Buckwalter Lab Publications

Professor of Neurology and of Neurosurgery


  • The 2022 FASEB virtual Catalyst Conference on B Cells in Injury and Regeneration, March 30, 2022. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Dwyer, L. J., Stowe, A. M., Doyle, K., Popovich, P., Engler-Chiurazzi, E., LeGuern, C., Buckwalter, M. S., Poznansky, M. C., Sirbulescu, R. F. 2022; 36 (8): e22459

    View details for DOI 10.1096/fj.202201027

    View details for PubMedID 35857314

  • An RNA-sequencing transcriptome of the rodent Schwann cell response to peripheral nerve injury. Journal of neuroinflammation Brosius Lutz, A., Lucas, T. A., Carson, G. A., Caneda, C., Zhou, L., Barres, B. A., Buckwalter, M. S., Sloan, S. A. 2022; 19 (1): 105


    BACKGROUND: The important contribution of glia to mechanisms of injury and repair of the nervous system is increasingly recognized. In stark contrast to the central nervous system (CNS), the peripheral nervous system (PNS) has a remarkable capacity for regeneration after injury. Schwann cells are recognized as key contributors to PNS regeneration, but the molecular underpinnings of the Schwann cell response to injury and how they interact with the inflammatory response remain incompletely understood.METHODS: We completed bulk RNA-sequencing of Schwann cells purified acutely using immunopanning from the naive and injured rodent sciatic nerve at 3, 5, and 7days post-injury. We used qRT-PCR and in situ hybridization to assess cell purity and probe dataset integrity. Finally, we used bioinformatic analysis to probe Schwann cell-specific injury-induced modulation of cellular pathways.RESULTS: Our data confirm Schwann cell purity and validate RNAseq dataset integrity. Bioinformatic analysis identifies discrete modules of genes that follow distinct patterns of regulation in the 1st days after injury and their corresponding molecular pathways. These findings enable improved differentiation of myeloid and glial components of neuroinflammation after peripheral nerve injury and highlight novel molecular aspects of the Schwann cell injury response such as acute downregulation of the AGE/RAGE pathway and of secreted molecules Sparcl1 and Sema5a.CONCLUSIONS: We provide a helpful resource for further deciphering the Schwann cell injury response and a depth of transcriptional data that can complement the findings of recent single cell sequencing approaches. As more data become available on the response of CNS glia to injury, we anticipate that this dataset will provide a valuable platform for understanding key differences in the PNS and CNS glial responses to injury and for designing approaches to ameliorate CNS regeneration.

    View details for DOI 10.1186/s12974-022-02462-6

    View details for PubMedID 35501870

  • Immune Pathways in Etiology, Acute Phase, and Chronic Sequelae of Ischemic Stroke. Circulation research Endres, M., Moro, M. A., Nolte, C. H., Dames, C., Buckwalter, M. S., Meisel, A. 2022; 130 (8): 1167-1186


    Inflammation and immune mechanisms are crucially involved in the pathophysiology of the development, acute damage cascades, and chronic course after ischemic stroke. Atherosclerosis is an inflammatory disease, and, in addition to classical risk factors, maladaptive immune mechanisms lead to an increased risk of stroke. Accordingly, individuals with signs of inflammation or corresponding biomarkers have an increased risk of stroke. Anti-inflammatory drugs, such as IL (interleukin)-1beta blockers, methotrexate, or colchicine, represent attractive treatment strategies to prevent vascular events and stroke. Lately, the COVID-19 pandemic shows a clear association between SARS-CoV2 infections and increased risk of cerebrovascular events. Furthermore, mechanisms of both innate and adaptive immune systems influence cerebral damage cascades after ischemic stroke. Neutrophils, monocytes, and microglia, as well as T and B lymphocytes each play complex interdependent roles that synergize to remove dead tissue but also can cause bystander injury to intact brain cells and generate maladaptive chronic inflammation. Chronic systemic inflammation and comorbid infections may unfavorably influence both outcome after stroke and recurrence risk for further stroke. In addition, stroke triggers specific immune depression, which in turn can promote infections. Recent research is now increasingly addressing the question of the extent to which immune mechanisms may influence long-term outcome after stroke and, in particular, cause specific complications such as poststroke dementia or even poststroke depression.

    View details for DOI 10.1161/CIRCRESAHA.121.319994

    View details for PubMedID 35420915

  • Prognostication of ICU Patients by Providers with and without Neurocritical Care Training. Neurocritical care Finley Caulfield, A., Mlynash, M., Eyngorn, I., Lansberg, M. G., Afjei, A., Venkatasubramanian, C., Buckwalter, M. S., Hirsch, K. G. 2022


    BACKGROUND: Predictions of functional outcome in neurocritical care (NCC) patients impact care decisions. This study compared the predictive values (PVs) of good and poor functional outcome among health care providers with and without NCC training.METHODS: Consecutive patients who were intubated for≥72h with primary neurological illness or neurological complications were prospectively enrolled and followed for 6-month functional outcome. Medical intensive care unit (MICU) attendings, NCC attendings, residents (RES), and nurses (RN) predicted 6-month functional outcome on the modified Rankin scale (mRS). The primary objective was to compare these four groups' PVs of a good (mRS score 0-3) and a poor (mRS score 4-6) outcome prediction.RESULTS: Two hundred eighty-nine patients were enrolled. One hundred seventy-six had mRS scores predicted by a provider from each group and were included in the primary outcome analysis. At 6months, 54 (31%) patients had good outcome and 122 (69%) had poor outcome. Compared with other providers, NCC attendings expected better outcomes (p<0.001). Consequently, the PV of a poor outcome prediction by NCC attendings was higher (96%[95% confidence interval [CI] 89-99%]) than that by MICU attendings (88% [95% CI 80-93%]), RES (82% [95% CI 74-88%]), and RN (85% [95% CI 77-91%]) (p=0.047, 0.002, and 0.012, respectively). When patients who had withdrawal of life-sustaining therapy (n=67) were excluded, NCC attendings remained better at predicting poor outcome (NCC 90% [95% CI 75-97%] vs. MICU 73% [95% CI 59-84%], p=0.064). The PV of a good outcome prediction was similar among groups (MICU 65% [95% CI 52-76%], NCC 63% [95% CI 51-73%], RES 71% [95% CI 55-84%], and RN 64% [95% CI 50-76%]).CONCLUSIONS: Neurointensivists expected better outcomes than other providers and were better at predicting poor functional outcomes. The PV of a good outcome prediction was modest among all providers.

    View details for DOI 10.1007/s12028-022-01467-6

    View details for PubMedID 35314970

  • Self-report Does Not Align With Objective Assessments Of Memory And Fine Motor Functioning In Stroke Survivors Mendez, M. P., Drag, L. L., Mlynash, M., Musabbir, M., Kim, D. K., Lansberg, M. G., Buckwalter, M. S. LIPPINCOTT WILLIAMS & WILKINS. 2022
  • Depression And Not Cognitive Ability Is Most Strongly Associated With Long-term Functional Outcomes Following Stroke. Drag, L. L., Musabbir, M., Mlynash, M., Mendez, M. P., Kim, D. K., Aghaeepour, N., Lansberg, M. G., Buckwalter, M. S. LIPPINCOTT WILLIAMS & WILKINS. 2022
  • Higher White Blood Cell Count In The First Week After Stroke Predicts Worse Cognitive Outcomes In A Population With Smaller Ischemic Strokes Musabbir, M., Kim, D., Drag, L., Mlynash, M., Mendez, M., Lansberg, M. G., Smith, C. J., Buckwalter, M. S. LIPPINCOTT WILLIAMS & WILKINS. 2022
  • Targeting VCAM1 to reduce neuroinflammation in ischemia-triggered vascular dementia. Alzheimer's & dementia : the journal of the Alzheimer's Association Zera, K. A., Peterson, T., Yousef, H., Lee, D., Wyss-Coray, T., Buckwalter, M. S. 1800; 17 Suppl 3: e053849


