Helen M. Bramlett’s research while affiliated with University of Miami and other places

Traumatic brain injury (TBI) initiates a cascade of cellular and molecular events that promote acute and long-term patterns of neuronal, glial, vascular, and synaptic vulnerability leading to lasting neurological deficits. These complex responses lead to patterns of programmed cell death, diffuse axonal injury, increased blood-brain barrier disruption, neuroinflammation, and reactive gliosis, each a potential target for therapeutic interventions. Posttraumatic therapeutic hypothermia (TH) has been reported to be highly protective after brain and spinal cord injury and studies have investigated molecular mechanisms underlying mild hypothermic protection while commonly assessing heterogenous cell populations. In this study we conducted single-cell RNA sequencing (scRNA-seq) on cerebral cortical tissues after experimental TBI followed by a period of normothermia or hypothermia to comprehensively assess multiple cell type-specific transcriptional responses. C57BL/6 mice underwent moderate controlled cortical impact (CCI) injury or sham surgery and then placed under sustained normothermia (37⁰C) or hypothermia (33⁰C) for 2 h. After 24 h, cortical tissues including peri-contused regions were processed for scRNA-seq. Unbiased clustering revealed cellular heterogeneity among glial and immune cells at this subacute posttraumatic time point. The analysis also revealed vascular and immune subtypes associated with neovascularization and debris clearance, respectively. Compared to normothermic conditions, TH treatment altered the abundance of specific cell subtypes and induced reactive astrocyte-specific modulation of neurotropic factor gene expression. In addition, an increase in the proportion of endothelial tip cells in the hypothermic TBI group was documented compared to normothermia. These data emphasize the importance of early temperature-sensitive glial and vascular cell processes in producing potentially neuroprotective downstream signaling cascades in a cell-type-dependent manner. The use of scRNA-seq to address cell type-specific mechanisms underlying therapeutic treatments provides a valuable resource for identifying targetable biological pathways for the development of neuroprotective and reparative interventions.

Traumatic brain injury (TBI) pathology is significantly mediated by an inflammatory response involving inflammasome activation, resulting in the release of interleukin (IL)-1β and pyroptotic cell death through gasdermin-D (GSDMD) cleavage. Inflammasome components are transported through extracellular vesicles (EVs) to mediate systemic inflammation in peripheral organs, including the gut. The purpose of this study was to determine the protective effect of GSDMD knockout (KO) on TBI-induced inflammasome activation, EV signaling, and gut function. GSDMD-KO and C57BL6 (WT) mice were subjected to the controlled cortical impact model of TBI. Cytokine expression was assessed with electrochemiluminescent immunoassay and immunoblotting of the cerebral cortex and gut. EVs were examined for pathology-associated markers using flow cytometry, and gut permeability was determined. GSDMD-KO attenuated IL-1β and IL-6 expression in the cerebral cortex and reduced IL-1β and IL-18 in the gut 3 days post-injury. GSDMD-KO mice had decreased neuronal- and gut-derived EVs compared to WT mice post-TBI. GSDMD-KO EVs also had decreased IL-1β and different surface marker expression post-TBI. GSDMD-KO mice had decreased gut permeability after TBI. These data demonstrate that GSDMD ablation improves post-TBI inflammation and gut pathology, suggesting that GSDMD may serve as a potential therapeutic target for the improvement of TBI-associated pathologies.

Traumatic brain injury (TBI) triggers a series of pathophysiological events, contributing significantly to secondary injury and long-term functional deficits. While exosome therapy is beginning to emerge as a promising avenue for various injuries, its efficacy in TBI, using preclinical models that mimic the biomechanics of human acceleration/deceleration TBI, remains largely unexplored. This study investigated the capacity of human Schwann cell-derived exosomes (hSC-Exo) to improve outcomes in a model of moderate fluid percussion injury (FPI). We found that jugular infusion of hSC-Exo 30 min after trauma attenuated acute proinflammatory responses in the ipsilateral cortex and hippocampus 24 h post-TBI, as demonstrated by a reduction in levels of key inflammasome components, and decreased activation of the STAT3/pSTAT3/SOCS3 pathway. Furthermore, exosome treatment mitigated subacute histopathological changes, including a significant decrease in cerebral edema and contusion volumes at 72 h post-injury. Immunohistochemical analysis revealed a decrease in microglial activation, characterized by a shift toward a more ramified morphology. Importantly, hSC-exosome therapy led to the preservation of both sensorimotor function subacutely and cognitive performance at chronic time points. Flow cytometry analysis of peripheral blood at 21 days post-TBI demonstrated a reduction in circulating neutrophils, indicating an attenuation of chronic systemic inflammation. These findings highlight the multifaceted therapeutic benefits of hSC-Exo in a clinically-relevant FPI model, targeting both acute and chronic neuroinflammatory processes to promote functional recovery. This study provides new evidence to support hSC-exosomes as a therapeutic strategy for TBI, and emphasizes the translational potential of human exosomes for treating acute and progressive neurological injury.

