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Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues—such as incomplete mechanistic knowledge and faulty clinical trial design—a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.

Ischemic stroke is a serious cerebrovascular event that results in cell death, blood-brain barrier dysfunction, tissue degradation, and inflammation, often leading to permanent disability or death. As the incidence of ischemic stroke continues to rise globally, it is crucial to examine the mechanisms of the various proteins and molecules contributing to worsened patient outcome and recovery. Cathepsin L, a cysteine protease known for degrading tissues in lysosomes and elsewhere, may play a role in brain tissue loss and inflammation after stroke. Studies have suggested that cathepsin L appears in the ischemic core shortly after stroke is induced. Using immunohistochemical staining, mass spectrometry, and other assays, the increase of cathepsin L in the brain was correlated with extracellular matrix and perlecan degradation after ischemic stroke. Additionally, injection of a cathepsin L inhibitor significantly reduced brain infarct size and improved functional scores. More research is needed to elucidate cathepsin L's role in post-stroke inflammation and brain damage, in order to further explore the factors contributing to worsened patient outcome after ischemic stroke and work toward finding better therapeutic interventions.

Bone marrow mononuclear cells (MNCs) attenuate secondary degeneration and enhance recovery in stroke animal models. In a nonrandomized clinical trial, we imaged 37 patients with stroke: 17 patients treated with MNCs (treated) and 20 patients who received standard of care (nontreated) at 1, 3, and 12 months onset of stroke on 3.0T MRI system. Three‐dimensional anatomical and diffusion tensor images were obtained. The integrity of the corticospinal tract was assessed by measuring absolute and relative fractional anisotropy (FA) and mean diffusivity (MD) in the rostral pons (RP), posterior limb of the internal capsule, and corona radiata by drawing regions of interest. Infarct volume and stroke severity, which was assessed via the NIH Stroke Scale (NIHSS), were higher in the MNC group compared with the nontreated patients, which is a major limitation. Overall, the relative FA (rFA) of the nontreated patients exhibited continued reduction and an increase in relative MD (rMD) from 1 to 12 months, whereas despite larger infarcts and higher severity, treated patients displayed an increase in rFA from 3 to 12 months and no change in rMD. Contrary to the nontreated group, the treated patients' rFA was also significantly correlated (P, Autologous bone‐marrow mononuclear cells were intravenously administered in patients with acute ischemic stroke as procedure outline in panel (A). The nontreated patients were recruited separately. Both groups were imaged three times over year and neuroimaging biomarkers were developed. Integrity of the ipsilesional and contralesional cortical spinal tracts (CST) were evaluated via diffusion tensor imaging (DTI), in the rostral pons (RP), posterior limb of internal capsule (PLIC), and corona radiata (CR) as illustrated in panel (B). The relative fractional anisotropy (rFA), an imaging marker of white matter integrity, was serially quantified in these three regions in each group as shown in panel (C1), (C2), and (C3). Despite larger infarct size and severity of the treated group, the rFA either increased or stabilized as compared to the nontreated patients.

Aneurysmal subarachnoid hemorrhage (aSAH) causes a robust inflammatory response which leads worse brain injury and poor outcomes. We investigated if stimulation of nicotinic acetylcholine α7 receptors (α7-AChR) (receptors shown to have anti-inflammatory effects) would reduce inflammation and improve outcomes. To investigate the level of peripheral inflammation after aSAH, inflammatory markers were measured in plasma samples collected in a cohort of aSAH patients. To study the effect of α7-AChR stimulation, SAH was induced in adult mice which were then treated with a α7-AChR agonist, galantamine, or vehicle. A battery of motor and cognitive tests were performed 24 h after subarachnoid hemorrhage. Mice were euthanized and tissue collected for analysis of markers of inflammation or activation of α7-AChR-mediated transduction cascades. A separate cohort of mice was allowed to survive for 28 days to assess long-term neurological deficits and histological outcome. Microglia cell culture subjected to hemoglobin toxicity was used to assess the effects of α7-AChR agonism. Analysis of eighty-two patient plasma samples confirmed enhanced systemic inflammation after aSAH. α7-AChR agonism reduced neuroinflammation at 24 h after SAH in male and female mice, which was associated with improved outcomes. This coincided with JAK2/STAT3 and IRAK-M activity modulations and a robust improvement in neurological/cognitive status that was effectively reversed by interfering with various components of these signaling pathways. Pharmacologic inhibition partially reversed the α7-AChR agonist’s benefits, supporting α7-AChR as a target of the agonist’s therapeutic effect. The cell culture experiment showed that α7-AChR agonism is directly beneficial to microglia. Our results demonstrate that activation of α7-AChR represents an attractive target for treatment of SAH. Our findings suggest that α7-AChR agonists, and specifically galantamine, might provide therapeutic benefit to aSAH patients.