    BACKGROUND: Ischemia is a well-established contributor to vascular dementia. Indeed, the most common pathology in people with dementia is mixed, and over half of patients diagnosed with AD have demonstrable vascular pathologies. Ischemia induces an immune response which triggers secondary neurodegeneration remote to the initial lesion, and consequent cognitive decline. Ischemia-triggered vascular dementia is dependent on B-lymphocytes driving chronic neuroinflammation in adult mice. However, vascular dementia is most common in the aged, and there are key differences in inflammatory responses with age. Vascular cell adhesion molecule 1 (VCAM1) is an endothelial protein that facilitates vascular-immune crosstalk via interaction with very late antigen-4 (VLA-4). Soluble VCAM1 is elevated in stroke, vascular dementia, and normal aging in both people and mice. In aging mice, anti-VCAM1 ameliorates age-induced neuroinflammation and cognitive impairment. Although the mechanism is unclear, this is likely mediated via changes in endothelial cell activation and secretion of pro-inflammatory mediators. Therefore, we hypothesized that acute anti-VCAM1 treatment would reduce microgliosis and astrogliosis, while delayed treatment would reduce B and T lymphocyte infiltration in a mouse model of ischemia-triggered vascular dementia.METHOD: Adult (3-month-old) or aged (10-month-old) C57BL/6J mice (n=10-15/group) underwent permanent distal middle cerebral artery occlusion. Mice were dosed with anti-VCAM1 antibody either 4 hours or 4 days post-ischemia, and then sacrificed at 72 hours, 3 weeks or 6 weeks post-ischemia. Microgliosis and astrogliosis were quantified as percent area immunostained in the lesion border by CD68 and GFAP, respectively. B and T cell infiltration were quantified as percent lesion immunostained by B220, and CD3+ cells in the ischemic lesion, respectively.RESULT: Acute treatment reduced microgliosis 30% (p=0.0476) and astrogliosis 39% (p<0.03). In adults, delayed anti-VCAM1 significantly reduced B and T cell infiltration approximately 25% (p=0.0015) and 50% (p=0.0192), respectively. Similarly, in aged mice, delayed anti-VCAM1 significantly reduced B and T cell infiltration approximately 50% (p=0.0037) and 30% (p=0.0036), respectively. In contrast, early anti-VCAM1 had little or no effect on B or T cell infiltration.CONCLUSION: Together, these findings establish VCAM1 as a possible therapeutic target to ameliorate ischemia-induced neuroinflammation and consequent cognitive decline in a mouse model of vascular dementia.

    View details for DOI 10.1002/alz.053849

    View details for PubMedID 35108898

  • Brain profiling in murine colitis and human epilepsy reveals neutrophils and TNFalpha as mediators of neuronal hyperexcitability. Journal of neuroinflammation Barnes, S. E., Zera, K. A., Ivison, G. T., Buckwalter, M. S., Engleman, E. G. 2021; 18 (1): 199


    BACKGROUND: Patients with chronic inflammatory disorders such as inflammatory bowel disease frequently experience neurological complications including epilepsy, depression, attention deficit disorders, migraines, and dementia. However, the mechanistic basis for these associations is unknown. Given that many patients are unresponsive to existing medications or experience debilitating side effects, novel therapeutics that target the underlying pathophysiology of these conditions are urgently needed.METHODS: Because intestinal disorders such as inflammatory bowel disease are robustly associated with neurological symptoms, we used three different mouse models of colitis to investigate the impact of peripheral inflammatory disease on the brain. We assessed neuronal hyperexcitability, which is associated with many neurological symptoms, by measuring seizure threshold in healthy and colitic mice. We profiled the neuroinflammatory phenotype of colitic mice and used depletion and neutralization assays to identify the specific mediators responsible for colitis-induced neuronal hyperexcitability. To determine whether our findings in murine models overlapped with a human phenotype, we performed gene expression profiling, pathway analysis, and deconvolution on microarray data from hyperexcitable human brain tissue from patients with epilepsy.RESULTS: We observed that murine colitis induces neuroinflammation characterized by increased pro-inflammatory cytokine production, decreased tight junction protein expression, and infiltration of monocytes and neutrophils into the brain. We also observed sustained neuronal hyperexcitability in colitic mice. Colitis-induced neuronal hyperexcitability was ameliorated by neutrophil depletion or TNFalpha blockade. Gene expression profiling of hyperexcitable brain tissue resected from patients with epilepsy also revealed a remarkably similar pathology to that seen in the brains of colitic mice, including neutrophil infiltration and high TNFalpha expression.CONCLUSIONS: Our results reveal neutrophils and TNFalpha as central regulators of neuronal hyperexcitability of diverse etiology. Thus, there is a strong rationale for evaluating anti-inflammatory agents, including clinically approved TNFalpha inhibitors, for the treatment of neurological and psychiatric symptoms present in, and potentially independent of, a diagnosed inflammatory disorder.

    View details for DOI 10.1186/s12974-021-02262-4

    View details for PubMedID 34511110

  • T cells direct microglial repair of white matter after stroke. Trends in neurosciences Zera, K. A., Buckwalter, M. S. 2021


    A recent paper by Shi et al. defines the role of regulatory T cells (Tregs) in white matter recovery after ischemic stroke. This study elucidates the mechanisms by which Tregs direct microglia to alter their phenotype to support oligodendrogenesis, thereby improving white matter integrity and functional recovery after stroke in mice.

    View details for DOI 10.1016/j.tins.2021.07.005

    View details for PubMedID 34332802

  • Spleen glia are a transcriptionally unique glial subtype interposed between immune cells and sympathetic axons. Glia Lucas, T. A., Zhu, L., Buckwalter, M. S. 2021


    Glia are known to play important roles in the brain, the gut, and around the sciatic nerve. While the gut has its own specialized nervous system, other viscera are innervated solely by autonomic nerves. The functions of glia that accompany autonomic innervation are not well known, even though they are one of the most abundant cell types in the peripheral nervous system. Here, we focused on non-myelinating Schwann cells in the spleen, spleen glia. The spleen is a major immune organ innervated by the sympathetic nervous system, which modulates immune function. This interaction is known as neuroimmune communication. We establish that spleen glia can be visualized using both immunohistochemistry for S100B and GFAP and with a reporter mouse. Spleen glia ensheath sympathetic axons and are localized to the lymphocyte-rich white pulp areas of the spleen. We sequenced the spleen glia transcriptome and identified genes that are likely involved in axonal ensheathment and communication with both nerves and immune cells. Spleen glia express receptors for neurotransmitters made by sympathetic axons (adrenergic, purinergic, and Neuropeptide Y), and also cytokines, chemokines, and their receptors that may communicate with immune cells in the spleen. We also established similarities and differences between spleen glia and other glial types. While all glia share many genes in common, spleen glia differentially express genes associated with immune responses, including genes involved in cytokine-cytokine receptor interactions, phagocytosis, and the complement cascade. Thus, spleen glia are a unique glial type, physically and transcriptionally poised to participate in neuroimmune communication in the spleen.

    View details for DOI 10.1002/glia.23993

    View details for PubMedID 33710690

  • Home-based Virtual Reality Therapy for Hand Recovery After Stroke. PM & R : the journal of injury, function, and rehabilitation Lansberg, M. G., Legault, C. n., MacLellan, A. n., Parikh, A. n., Muccini, J. n., Mlynash, M. n., Kemp, S. n., Buckwalter, M. S., Flavin, K. n. 2021


    Many stroke survivors suffer from arm and hand weakness, but there are only limited efficacious options for arm therapy available.To assess the feasibility of unsupervised home-based use of a virtual reality device (Smart Glove) for hand rehabilitation post stroke.Prospective single-arm study consisting of a 2-week run-in phase with no device use followed by an 8-week intervention period.Participants were recruited at the Stanford Neuroscience Outpatient Clinic.Twenty chronic stroke patients with upper extremity impairment.Participants were instructed to use the Smart Glove 50 minutes per day, 5 days per week for 8 weeks.We measured (1) compliance, (2) patients' impression of the intervention, and (3) efficacy measures including the upper extremity Fugl-Meyer (UE-FM), the Jebsen-Taylor hand function test (JTHFT) and the Stroke Impact Scale (SIS).Of 20 subjects, 7 (35%) met target compliance of 40 days use, and 6 (30%) used the device for 20-39 days. Eighty-five percent of subjects were satisfied with the therapy, with 80% reporting improvement in hand function. During the run-in phase there were no improvements in hand function. During the intervention, patients improved by a mean of 26.6 ± 48.8 seconds on the JTHFT (P = 0.03), by 16.1 ± 15.3 points on the hand-domain of the SIS (P < 0.01) and there was a trend towards improvement on the UE-FM (2.2 ± 5.5 points, P = 0.10).Unsupervised use of the Smart Glove in the home environment may improve hand/arm function in subacute/chronic stroke patients. A randomized controlled trial is needed to confirm these results. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/pmrj.12598