Supplemental Materials: Human Schwann cell exosome treatment attenuates secondary injury mechanisms, histopathological consequences, and behavioral deficits after traumatic brain injury

Introduction: Rehabilitative physical therapy is essential for reducing stroke-related functional deficits; however, comorbidities may limit patient participation. Whole body vibration (WBV; 40 Hz) offers an exercise-like alternative. Our studies show that one month of post-stroke WBV reduces ischemic damage and improves motor and cognitive function in middle-aged rats. Notably, WBV significantly increased circulating irisin, a muscle-derived hormone. We hypothesize that post-stroke WBV modifies the cerebral transcriptome and irisin treatment improves stroke outcome in middle-aged female rats. Methods: Middle-aged Sprague-Dawley rats were randomized to sham or transient middle cerebral artery occlusion (tMCAO; 90 min) surgery and divided into two cohorts. A cohort received either no-WBV (steady platform) or WBV (platform vibrating at 40 Hz) for 15 minutes twice a day for a week. Cortical tissue was then collected for RNA sequencing (RNAseq) and gene enrichment analysis. The second cohort received either saline or irisin (PeproTech, 0.2 µg/g BW) treatment at 4.5 hours post-tMCAO and then once a week for a month. At 21 days post-tMCAO, rats were assessed for cognitive deficits via the Morris water maze. At 1-month post-tMCAO, brains were collected for histological analysis. Results: RNAseq revealed significant (p<0.05) differential expression of 581 genes in the cortex due to WBV. Specifically, there was downregulation (log 2 (fold change) = -2.9; p= 0.03943) of Calcium Voltage-Gated Channel Auxiliary Subunit Gamma 7 ( Cacng7 ) and calcium-release channel activity (GO:0015278), which are both implicated in excitotoxicity following stroke. The expression of Vacuolar Protein Sorting-Associated Protein 37C ( Vps37c ), a component of an endosomal sorting complex that mediates apoptosis, was also downregulated (log 2 (fold change) = -7.1; p= 0.01888). In our second cohort, rats treated with irisin for 1-month post-tMCAO, as compared to saline, demonstrated significantly (p<0.05) reduced cognitive deficits in spatial learning (hidden platform) and memory (probe trial). Additionally, irisin treatment significantly (p<0.05) reduced infarct damage at 1-month post-tMCAO. Conclusion: Post-stroke WBV increases the expression of genes responsible for ischemic tolerance and irisin treatment improves cognitive outcomes in middle-aged rats. Future studies are needed to understand the underlying mechanism(s) responsible for irisin-conferred ischemic protection.

Background Stroke and AD patients with gut complications often present worsened neurological outcomes. The goal of this study was to examine the role of extracellular vesicle (EV)‐mediated pyroptosis in the bi‐directional gut‐brain axis after photothrombotic stroke (PTS) in aged 3xTg mice and wildtype (WT) controls. Method Twelve‐month 3xTg and WT male and female mice underwent PTS using a YAG laser. Lesion volume analysis was performed at 1‐month post‐PTS. At 24 hours after PTS, intestinal and cortical tissue was collected for Western Blot analysis for the following inflammasome proteins: caspase‐1, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), interleukin‐1b, and Gasdermin‐D (GSDMD). Additionally, brain and intestines were sectioned for immunohistochemical analysis of GSDMD and Amyloid‐Beta (Ab). In order to show changes in gut function, gut permeability was measured 72 hours post PTS using a FITC‐dextran assay. Moreover, we performed an adoptive transfer in which stool‐derived EVs collected from 3xTg‐ PTS and WT‐PTS mice were injected into naïve 3xTg and WT mice. Cortical lysates were examined for expression of inflammasome proteins, GSDMD, and Ab using Western Blot analysis. Results 3xTg mice presented increased infarct volume compared to WT‐PTS mice one month after PTS. Based on Western blot analysis, we found that inflammasome proteins, GSDMD, and Ab were significantly increased in both intestinal and cortical tissue in 3xTg‐ PTS mice compared to WT‐ PTS mice at 24 hours post‐stroke. Results from the FITC‐Dextran assay showed that gut permeability was significantly increased in the aged 3xTg‐PTS mice compared to WT‐ PTS mice 72 hours post‐PTS. Additionally, our immunohistochemical analysis in both WT and 3xTg PTS mice presented evidence of activated/ameboid microglial morphology as well as the presence of GSDMD and Ab in the brain and intestines 72 hours after stroke. Lastly, we demonstrated increased levels of inflammasome proteins, GSDMD, and Ab after adoptive transfer. Conclusion Taken together these results indicate an important role for EV signaling and pyroptosis in disruption of the bidirectional gut‐brain axis after stroke in aged 3xTg mice in an animal model of stroke.

