or worsening cognitive impairment late after stroke. Their cognitive symptoms are associated with chronic alterations of brain structure in regions distant from the infarct, including in the uninjured hemisphere. However, the mechanisms that cause childrens late cognitive symptoms and disrupted brain growth outside the infarcted tissue remain unknown. We therefore developed and validated a mouse model of pediatric ischemic stroke that causes infarct-induced, delayed cognitive decline accompanied by chronic innate and adaptive neuroinflammation in the infarct and in uninjured subcortical regions that are connected to the infarct. MethodsMale and female C57BL/6J mice were randomized to stroke or sham surgery at p28 to model stroke in late childhood and compared to adult 6-month-old mice. We used permanent distal MCAO followed by 60 minutes of hypoxia, an established model for focal ischemia that generates a purely cortical stroke. Mice underwent behavioral testing at 1- and 7-weeks post stroke Barnes Maze protocol adapted for reversal learning. We performed immunostaining to quantify stroke size, atrophy, and innate and adaptive neuroinflammation at 3 days and 7 weeks after surgery. ResultsAt 7 weeks post-stroke, juvenile stroke mice performed significantly worse on reversal learning on Barnes Maze testing than sham mice (n = 7 stroke, 7 sham, p = 0.0265 2-way ANOVA with repeated measures). At 3 days after stroke, juvenile mice had greater innate immune cell activation at subcortical sites of secondary neurodegeneration; however, by 7 weeks after surgery, this relationship was reversed, and adults had greater chronic innate immune cell activation at these uninjured subcortical sites (corpus callosum, corticospinal tract, thalamus). Like adult mice, pediatric mice exhibited chronic innate and adaptive neuroimmunity in the infarct scar; the inflammation in the stroke scar did not differ by age. ConclusionsIn a model of pediatric ischemic stroke, juvenile mice develop a delayed cognitive deficit that is associated with chronic innate and adaptive neuroinflammation in uninjured subcortical structures that undergo secondary neurodegeneration. Our results suggest that infarct-induced neurodegeneration occurs after stroke in juvenile mice and that juvenile and adult mice have divergent trajectories in the innate immune response to stroke.

neuroscience↗

bioRxiv2024

TREM1 disrupts myeloid bioenergetics and cognitive function in aging and Alzheimer disease models

Human genetics implicate defective myeloid responses in the development of late onset, age-associated Alzheimers disease (AD). Aging is characterized by a decline in myeloid metabolism that triggers maladaptive, neurotoxic immune responses. TREM1 is an amplifier of pro-inflammatory myeloid responses, and here we find that Trem1 deficiency prevents age-dependent changes in myeloid metabolism, inflammation, and hippocampal memory function. Trem1 deficiency rescues age-associated declines in ribose-5P, a glycolytic intermediate and the precursor for purine, pyrimidine, and NAD+ biosynthesis. In vitro, Trem1 deficient microglia are resistant to bioenergetic changes induced by amyloid-{beta}42 oligomers (A{beta}42), suggesting that A{beta}42 stimulation disrupts homeostatic microglial metabolism and immune function via TREM1. In the 5XFAD model of amyloid accumulation, Trem1 haploinsufficiency prevents spatial memory loss, preserves homeostatic microglial morphology, and reduces neuritic dystrophy independent of amyloid accumulation or changes in the disease-associated microglial transcriptomic signature. In aging APPSwe mice, Trem1 deficiency restores synaptic mitochondrial function and cerebral glucose uptake and prevents hippocampal memory decline. In post-mortem human brain, microglial TREM1 expression increases with clinical and neuropathological severity. Thus, TREM1-mediated disruption of myeloid metabolism, both in the periphery and brain, promotes cognitive decline in aging and amyloid accumulation, two major risk factors for AD development.

neuroscience↗

bioRxiv2023

Blocking Formation of Neurotoxic Reactive Astrocytes is Beneficial Following Stroke