    View details for PubMedID 33773059

  • New Mechanistic Insights, Novel Treatment Paradigms, and Clinical Progress in Cerebrovascular Diseases. Frontiers in aging neuroscience Boltze, J., Aronowski, J. A., Badaut, J., Buckwalter, M. S., Caleo, M., Chopp, M., Dave, K. R., Didwischus, N., Dijkhuizen, R. M., Doeppner, T. R., Dreier, J. P., Fouad, K., Gelderblom, M., Gertz, K., Golubczyk, D., Gregson, B. A., Hamel, E., Hanley, D. F., Hartig, W., Hummel, F. C., Ikhsan, M., Janowski, M., Jolkkonen, J., Karuppagounder, S. S., Keep, R. F., Koerte, I. K., Kokaia, Z., Li, P., Liu, F., Lizasoain, I., Ludewig, P., Metz, G. A., Montagne, A., Obenaus, A., Palumbo, A., Pearl, M., Perez-Pinzon, M., Planas, A. M., Plesnila, N., Raval, A. P., Rueger, M. A., Sansing, L. H., Sohrabji, F., Stagg, C. J., Stetler, R. A., Stowe, A. M., Sun, D., Taguchi, A., Tanter, M., Vay, S. U., Vemuganti, R., Vivien, D., Walczak, P., Wang, J., Xiong, Y., Zille, M. 2021; 13: 623751


    The past decade has brought tremendous progress in diagnostic and therapeutic options for cerebrovascular diseases as exemplified by the advent of thrombectomy in ischemic stroke, benefitting a steeply increasing number of stroke patients and potentially paving the way for a renaissance of neuroprotectants. Progress in basic science has been equally impressive. Based on a deeper understanding of pathomechanisms underlying cerebrovascular diseases, new therapeutic targets have been identified and novel treatment strategies such as pre- and post-conditioning methods were developed. Moreover, translationally relevant aspects are increasingly recognized in basic science studies, which is believed to increase their predictive value and the relevance of obtained findings for clinical application.This review reports key results from some of the most remarkable and encouraging achievements in neurovascular research that have been reported at the 10th International Symposium on Neuroprotection and Neurorepair. Basic science topics discussed herein focus on aspects such as neuroinflammation, extracellular vesicles, and the role of sex and age on stroke recovery. Translational reports highlighted endovascular techniques and targeted delivery methods, neurorehabilitation, advanced functional testing approaches for experimental studies, pre-and post-conditioning approaches as well as novel imaging and treatment strategies. Beyond ischemic stroke, particular emphasis was given on activities in the fields of traumatic brain injury and cerebral hemorrhage in which promising preclinical and clinical results have been reported. Although the number of neutral outcomes in clinical trials is still remarkably high when targeting cerebrovascular diseases, we begin to evidence stepwise but continuous progress towards novel treatment options. Advances in preclinical and translational research as reported herein are believed to have formed a solid foundation for this progress.

    View details for DOI 10.3389/fnagi.2021.623751

    View details for PubMedID 33584250

  • B and T Lymphocyte Densities Remain Stable With Age in Human Cortex. ASN neuro Berry, K., Farias-Itao, D. S., Grinberg, L. T., Plowey, E. D., Schneider, J. A., Rodriguez, R. D., Suemoto, C. K., Buckwalter, M. S. 2021; 13: 17590914211018117


    One hallmark of human aging is increased brain inflammation represented by glial activation. With age, there is also diminished function of the adaptive immune system, and modest decreases in circulating B- and T-lymphocytes. Lymphocytes traffic through the human brain and reside there in small numbers, but it is unknown how this changes with age. Thus we investigated whether B- and T-lymphocyte numbers change with age in the normal human brain. We examined 16 human subjects in a pilot study and then 40 human subjects from a single brain bank, ranging in age from 44-96years old, using rigorous criteria for defining neuropathological changes due to age alone. We immunostained post-mortem cortical tissue for B- and T-lymphocytes using antibodies to CD20 and CD3, respectively. We quantified cell density and made a qualitative assessment of cell location in cortical brain sections, and reviewed prior studies. We report that density and location of both B- and T-lymphocytes do not change with age in the normal human cortex. Solitary B-lymphocytes were found equally in intravascular, perivascular, and parenchymal locations, while T-lymphocytes appeared primarily in perivascular clusters. Thus, any change in number or location of lymphocytes in an aging brain may indicate disease rather than normal aging.

    View details for DOI 10.1177/17590914211018117

    View details for PubMedID 34056948

  • Mapping causal circuit dynamics in stroke using simultaneous electroencephalography and transcranial magnetic stimulation. BMC neurology Rolle, C. E., Baumer, F. M., Jordan, J. T., Berry, K., Garcia, M., Monusko, K., Trivedi, H., Wu, W., Toll, R., Buckwalter, M. S., Lansberg, M., Etkin, A. 2021; 21 (1): 280


    Motor impairment after stroke is due not only to direct tissue loss but also to disrupted connectivity within the motor network. Mixed results from studies attempting to enhance motor recovery with Transcranial Magnetic Stimulation (TMS) highlight the need for a better understanding of both connectivity after stroke and the impact of TMS on this connectivity. This study used TMS-EEG to map the causal information flow in the motor network of healthy adult subjects and define how stroke alters these circuits.Fourteen stroke patients and 12 controls received TMS to two sites (bilateral primary motor cortices) during two motor tasks (paretic/dominant hand movement vs. rest) while EEG measured the cortical response to TMS pulses. TMS-EEG based connectivity measurements were derived for each hemisphere and the change in connectivity (ΔC) between the two motor tasks was calculated. We analyzed if ΔC for each hemisphere differed between the stroke and control groups or across TMS sites, and whether ΔC correlated with arm function in stroke patients.Right hand movement increased connectivity in the left compared to the right hemisphere in controls, while hand movement did not significantly change connectivity in either hemisphere in stroke. Stroke patients with the largest increase in healthy hemisphere connectivity during paretic hand movement had the best arm function.TMS-EEG measurements are sensitive to movement-induced changes in brain connectivity. These measurements may characterize clinically meaningful changes in circuit dynamics after stroke, thus providing specific targets for trials of TMS in post-stroke rehabilitation.

    View details for DOI 10.1186/s12883-021-02319-0

    View details for PubMedID 34271872

  • Immune responses to stroke: mechanisms, modulation, and therapeutic potential. The Journal of clinical investigation Iadecola, C., Buckwalter, M. S., Anrather, J. 2020


    Stroke is the second leading cause of death worldwide and a leading cause of disability. Most strokes are caused by occlusion of a major cerebral artery, and substantial advances have been made in elucidating how ischemia damages the brain. In particular, increasing evidence points to a double-edged role of the immune system in stroke pathophysiology. In the acute phase, innate immune cells invade brain and meninges and contribute to ischemic damage, but may also be protective. At the same time, danger signals released into the circulation by damaged brain cells lead to activation of systemic immunity, followed by profound immunodepression that promotes life-threatening infections. In the chronic phase, antigen presentation initiates an adaptive immune response targeted to the brain, which may underlie neuropsychiatric sequelae, a considerable cause of poststroke morbidity. Here, we briefly review these pathogenic processes and assess the potential therapeutic value of targeting immunity in human stroke.

    View details for DOI 10.1172/JCI135530

    View details for PubMedID 32391806

  • Home-Based Virtual Reality Therapy for Hand Recovery After Stroke MacLellan, A., Legault, C., Parikh, A., Lugo, L., Kemp, S., Mlynash, M., Buckwalter, M., Flavin, K., Lansberg, M. LIPPINCOTT WILLIAMS & WILKINS. 2020
  • Development of a Comprehensive Neuropsychological Battery to Assess Post-Stroke Cognitive Functioning Drag, L., Aghaeepour, N., Mlynash, M., Osborn, E., Rah, E., Buckwalter, M., Lansberg, M. LIPPINCOTT WILLIAMS & WILKINS. 2020
  • Obesity Drives Delayed Infarct Expansion, Inflammation, and Distinct Gene Networks in a Mouse Stroke Model. Translational stroke research Peterson, T. C., Lechtenberg, K. J., Piening, B. D., Lucas, T. A., Wei, E. n., Chaib, H. n., Dowdell, A. K., Snyder, M. n., Buckwalter, M. S. 2020


    Obesity is associated with chronic peripheral inflammation, is a risk factor for stroke, and causes increased infarct sizes. To characterize how obesity increases infarct size, we fed a high-fat diet to wild-type C57BL/6J mice for either 6 weeks or 15 weeks and then induced distal middle cerebral artery strokes. We found that infarct expansion happened late after stroke. There were no differences in cortical neuroinflammation (astrogliosis, microgliosis, or pro-inflammatory cytokines) either prior to or 10 h after stroke, and also no differences in stroke size at 10 h. However, by 3 days after stroke, animals fed a high-fat diet had a dramatic increase in microgliosis and astrogliosis that was associated with larger strokes and worsened functional recovery. RNA sequencing revealed a dramatic increase in inflammatory genes in the high-fat diet-fed animals 3 days after stroke that were not present prior to stroke. Genetic pathways unique to diet-induced obesity were primarily related to adaptive immunity, extracellular matrix components, cell migration, and vasculogenesis. The late appearance of neuroinflammation and infarct expansion indicates that there may be a therapeutic window between 10 and 36 h after stroke where inflammation and obesity-specific transcriptional programs could be targeted to improve outcomes in people with obesity and stroke.