Background Neurogranin (Ng) is considered a biomarker for synaptic dysfunction in Alzheimer’s disease (AD). In contrast, the inflammasome complex has been shown to exacerbate AD pathology. Method We investigated the protein expression, morphological differences of Ng and correlated Ng to hyperphosphorylated tau in the postmortem brains of 17 AD cases and 17 age and sex‐matched controls. In addition, we correlated the Ng expression with two different epitopes of apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC). Result We show a reduction of Ng immunopositive neurons and morphological differences in AD compared to controls. Ng immunostaining was negatively correlated with neurofibrillary tangles, humanized anti‐ASC (IC100) positive neurons and anti‐ ASC positive microglia, in AD. Conclusion The finding of a negative correlation between Ng and ASC speck protein expression in postmortem brains of AD suggests that the activation of inflammasome/ASC speck pathway may play an important role in synaptic degeneration in AD.

Background The global ageing population is rising with each year, and with that, the percentage of individuals with Alzheimer’s disease (AD) is expected to rise in parallel. Along with age, traumatic brain injury (TBI) is another risk factor for AD. TBI and AD patients demonstrate abnormal inflammatory responses, including that of the inflammasome. The inflammasome is a multi‐protein complex of the innate immune system, that leads to the release of the pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐18 through caspase‐1 and apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC). Pyroptosis, a form of inflammatory cell death, also occurs via gasdermin‐D cleavage by caspase‐1. Our laboratory has previously demonstrated that genetic predisposition to AD can worsen acute inflammasome activation and outcome after TBI, but the chronic effects of TBI with AD have not been studied. Method The 3xTg model of early‐onset familial AD was used, along with their B6129SF2 (WT) controls. AD and WT mice underwent either sham surgery or controlled cortical impact (CCI) and were sacrificed 3‐months post‐injury. Cortical lysates were collected and probed for inflammasome proteins and pro‐inflammatory cytokines via immunoblotting and ECLIAs. Brain sections were utilized for volumetric analysis and immunohistochemistry for inflammasome proteins and degenerative markers. Result There was a significant increase in chronic IL‐1β in AD/TBI mice that was not present in WT/TBI mice. Furthermore, there were also increased levels of the inflammasome proteins NLRP3, caspase‐8, and ASC at 3‐months post‐injury in AD/TBI vs WT/TBI mice. AD/TBI demonstrated elevated expression of GFAP, a marker of astrogliosis, which co‐localized with ASC in immunohistochemical analyses. Injured mice showed increased neurofilament light protein, which also co‐localized with ASC. Finally, AD/TBI mice had significant loss of total cortical and hippocampal volume compared to WT/TBI mice. Conclusion Our findings establish a chronic dysfunctional inflammatory response in TBI with a genetic predisposition to AD that is not seen in TBI alone. This suggests a unique injury progression that is affected by genetic predisposition to AD that calls for specialized treatment. In addition, due to the increased inflammasome response seen in our results, our conclusions indicate a promising role for the inflammasome as a treatment target.

We previously reported an ability of low-intensity vibration (LIV) to improve selected biomarkers of bone turnover and gene expression and reduce osteoclastogenesis but lacking of evident bone accrual. In this study, we demonstrate that a prolonged course of LIV that initiated at 2 weeks post-injury and continued for 8 weeks can protect against bone loss after SCI in rats. LIV stimulates bone formation and improves osteoblast differentiation potential of bone marrow stromal stem cells while inhibiting osteoclast differentiation potential of marrow hematopoietic progenitors to reduce bone resorption. We further demonstrate that the combination of LIV and RANKL antibody reduces SCI-related bone loss more than each intervention alone. Our findings that LIV is efficacious in maintaining sublesional bone mass suggests that such physical-based intervention approach would be a noninvasive, simple, inexpensive and practical intervention to treat bone loss after SCI. Because the combined administration of LIV and RANKL inhibition better preserved sublesional bone after SCI than either intervention alone, this work provides the impetus for the development of future clinical protocols based on the potential greater therapeutic efficacy of combining non-pharmacological (e.g., LIV) and pharmacological (e.g., RANKL inhibitor or other agents) approaches to treat osteoporosis after SCI or other conditions associated with severe immobilization.