Microglia and astrocytes play an important role in the neuroinflammatory response and contribute to both the destruction of neighboring tissue as well as the resolution of inflammation following stroke. These reactive glial cells are highly heterogeneous at both the transcriptomic and functional level. Depending upon the stimulus, microglia and astrocytes mount a complex, and specific response composed of distinct microglial and astrocyte substates. These substates ultimately drive the landscape of the initiation and recovery from the adverse stimulus. In one state, inflammation- and damage-induced microglia release tumor necrosis factor (TNF), interleukin 1 (IL1), and complement component 1q (C1q), together TIC. This cocktail of cytokines drives astrocytes into a neurotoxic reactive astrocyte (nRA) substate. This nRA substate is associated with loss of many physiological astrocyte functions (e.g., synapse formation and maturation, phagocytosis, among others), as well as a gain-of-function release of neurotoxic long-chain fatty acids which kill neighboring cells. Here we report that transgenic removal of TIC led to reduction of gliosis, infarct expansion, and worsened functional deficits in the acute and delayed stages following stroke. Our results suggest that TIC cytokines, and likely nRAs play an important role that may maintain neuroinflammation and inhibit functional motor recovery after ischemic stroke. This is the first report that this paradigm is relevant in stroke and that therapies against nRAs may be a novel means to treat patients. Since nRAs are evolutionarily conserved from rodents to humans and present in multiple neurodegenerative diseases and injuries, further identification of mechanistic role of nRAs will lead to a better understanding of the neuroinflammatory response and the development of new therapies.

neuroscience↗

bioRxiv2023

Translatome analysis reveals microglia and astrocytes to be distinct regulators of inflammation in the hyperacute and acute phases after stroke

Neuroinflammation is a hallmark of ischemic stroke, which is a leading cause of death and long-term disability. Understanding the exact cellular signaling pathways that initiate and propagate neuroinflammation after stroke will be critical for developing immunomodulatory stroke therapies. In particular, the precise mechanisms of inflammatory signaling in the clinically relevant hyperacute period, hours after stroke, have not been elucidated. We used the RiboTag technique to obtain astrocyte and microglia-derived mRNA transcripts in a hyperacute (4 hours) and acute (3 days) period after stroke, as these two cell types are key modulators of acute neuroinflammation. Microglia initiated a rapid response to stroke at 4 hours by adopting an inflammatory profile associated with the recruitment of immune cells. The hyperacute astrocyte profile was marked by stress response genes and transcription factors, such as Fos and Jun, involved in pro-inflammatory pathways such as TNF-. By 3 days, microglia shift to a proliferative state and astrocytes strengthen their inflammatory response. The astrocyte pro-inflammatory response at 3 days is partially driven by the upregulation of the transcription factors C/EBP{beta}, Spi1, and Rel, which comprise 25% of upregulated transcription factor-target interactions. Surprisingly, few sex differences across all groups were observed. Expression and log2 fold data for all sequenced genes are available on a user-friendly website for researchers to examine gene changes and generate hypotheses for stroke targets. Taken together our data comprehensively describe the astrocyte and microglia-specific translatome response in the hyperacute and acute period after stroke and identify pathways critical for initiating neuroinflammation.

neuroscience↗

bioRxiv2022

IPSILESIONAL HIPPOCAMPAL GABA CORRELATES WITH COGNITIVE IMPAIRMENT AND MALADAPTIVE NEUROGENESIS AFTER STROKE IN MICE

BackgroundCognitive dysfunction is a frequent stroke sequela but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodelling has been proposed but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. MethodsStroke was induced by permanent occlusion of the middle cerebral artery (MCAO) in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analysed longitudinally by in vivo magnetic resonance spectroscopy (MRS). Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts and interneurons were analysed by immunofluorescence. ResultsApproximately 50% of mice exhibited progressive post-MCAO cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the non-impaired group. Using MRS, significant bilateral changes in hippocampal metabolites such as myo-Inositol (mIns) or N-acetylaspartic acid (NAA) were found that correlated, respectively, with numbers of glia and immature neuroblasts in the ischemic group. Importantly, some metabolites were specifically altered in the ipsilateral hippocampus suggesting its involvement in aberrant neurogenesis and remodelling processes. Specifically, MCAO animals with higher hippocampal GABA levels displayed worse cognitive outcome. Implication of GABA in this setting was supported by the amelioration of ischemia-induced memory deficits and aberrant hippocampal neurogenesis after blocking pharmacologically GABAergic neurotransmission. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. ConclusionsHippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for post-stroke cognitive impairment.

animal behavior and cognition↗