    View details for DOI 10.1007/s12975-020-00826-9

    View details for PubMedID 32588199

  • Development of a CD19 PET tracer for detecting B cells in a mouse model of multiple sclerosis. Journal of neuroinflammation Stevens, M. Y., Cropper, H. C., Lucot, K. L., Chaney, A. M., Lechtenberg, K. J., Jackson, I. M., Buckwalter, M. S., James, M. L. 2020; 17 (1): 275


    B cells play a central role in multiple sclerosis (MS) through production of injurious antibodies, secretion of pro-inflammatory cytokines, and antigen presentation. The therapeutic success of monoclonal antibodies (mAbs) targeting B cells in some but not all individuals suffering from MS highlights the need for a method to stratify patients and monitor response to treatments in real-time. Herein, we describe the development of the first CD19 positron emission tomography (PET) tracer, and its evaluation in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE).Female C57BL/6 J mice were induced with EAE through immunization with myelin oligodendrocyte glycoprotein (MOG1-125). PET imaging of naïve and EAE mice was performed 19 h after administration of [64Cu]CD19-mAb. Thereafter, radioactivity in organs of interest was determined by gamma counting, followed by ex vivo autoradiography of central nervous system (CNS) tissues. Anti-CD45R (B220) immunostaining of brain tissue from EAE and naïve mice was also conducted.Radiolabelling of DOTA-conjugated CD19-mAb with 64Cu was achieved with a radiochemical purity of 99% and molar activity of 2 GBq/μmol. Quantitation of CD19 PET images revealed significantly higher tracer binding in whole brain of EAE compared to naïve mice (2.02 ± 0.092 vs. 1.68 ± 0.06 percentage of injected dose per gram, % ID/g, p = 0.0173). PET findings were confirmed by ex vivo gamma counting of perfused brain tissue (0.22 ± 0.020 vs. 0.12 ± 0.003 % ID/g, p = 0.0010). Moreover, ex vivo autoradiography of brain sections corresponded with PET imaging results and the spatial distribution of B cells observed in B220 immunohistochemistry-providing further evidence that [64Cu]CD19-mAb enables visualization of B cell infiltration into the CNS of EAE mice.CD19-PET imaging can be used to detect elevated levels of B cells in the CNS of EAE mice, and has the potential to impact the way we study, monitor, and treat clinical MS.

    View details for DOI 10.1186/s12974-020-01880-8

    View details for PubMedID 32948198

  • Infection as a Stroke Risk Factor and Determinant of Outcome After Stroke. Stroke Elkind, M. S., Boehme, A. K., Smith, C. J., Meisel, A. n., Buckwalter, M. S. 2020: STROKEAHA120030429


    Understanding the relationship between infection and stroke has taken on new urgency in the era of the coronavirus disease 2019 (COVID-19) pandemic. This association is not a new concept, as several infections have long been recognized to contribute to stroke risk. The association of infection and stroke is also bidirectional. Although infection can lead to stroke, stroke also induces immune suppression which increases risk of infection. Apart from their short-term effects, emerging evidence suggests that poststroke immune changes may also adversely affect long-term cognitive outcomes in patients with stroke, increasing the risk of poststroke neurodegeneration and dementia. Infections at the time of stroke may also increase immune dysregulation after the stroke, further exacerbating the risk of cognitive decline. This review will cover the role of acute infections, including respiratory infections such as COVID-19, as a trigger for stroke; the role of infectious burden, or the cumulative number of infections throughout life, as a contributor to long-term risk of atherosclerotic disease and stroke; immune dysregulation after stroke and its effect on the risk of stroke-associated infection; and the impact of infection at the time of a stroke on the immune reaction to brain injury and subsequent long-term cognitive and functional outcomes. Finally, we will present a model to conceptualize the many relationships among chronic and acute infections and their short- and long-term neurological consequences. This model will suggest several directions for future research.

    View details for DOI 10.1161/STROKEAHA.120.030429

    View details for PubMedID 32897811

  • A longitudinal study of the post-stroke immune response and cognitive functioning: the StrokeCog study protocol. BMC neurology Drag, L. L., Mlynash, M. n., Nassar, H. n., Osborn, E. n., Kim, D. E., Angst, M. S., Aghaeepour, N. n., Buckwalter, M. n., Lansberg, M. G. 2020; 20 (1): 313


    Stroke increases the risk of cognitive impairment even several years after the stroke event. The exact mechanisms of post-stroke cognitive decline are unclear, but the immunological response to stroke might play a role. The aims of the StrokeCog study are to examine the associations between immunological responses and long-term post-stroke cognitive trajectories in individuals with ischemic stroke.StrokeCog is a single-center, prospective, observational, cohort study. Starting 6-12 months after stroke, comprehensive neuropsychological assessment, plasma and serum, and psychosocial variables will be collected at up to 4 annual visits. Single cell sequencing of peripheral blood monocytes and plasma proteomics will be conducted. The primary outcome will be the change in global and domain-specific neuropsychological performance across annual evaluations. To explain the differences in cognitive change amongst participants, we will examine the relationships between comprehensive immunological measures and these cognitive trajectories. It is anticipated that 210 participants will be enrolled during the first 3 years of this 4-year study. Accounting for attrition, an anticipated final sample size of 158 participants with an average of 3 annual study visits will be available at the completion of the study. Power analyses indicate that this sample size will provide 90% power to detect an average cognitive change of at least 0.23 standard deviations in either direction.StrokeCog will provide novel insight into the relationships between immune events and cognitive change late after stroke.

    View details for DOI 10.1186/s12883-020-01897-9

    View details for PubMedID 32847540

  • The Local and Peripheral Immune Responses to Stroke: Implications for Therapeutic Development. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics Zera, K. A., Buckwalter, M. S. 2020


    The immune response to stroke is an exciting target for future stroke therapies. Stroke is a leading cause of morbidity and mortality worldwide, and clot removal (mechanical or pharmacological) to achieve tissue reperfusion is the only therapy currently approved for patient use. Due to a short therapeutic window and incomplete effectiveness, however, many patients are left with infarcted tissue that stimulates inflammation. Although this is critical to promote repair, it can also damage surrounding healthy brain tissue. In addition, acute immunodepression and subsequent infections are common and are associated with worse patient outcomes. Thus, the acute immune response is a major focus of researchers attempting to identify ways to amplify its benefits and suppress its negative effects to improve short-term recovery of patients. Here we review what is known about this powerful process. This includes the role of brain resident cells such as microglia, peripherally activated cells such as macrophages and neutrophils, and activated endothelium. The role of systemic immune activation and subsequent immunodepression in the days after stroke is also discussed, as is the chronic immune responses and its effects on cognitive function. The biphasic role of inflammation, as well as complex timelines of cell production, differentiation, and trafficking, suggests that the relationship between the acute and chronic phases of stroke recovery is complex. Gaining a more complete understanding of this intricate process by which inflammation is initiated, propagated, and terminated may potentially lead to therapeutics that can treat a larger population of stroke patients than what is currently available. The immune response plays a critical role in patient recovery in both the acute and chronic phases after stroke. In patients, the immune response can be beneficial by promoting repair and recovery, and also detrimental by propagating a pro-inflammatory microenvironment. Thus, it is critical to understand the mechanisms of immune activation following stroke in order to successfully design therapeutics.

    View details for DOI 10.1007/s13311-020-00844-3

    View details for PubMedID 32193840

  • Immunological mechanisms in poststroke dementia. Current opinion in neurology Doyle, K. P., Buckwalter, M. S. 2019


    PURPOSE OF REVIEW: To review new evidence on links between poststroke dementia and inflammation.RECENT FINDINGS: Although there are still no treatments for poststroke dementia, recent evidence has improved our understanding that stroke increases the risk of incident dementia and worsens cognitive trajectory for at least a decade afterwards. Within approximately the first year dementia onset is associated with stroke severity and location, whereas later absolute risk is associated with more traditional dementia risk factors, such as age and imaging findings. The molecular mechanisms that underlie increased risk of incident dementia in stroke survivors remain unproven; however new data in both human and animal studies suggests links between cognitive decline and inflammation. These point to a model where chronic brain inflammation, provoked by inefficient clearance of myelin debris and a prolonged innate and adaptive immune response, causes poststroke dementia. These localized immune events in the brain may themselves be influenced by the peripheral immune state at key times after stroke.SUMMARY: This review recaps clinical evidence on poststroke dementia, new mechanistic links between the chronic inflammatory response to stroke and poststroke dementia, and proposes a model of immune-mediated neurodegeneration after stroke.

    View details for DOI 10.1097/WCO.0000000000000783

    View details for PubMedID 31789707

  • Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1 NATURE MEDICINE Yousef, H., Czupalla, C. J., Lee, D., Chen, M. B., Burke, A. N., Zera, K. A., Zandstra, J., Berber, E., Lehallier, B., Mathur, V., Nair, R. V., Bonanno, L. N., Yang, A. C., Peterson, T., Hadeiba, H., Merkel, T., Koerbelin, J., Schwaninger, M., Buckwalter, M. S., Quake, S. R., Butcher, E. C., Wyss-Coray, T. 2019; 25 (6): 988-+
  • A YEAR-LONG IMMUNE PROFILE OF THE SYSTEMIC RESPONSE IN ACUTE STROKE SURVIVORS Tsai, A., Berry, K., Beneyto, M. M., Gaudilliere, D., Ganio, E. A., Culos, A., Ghaemi, M. S., Choisy, B., Djebali, K., Einhaus, J. F., Bertrand, B., Tanada, A., Stanley, N., Fallahzadeh, R., Baca, Q., Quach, L. N., Osborn, E., Drag, L., Lansberg, M., Angst, M., Gaudilliere, B., Buckwalter, M. S., Aghaeepour, N. LIPPINCOTT WILLIAMS & WILKINS. 2019: 155
  • Augmented beta2-adrenergic signaling dampens the neuroinflammatory response following ischemic stroke and increases stroke size. Journal of neuroinflammation Lechtenberg, K. J., Meyer, S. T., Doyle, J. B., Peterson, T. C., Buckwalter, M. S. 2019; 16 (1): 112


    BACKGROUND: Ischemic stroke provokes a neuroinflammatory response and simultaneously promotes release of epinephrine and norepinephrine by the sympathetic nervous system. This increased sympathetic outflow can act on beta2-adrenergic receptors expressed by immune cells such as brain-resident microglia and monocyte-derived macrophages (MDMs), but the effect on post-stroke neuroinflammation is unknown. Thus, we investigated how changes in beta2-adrenergic signaling after stroke onset influence the microglia/MDM stroke response, and the specific importance of microglia/MDM beta2-adrenergic receptors to post-stroke neuroinflammation.METHODS: To investigate the effects of beta2-adrenergic receptor manipulation on post-stroke neuroinflammation, we administered the beta2-adrenergic receptor agonist clenbuterol to mice 3h after the onset of photothrombotic stroke. We immunostained to quantify microglia/MDM numbers and proliferation and to assess morphology and activation 3days later. We assessed stroke outcomes by measuring infarct volume and functional motor recovery and analyzed gene expression levels of neuroinflammatory molecules. Finally, we evaluated changes in cytokine expression and microglia/MDM response in brains of mice with selective knockout of the beta2-adrenergic receptor from microglia and monocyte-lineage cells.RESULTS: We report that clenbuterol treatment after stroke onset causes enlarged microglia/MDMs and impairs their proliferation, resulting in reduced numbers of these cells in the peri-infarct cortex by 1.7-fold at 3days after stroke. These changes in microglia/MDMs were associated with increased infarct volume in clenbuterol-treated animals. In mice that had the beta2-adrenergic receptor specifically knocked out of microglia/MDMs, there was no change in morphology or numbers of these cells after stroke. However, knockdown of beta2-adrenergic receptors in microglia and MDMs resulted in increased expression of TNFalpha and IL-10 in peri-infarct tissue, while stimulation of beta2-adrenergic receptors with clenbuterol had the opposite effect, suppressing TNFalpha and IL-10 expression.CONCLUSIONS: We identified beta2-adrenergic receptor signaling as an important regulator of the neuroimmune response after ischemic stroke. Increased beta2-adrenergic signaling after stroke onset generally suppressed the microglia/MDM response, reducing upregulation of both pro- and anti-inflammatory cytokines, and increasing stroke size. In contrast, diminished beta2-adrenergic signaling in microglia/MDMs augmented both pro- and anti-inflammatory cytokine expression after stroke. The beta2-adrenergic receptor may therefore present a therapeutic target for improving the post-stroke neuroinflammatory and repair process.

    View details for DOI 10.1186/s12974-019-1506-4

    View details for PubMedID 31138227

  • Radiolabeling and pre-clinical evaluation of a first-in-class CD19 PET Tracer for imaging B cells in multiple sclerosis Stevens, M., Cropper, H., Jackson, I., Chaney, A., Lechtenberg, K., Buckwalter, M., James, M. L. SOC NUCLEAR MEDICINE INC. 2019
  • A year-long immune profile of the systemic response in acute stroke survivors. Brain : a journal of neurology Tsai, A. S., Berry, K., Beneyto, M. M., Gaudilliere, D., Ganio, E. A., Culos, A., Ghaemi, M. S., Choisy, B., Djebali, K., Einhaus, J. F., Bertrand, B., Tanada, A., Stanley, N., Fallahzadeh, R., Baca, Q., Quach, L. N., Osborn, E., Drag, L., Lansberg, M. G., Angst, M. S., Gaudilliere, B., Buckwalter, M. S., Aghaeepour, N. 2019


    Stroke is a leading cause of cognitive impairment and dementia, but the mechanisms that underlie post-stroke cognitive decline are not well understood. Stroke produces profound local and systemic immune responses that engage all major innate and adaptive immune compartments. However, whether the systemic immune response to stroke contributes to long-term disability remains ill-defined. We used a single-cell mass cytometry approach to comprehensively and functionally characterize the systemic immune response to stroke in longitudinal blood samples from 24 patients over the course of 1 year and correlated the immune response with changes in cognitive functioning between 90 and 365 days post-stroke. Using elastic net regularized regression modelling, we identified key elements of a robust and prolonged systemic immune response to ischaemic stroke that occurs in three phases: an acute phase (Day 2) characterized by increased signal transducer and activator of transcription 3 (STAT3) signalling responses in innate immune cell types, an intermediate phase (Day 5) characterized by increased cAMP response element-binding protein (CREB) signalling responses in adaptive immune cell types, and a late phase (Day 90) by persistent elevation of neutrophils, and immunoglobulin M+ (IgM+) B cells. By Day 365 there was no detectable difference between these samples and those from an age- and gender-matched patient cohort without stroke. When regressed against the change in the Montreal Cognitive Assessment scores between Days 90 and 365 after stroke, the acute inflammatory phase Elastic Net model correlated with post-stroke cognitive trajectories (r = -0.692, Bonferroni-corrected P = 0.039). The results demonstrate the utility of a deep immune profiling approach with mass cytometry for the identification of clinically relevant immune correlates of long-term cognitive trajectories.

    View details for DOI 10.1093/brain/awz022

    View details for PubMedID 30860258

  • Deep Immune Profiling of the Post-Stroke Peripheral Immune Response Reveals Tri-phasic Response and Correlations With Long-Term Cognitive Outcomes Tsai, A. S., Berry, K., Beneyto, M. M., Gaudilliere, D., Ganio, E. A., Choisy, B., Djebali, K., Baca, Q., Quach, L., Drag, L., Lansberg, M. G., Angst, M. S., Gaudilliere, B., Buckwalter, M. S., Aghaeepour, N. LIPPINCOTT WILLIAMS & WILKINS. 2019
  • Feasibility and Utility of Home-Based Gait Analysis Using Body-Worn Sensors Huang, E., Sharp, M. T., Osborn, E., MacLellan, A., Mlynash, M., Kemp, S., Buckwalter, M. S., Lansberg, M. G. LIPPINCOTT WILLIAMS & WILKINS. 2019
  • Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1. Nature medicine Yousef, H. n., Czupalla, C. J., Lee, D. n., Chen, M. B., Burke, A. N., Zera, K. A., Zandstra, J. n., Berber, E. n., Lehallier, B. n., Mathur, V. n., Nair, R. V., Bonanno, L. N., Yang, A. C., Peterson, T. n., Hadeiba, H. n., Merkel, T. n., Körbelin, J. n., Schwaninger, M. n., Buckwalter, M. S., Quake, S. R., Butcher, E. C., Wyss-Coray, T. n. 2019


    An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier as a possible target to treat age-related neurodegeneration.

    View details for PubMedID 31086348

  • C-11-DPA-713 Versus F-18-GE-180: A Preclinical Comparison of Translocator Protein 18 kDa PET Tracers to Visualize Acute and Chronic Neuroinflammation in a Mouse Model of Ischemic Stroke JOURNAL OF NUCLEAR MEDICINE Chaney, A., Cropper, H. C., Johnson, E. M., Lechtenberg, K. J., Peterson, T. C., Stevens, M. Y., Buckwalter, M. S., James, M. L. 2019; 60 (1): 122–28
  • Clopidogrel and Aspirin in Acute Ischemic Stroke and High-Risk TIA. The New England journal of medicine Johnston, S. C., Easton, J. D., Farrant, M. n., Barsan, W. n., Conwit, R. A., Elm, J. J., Kim, A. S., Lindblad, A. S., Palesch, Y. Y. 2018


    Background Combination antiplatelet therapy with clopidogrel and aspirin may reduce the rate of recurrent stroke during the first 3 months after a minor ischemic stroke or transient ischemic attack (TIA). A trial of combination antiplatelet therapy in a Chinese population has shown a reduction in the risk of recurrent stroke. We tested this combination in an international population. Methods In a randomized trial, we assigned patients with minor ischemic stroke or high-risk TIA to receive either clopidogrel at a loading dose of 600 mg on day 1, followed by 75 mg per day, plus aspirin (at a dose of 50 to 325 mg per day) or the same range of doses of aspirin alone. The dose of aspirin in each group was selected by the site investigator. The primary efficacy outcome in a time-to-event analysis was the risk of a composite of major ischemic events, which was defined as ischemic stroke, myocardial infarction, or death from an ischemic vascular event, at 90 days. Results A total of 4881 patients were enrolled at 269 international sites. The trial was halted after 84% of the anticipated number of patients had been enrolled because the data and safety monitoring board had determined that the combination of clopidogrel and aspirin was associated with both a lower risk of major ischemic events and a higher risk of major hemorrhage than aspirin alone at 90 days. Major ischemic events occurred in 121 of 2432 patients (5.0%) receiving clopidogrel plus aspirin and in 160 of 2449 patients (6.5%) receiving aspirin plus placebo (hazard ratio, 0.75; 95% confidence interval [CI], 0.59 to 0.95; P=0.02), with most events occurring during the first week after the initial event. Major hemorrhage occurred in 23 patients (0.9%) receiving clopidogrel plus aspirin and in 10 patients (0.4%) receiving aspirin plus placebo (hazard ratio, 2.32; 95% CI, 1.10 to 4.87; P=0.02). Conclusions In patients with minor ischemic stroke or high-risk TIA, those who received a combination of clopidogrel and aspirin had a lower risk of major ischemic events but a higher risk of major hemorrhage at 90 days than those who received aspirin alone. (Funded by the National Institute of Neurological Disorders and Stroke; POINT ClinicalTrials.gov number, NCT00991029 .).

    View details for PubMedID 29766750

  • 11C-DPA-713 versus 18F-GE-180: A preclinical comparison of TSPO-PET tracers to visualize acute and chronic neuroinflammation in a mouse model of ischemic stroke. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Chaney, A. n., Cropper, H. C., Johnson, E. M., Lechtenberg, K. J., Peterson, T. C., Stevens, M. Y., Buckwalter, M. S., James, M. L. 2018


    Neuroinflammation plays a key role in neuronal injury following ischemic stroke. Positron emission tomography (PET) imaging of translocator protein 18 kDa (TSPO) permits longitudinal, non-invasive visualization of neuroinflammation in both pre-clinical and clinical settings. Many TSPO tracers have been developed, however it is unclear which tracer is the most sensitive and accurate for monitoring the in vivo spatiotemporal dynamics of neuroinflammation across applications. Hence, there is a need for head-to-head comparisons of promising TSPO-PET tracers across different disease states. Accordingly, the aim of this study was to directly compare two promising second-generation TSPO tracers; 11C-DPA-713 and 18F-GE-180, for the first time at acute and chronic time-points following ischemic stroke. Methods: Following distal middle cerebral artery occlusion (dMCAO) or sham surgery, mice underwent consecutive PET/CT imaging with 11C-DPA-713 and 18F-GE-180 at 2, 6, and 28 days after stroke. T2-weighted magnetic resonance (MR) images were acquired to enable delineation of ipsilateral (infarct) and contralateral brain regions of interest (ROIs). PET images were analyzed by calculating % injected dose per gram (%ID/g) in MR-guided ROIs. Standardized uptake value ratios were determined using the contralateral thalamus as a pseudo-reference region (SUVTh). Ex vivo autoradiography and immunohistochemistry were performed to verify in vivo findings. Results: Significantly increased tracer uptake was observed in the ipsilateral compared to contralateral ROI (SUVTh, 50-60 min summed data) at acute and chronic time-points using 11C-DPA-713 and 18F-GE-180. Ex vivo autoradiography confirmed in vivo findings demonstrating increased TSPO-tracer uptake in infarcted versus contralateral brain tissue. Importantly, a significant correlation was identified between microglial/macrophage activation (CD68 immunostaining) and 11C-DPA-713-PET signal, that was not evident with 18F-GE-180. No significant correlations were observed between TSPO-PET and activated astrocytes (GFAP immunostaining). Conclusion: Both 11C-DPA-713 and 18F-GE-180-PET enable detection of neuroinflammation at early and chronic time-points following cerebral ischemia in mice. 11C-DPA-713-PET reflects the extent of microglial activation in infarcted dMCAO mouse brain tissue more accurately compared to 18F-GE-180, and appears to be slightly more sensitive. These results highlight the potential of 11C-DPA-713 for tracking microglial activation in vivo after stroke, and warrants further investigation in both pre-clinical and clinical settings.

    View details for PubMedID 29976695

  • Depression one year after hemorrhagic stroke is associated with late worsening of outcomes. NeuroRehabilitation Stern-Nezer, S., Eyngorn, I., Mlynash, M., Snider, R. W., Venkatsubramanian, C., Wijman, C. A., Buckwalter, M. S. 2017


    Poststroke depression is the most common psychiatric sequelae of stroke, and it's independently associated with increased morbidity and mortality. Few studies have examined depression after intracranial hemorrhage (ICH).To investigate the relationship between depression, ICH and outcomes.A substudy of the prospective Diagnostic Accuracy of MRI in Spontaneous Intracerebral Hemorrhage (DASH) study, we included 89 subjects assessed for depression 1 year after hemorrhage. A Hamilton Depression Rating Scale score >10 defined depression. Univariate, multivariable, and trend analyses evaluated relationships between depression, clinical, radiographic, and inflammatory factors and modified Rankin score (mRS) at 90 days and one year.Prevalence of depression at one year was 15%. Depression was not associated with hematoma volumes, presence of IVH or admission NIHSS, nor with demographic factors. Despite this, depressed patients had worse 1-year outcomes (p = 0.004) and were less likely to improve between 3 and 12 months, and more likely to worsen (p = 0.042).This is the first study to investigate depression one year after ICH. Post-ICH depression was common and associated with late worsening of disability unrelated to initial hemorrhage severity. Further research is needed to understand whether depression is caused by worsened disability, or whether the converse is true.

    View details for DOI 10.3233/NRE-171470

    View details for PubMedID 28505996

  • Neurotoxic reactive astrocytes are induced by activated microglia. Nature Liddelow, S. A., Guttenplan, K. A., Clarke, L. E., Bennett, F. C., Bohlen, C. J., Schirmer, L., Bennett, M. L., Münch, A. E., Chung, W., Peterson, T. C., Wilton, D. K., Frouin, A., Napier, B. A., Panicker, N., Kumar, M., Buckwalter, M. S., Rowitch, D. H., Dawson, V. L., Dawson, T. M., Stevens, B., Barres, B. A. 2017; 541 (7638): 481-487


    Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease, but their role is poorly understood. Here we show that a subtype of reactive astrocytes, which we termed A1, is induced by classically activated neuroinflammatory microglia. We show that activated microglia induce A1 astrocytes by secreting Il-1α, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A1 astrocytes. A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce the death of neurons and oligodendrocytes. Death of axotomized CNS neurons in vivo is prevented when the formation of A1 astrocytes is blocked. Finally, we show that A1 astrocytes are abundant in various human neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. Taken together these findings help to explain why CNS neurons die after axotomy, strongly suggest that A1 astrocytes contribute to the death of neurons and oligodendrocytes in neurodegenerative disorders, and provide opportunities for the development of new treatments for these diseases.

    View details for DOI 10.1038/nature21029

    View details for PubMedID 28099414

  • Imaging B cells in a mouse model of multiple sclerosis using (64)Cu-Rituximab-PET. Journal of nuclear medicine : official publication, Society of Nuclear Medicine James, M. L., Hoehne, A. n., Mayer, A. T., Lechtenberg, K. n., Moreno, M. n., Gowrishankar, G. n., Ilovich, O. n., Natarajan, A. n., Johnson, E. M., Nguyen, J. n., Quach, L. n., Han, M. n., Buckwalter, M. n., Chandra, S. n., Gambhir, S. S. 2017


    B lymphocytes are a key pathological feature of multiple sclerosis (MS), and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to non-invasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here we evaluated (64)Cu-Rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using positron emission tomography (PET). Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of Myelin Oligodendrocyte Glycoprotein fragment1-125 (MOG1-125) emulsified in complete Freund's adjuvant. Control mice received complete Freund's adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19h following (64)Cu-Rituximab administration. Mice were perfused and sacrificed after final PET scan, and radioactivity in dissected tissues was measured with a gamma-counter. CNS tissues from these mice were immunostained to quantify B cells or further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice compared to controls at all evaluated time points (e.g., 1h post-injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 %ID/g, p<0.05). (64)Cu-Rituximab-PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice compared to 1.25±0.08 and 2.24±0.11%ID/g for controls, p<0.05 for all regions except striatum and thalamus at 1h post-injection. Similarly, ex vivo biodistribution results revealed notably higher (64)Cu-Rituximab uptake in brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased (64)Cu-Rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using (64)Cu-Rituximab-PET. Results from these studies warrant further investigation of (64)Cu-Rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell-targeted therapeutics en route to the clinic.

    View details for PubMedID 28687602

  • Astrocytes: Integrative Regulators of Neuroinflammation in Stroke and Other Neurological Diseases. Neurotherapeutics Cekanaviciute, E., Buckwalter, M. S. 2016; 13 (4): 685-701


    Astrocytes regulate neuroinflammatory responses after stroke and in other neurological diseases. Although not all astrocytic responses reduce inflammation, their predominant function is to protect the brain by driving the system back to homeostasis after injury. They receive multidimensional signals within the central nervous system and between the brain and the systemic circulation. Processing this information allows astrocytes to regulate synapse formation and maintenance, cerebral blood flow, and blood-brain barrier integrity. Similarly, in response to stroke and other central nervous system disorders, astrocytes detect and integrate signals of neuronal damage and inflammation to regulate the neuroinflammatory response. Two direct regulatory mechanisms in the astrocyte arsenal are the ability to form both physical and molecular barriers that seal the injury site and localize the neuroinflammatory response. Astrocytes also indirectly regulate the inflammatory response by affecting neuronal health during the acute injury and axonal regrowth. This ability to regulate the location and degree of neuroinflammation after injury, combined with the long time course of neuroinflammation, makes astrocytic signaling pathways promising targets for therapies.

    View details for PubMedID 27677607

    View details for PubMedCentralID PMC5081110

  • Stroke, Inflammation and the Immune Response: Dawn of a New Era NEUROTHERAPEUTICS Becker, K. J., Buckwalter, M. 2016; 13 (4): 659–60

    View details for PubMedID 27677606

    View details for PubMedCentralID PMC5081111

  • Does B lymphocyte-mediated autoimmunity contribute to post-stroke dementia? Brain, behavior, and immunity Doyle, K. P., Buckwalter, M. S. 2016


    Post-stroke cognitive decline and dementia pose a significant public health problem, with 30% of stroke survivors suffering from dementia. The reason for this high prevalence is not well understood. Pathogenic B cell responses to the damaged CNS are one possible contributing factor. B-lymphocytes and antibodies are present in and around the stroke core of some human subjects who die with stroke and dementia, and mice that develop delayed cognitive dysfunction after stroke have clusters of B-lymphocytes in the stroke lesion, and antibody infiltration in the stroked hemisphere. The ablation of B-lymphocytes prevents post-stroke cognitive impairment in mice. Multiple drugs that target B cells are FDA approved, and so if pathogenic B cell responses are occurring in a subset of stroke patients, this is potentially treatable. However, it has also been demonstrated that regulatory B cells can be beneficial in mouse models of stroke. Consequently, it is important to understand the relative contribution of B-lymphocytes to recovery versus pathogenicity, and if this balance is heterogeneous in different individuals. Therefore, the purpose of this review is to summarize the current state of knowledge with regard to the role of B-lymphocytes in the etiology of post-stroke dementia.

    View details for DOI 10.1016/j.bbi.2016.08.009

    View details for PubMedID 27531189

  • Antibodies to myelin basic protein are associated with cognitive decline after stroke. Journal of neuroimmunology Becker, K. J., Tanzi, P., Zierath, D., Buckwalter, M. S. 2016; 295-296: 9-11


    B lymphocytes cause post-stroke cognitive decline in mice. We therefore evaluated the association between autoantibodies and post-stroke cognitive decline in a prospectively collected human cohort. The mini-mental state exam (MMSE) was administered 30, 90, 180, and 365days after stroke. Antibody titers to myelin basic protein (MBP), proteolipid protein, and several non-specific proteins were determined. Among 58 subjects with initial MMSE≥20 and at least 2 MMSE examinations in the year after stroke, cognitive decline (MMSE decrease ≥2) occurred in 10 (17%) subjects. In multivariate analysis, MBP antibody titers were the only independent predictor of cognitive decline (OR=9.02 [1.18, 68.90]; P=0.03).

    View details for DOI 10.1016/j.jneuroim.2016.04.001

    View details for PubMedID 27235342

  • Antibodies to myelin basic protein are associated with cognitive decline after stroke JOURNAL OF NEUROIMMUNOLOGY Becker, K. J., Tanzi, P., Zierath, D., Buckwalter, M. S. 2016; 295: 9-11


    B lymphocytes cause post-stroke cognitive decline in mice. We therefore evaluated the association between autoantibodies and post-stroke cognitive decline in a prospectively collected human cohort. The mini-mental state exam (MMSE) was administered 30, 90, 180, and 365days after stroke. Antibody titers to myelin basic protein (MBP), proteolipid protein, and several non-specific proteins were determined. Among 58 subjects with initial MMSE≥20 and at least 2 MMSE examinations in the year after stroke, cognitive decline (MMSE decrease ≥2) occurred in 10 (17%) subjects. In multivariate analysis, MBP antibody titers were the only independent predictor of cognitive decline (OR=9.02 [1.18, 68.90]; P=0.03).

    View details for DOI 10.1016/j.jneuroim.2016.04.001

    View details for Web of Science ID 000377822000002

    View details for PubMedCentralID PMC4884610

  • Glial Fibrillary Acidic Protein-Expressing Glia in the Mouse Lung ASN NEURO Suarez-Mier, G. B., Buckwalter, M. S. 2015; 7 (5)


    Autonomic nerves regulate important functions in visceral organs, including the lung. The postganglionic portion of these nerves is ensheathed by glial cells known as non-myelinating Schwann cells. In the brain, glia play important functional roles in neurotransmission, neuroinflammation, and maintenance of the blood brain barrier. Similarly, enteric glia are now known to have analogous roles in gastrointestinal neurotransmission, inflammatory response, and barrier formation. In contrast to this, very little is known about the function of glia in other visceral organs. Like the gut, the lung forms a barrier between airborne pathogens and the bloodstream, and autonomic lung innervation is known to affect pulmonary inflammation and lung function. Lung glia are described as non-myelinating Schwann cells but their function is not known, and indeed no transgenic tools have been validated to study them in vivo. The primary goal of this research was, therefore, to investigate the relationship between non-myelinating Schwann cells and pulmonary nerves in the airways and vasculature and to validate existing transgenic mouse tools that would be useful for studying their function. We focused on the glial fibrillary acidic protein promoter, which is a cognate marker of astrocytes that is expressed by enteric glia and non-myelinating Schwann cells. We describe the morphology of non-myelinating Schwann cells in the lung and verify that they express glial fibrillary acidic protein and S100, a classic glial marker. Furthermore, we characterize the relationship of non-myelinating Schwann cells to pulmonary nerves. Finally, we report tools for studying their function, including a commercially available transgenic mouse line.

    View details for DOI 10.1177/1759091415601636

    View details for Web of Science ID 000364740200001

    View details for PubMedID 26442852

    View details for PubMedCentralID PMC4601129

  • Albumin induces excitatory synaptogenesis through astrocytic TGF-beta/ALK5 signaling in a model of acquired epilepsy following blood-brain barrier dysfunction NEUROBIOLOGY OF DISEASE Weissberg, I., Wood, L., Kamintsky, L., Vazquez, O., Milikovsky, D. Z., Alexander, A., Oppenheim, H., Ardizzone, C., Becker, A., Frigerio, F., Vezzani, A., Buckwalter, M. S., Huguenard, J. R., Friedman, A., Kaufer, D. 2015; 78: 115-125


    Post injury epilepsy (PIE) is a common complication following brain insults, including ischemic and traumatic brain injuries. At present there are no means to identify the patients at-risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood-brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures, remains unknown. Here we demonstrate in-vitro and in-vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process, and highlight manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE.

    View details for DOI 10.1016/j.nbd.2015.02.029

    View details for PubMedID 25836421

  • Metronidazole-Induced Encephalopathy: Not Always a Reversible Situation NEUROCRITICAL CARE Hobbs, K., Stern-Nezer, S., Buckwalter, M. S., Fischbein, N., Caulfield, A. F. 2015; 22 (3): 429-436


    Metronidazole is a nitroimidazole antimicrobial drug prescribed to treat infections caused by anaerobic bacteria and protozoa. Uncommonly, it causes central nervous system (CNS) toxicity manifesting as metronidazole-induced encephalopathy (MIE).Case report.A 65-year-old woman with hepatitis B cirrhosis (Child-Pugh class C, MELD 21) developed progressive encephalopathy to GCS 4 during a 3-week course of metronidazole for cholecystitis. Initial MRI was consistent with CNS metronidazole toxicity, with symmetrical T2 hyperintensity and generally restricted diffusion in bilateral dentate nuclei, corpus callosum, midbrain, superior cerebellar peduncles, internal capsules, and cerebral white matter. Laboratory values did not demonstrate significant electrolyte shifts, and continuous EEG was without seizure. High-dose thiamine was empirically administered. Lumbar puncture was not performed due to coagulopathy and thrombocytopenia. Despite discontinuation of metronidazole and keeping ammonia levels near normal, the patient did not improve. MRI was repeated 1 week after discontinuation of metronidazole. Although there was decreased DWI hyperintensity in the dentate nuclei, diffuse T2 hyperintensity persisted and even progressed in the brainstem, basal ganglia, and subcortical white matter. Petechial hemorrhages developed in bilateral corticospinal tracts and subcortical white matter. T1 hypointensity appeared in the corpus callosum. She was transitioned to comfort measures only and died 12 days later.MIE is an uncommon adverse effect of treatment with metronidazole that characteristically affects the dentate nuclei but may also involve the brainstem, corpus callosum, subcortical white matter, and basal ganglia. While the clinical symptoms and neuroimaging changes are usually reversible, persistent encephalopathy with poor outcome may occur.

    View details for DOI 10.1007/s12028-014-0102-9

    View details for PubMedID 25561434

  • B-Lymphocyte-Mediated Delayed Cognitive Impairment following Stroke. journal of neuroscience Doyle, K. P., Quach, L. N., Solé, M., Axtell, R. C., Nguyen, T. V., Soler-Llavina, G. J., Jurado, S., Han, J., Steinman, L., Longo, F. M., Schneider, J. A., Malenka, R. C., Buckwalter, M. S. 2015; 35 (5): 2133-2145


    Each year, 10 million people worldwide survive the neurologic injury associated with a stroke. Importantly, stroke survivors have more than twice the risk of subsequently developing dementia compared with people who have never had a stroke. The link between stroke and the later development of dementia is not understood. There are reports of oligoclonal bands in the CSF of stroke patients, suggesting that in some people a B-lymphocyte response to stroke may occur in the CNS. Therefore, we tested the hypothesis that a B-lymphocyte response to stroke could contribute to the onset of dementia. We discovered that, in mouse models, activated B-lymphocytes infiltrate infarcted tissue in the weeks after stroke. B-lymphocytes undergo isotype switching, and IgM, IgG, and IgA antibodies are found in the neuropil adjacent to the lesion. Concurrently, mice develop delayed deficits in LTP and cognition. Genetic deficiency, and the pharmacologic ablation of B-lymphocytes using an anti-CD20 antibody, prevents the appearance of delayed cognitive deficits. Furthermore, immunostaining of human postmortem tissue revealed that a B-lymphocyte response to stroke also occurs in the brain of some people with stroke and dementia. These data suggest that some stroke patients may develop a B-lymphocyte response to stroke that contributes to dementia, and is potentially treatable with FDA-approved drugs that target B cells.

    View details for DOI 10.1523/JNEUROSCI.4098-14.2015

    View details for PubMedID 25653369

  • Ferumoxytol administration does not alter infarct volume or the inflammatory response to stroke in mice. Neuroscience letters Doyle, K. P., Quach, L. N., Arceuil, H. E., Buckwalter, M. S. 2015; 584: 236-240


    Ferumoxytol is an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle that is FDA-approved as an intravenous iron replacement therapy for the treatment of iron deficiency anemia in patients with chronic kidney disease. Ferumoxytol has also been used as a contrast agent for cerebral blood volume mapping by magnetic resonance imaging (MRI), which suggests it could be used for imaging hemodynamic abnormalities after stroke. However, circulating macrophages can internalize USPIOs, and recent data indicate that the accumulation of iron in macrophages can lead them to adopt the M1 pro-inflammatory phenotype. Therefore, the uptake of intravenously administered iron particles by circulating macrophages that home to the stroke core could potentially alter the inflammatory response to stroke. To test this possibility in vivo we administered a dose of ferumoxytol previously used to obtain cerebral blood volume maps in healthy humans by steady-state susceptibility contrast (SSC) MRI to BALB/cJ mice 48h after stroke and examined cytokine levels, microglial/macrophage activation, and lesion volume in the brain 5 days later. Treatment with ferumoxytol did not lead to any differences in these parameters. These data indicate that the use of ferumoxytol as a contrast agent for brain imaging after stroke does not alter the inflammatory response to stroke in mice, and is therefore unlikely to do so in human subjects.

    View details for DOI 10.1016/j.neulet.2014.10.041

    View details for PubMedID 25449870

  • Astrocytic transforming growth factor-beta signaling reduces subacute neuroinflammation after stroke in mice. Glia Cekanaviciute, E., Fathali, N., Doyle, K. P., Williams, A. M., Han, J., Buckwalter, M. S. 2014; 62 (8): 1227-1240


    Astrocytes limit inflammation after CNS injury, at least partially by physically containing it within an astrocytic scar at the injury border. We report here that astrocytic transforming growth factor-beta (TGFβ) signaling is a second, distinct mechanism that astrocytes utilize to limit neuroinflammation. TGFβs are anti-inflammatory and neuroprotective cytokines that are upregulated subacutely after stroke, during a clinically accessible time window. We have previously demonstrated that TGFβs signal to astrocytes, neurons and microglia in the stroke border days after stroke. To investigate whether TGFβ affects astrocyte immunoregulatory functions, we engineered "Ast-Tbr2DN" mice where TGFβ signaling is inhibited specifically in astrocytes. Despite having a similar infarct size to wildtype controls, Ast-Tbr2DN mice exhibited significantly more neuroinflammation during the subacute period after distal middle cerebral occlusion (dMCAO) stroke. The peri-infarct cortex of Ast-Tbr2DN mice contained over 60% more activated CD11b(+) monocytic cells and twice as much immunostaining for the activated microglia and macrophage marker CD68 than controls. Astrocytic scarring was not altered in Ast-Tbr2DN mice. However, Ast-Tbr2DN mice were unable to upregulate TGF-β1 and its activator thrombospondin-1 2 days after dMCAO. As a result, the normal upregulation of peri-infarct TGFβ signaling was blunted in Ast-Tbr2DN mice. In this setting of lower TGFβ signaling and excessive neuroinflammation, we observed worse motor outcomes and late infarct expansion after photothrombotic motor cortex stroke. Taken together, these data demonstrate that TGFβ signaling is a molecular mechanism by which astrocytes limit neuroinflammation, activate TGFβ in the peri-infarct cortex and preserve brain function during the subacute period after stroke.

    View details for DOI 10.1002/glia.22675

    View details for PubMedID 24733756

  • Astrocytic TGF-ß signaling limits inflammation and reduces neuronal damage during central nervous system Toxoplasma infection. Journal of immunology Cekanaviciute, E., Dietrich, H. K., Axtell, R. C., Williams, A. M., Egusquiza, R., Wai, K. M., Koshy, A. A., Buckwalter, M. S. 2014; 193 (1): 139-149


    The balance between controlling infection and limiting inflammation is particularly precarious in the brain because of its unique vulnerability to the toxic effects of inflammation. Astrocytes have been implicated as key regulators of neuroinflammation in CNS infections, including infection with Toxoplasma gondii, a protozoan parasite that naturally establishes a chronic CNS infection in mice and humans. In CNS toxoplasmosis, astrocytes are critical to controlling parasite growth. They secrete proinflammatory cytokines and physically encircle parasites. However, the molecular mechanisms used by astrocytes to limit neuroinflammation during toxoplasmic encephalitis have not yet been identified. TGF-β signaling in astrocytes is of particular interest because TGF-β is universally upregulated during CNS infection and serves master regulatory and primarily anti-inflammatory functions. We report in this study that TGF-β signaling is activated in astrocytes during toxoplasmic encephalitis and that inhibition of astrocytic TGF-β signaling increases immune cell infiltration, uncouples proinflammatory cytokine and chemokine production from CNS parasite burden, and increases neuronal injury. Remarkably, we show that the effects of inhibiting astrocytic TGF-β signaling are independent of parasite burden and the ability of GFAP(+) astrocytes to physically encircle parasites.

    View details for DOI 10.4049/jimmunol.1303284

    View details for PubMedID 24860191