In this minireview we discuss the role of lactoferrin (LTF) in detoxifying hematoma after intracerebral hemorrhage (ICH). Subsequent to ICH, neutrophils enter the ICH-affected brain, where they release various granule contents, including LTF. LTF is an iron-binding glycoprotein that binds Fe3+ with high affinity. Unlike other iron-binding proteins, LTF can retain Fe3+ at the low pH associated with inflamed tissue. LTF’s ability to sequester Fe3+ is of particular importance to ICH pathogenesis, because large quantities of free iron, which is pro-oxidative and pro-inflammatory, are generated in the ICH-affected brain owing to blood hemolysis. LTF delivered to ICH-affected brain, either as a therapeutic agent or through infiltrated polymorphonuclear neutrophils (cells containing high levels of LTF), could limit the pathogenesis of ICH. LTF is a protein with a high isoelectric point (8.7), a property that enables it to bind to negatively-charged apoptotic cells or proteins. Here, LTF could act as a bridging molecule that couples the apoptotic cells to LTF receptors on the cellular membranes of microglia/macrophages to facilitate the efferocytosis/erythrophagocytosis of apoptotic cells and damaged red blood cells. Thus, by virtue of sequestrating iron and facilitating efferocytosis, LTF may contribute to hematoma detoxification and hematoma/inflammation resolution after ICH.

Ischemic stroke is major cause of disability and mortality worldwide, and aging is strong risk factor for poor post-stroke outcome. Neutrophils traffic rapidly to the brain following ischemic stroke, and recent evidence has suggested that aging may alter neutrophil function after tissue injury. In this study, we hypothesize that aging enhances the pro-inflammatory function of neutrophils, directly contributing to the poorer outcomes seen in aging patients. We utilized demographic data and biological specimens from ischemic stroke patients and an experimental mouse model to determine the correlation between age, neutrophil function and stroke outcomes. In ischemic stroke patients, age was associated with increased mortality and morbidity and higher levels of neutrophil-activating cytokines. In mice, aged animals had higher stroke mortality and morbidity, higher levels of neutrophil-activating cytokines and enhanced generation of neutrophil reactive oxygen species compared to young mice. Finally, depletion of neutrophils via a specific monoclonal antibody after ischemic stroke led to long-term benefits in functional outcome in aged male and female animals, with no benefit observed in young. These results demonstrate that aging is associated with augmented neutrophil pathogenicity in ischemic stroke, and that neutrophil-targeted therapies may confer greater benefit in aged subjects.

Innate lymphoid cells are the innate counterpart to CD4+ T cells that are mediated by the same transcription factors and produce similar cytokines. ILCs are being investigated in many different disease states, but the field current lacks foundational information on ILC representation whether it be in tissues, between males and females, or in aging as these are all vital components in disease etiology and severity. Our descriptive study used flow cytometry to characterize ILCs compared to the entire CD45+ (e.g., lymphocyte) and lineage negative (e.g., ILC) compartments to understand their homeostatic balance and plasticity. Moreover, we defined ILC2 expression and subsets based on their cytokine production and created several mathematical models to elucidate the correlation of extra- and intra-cellular ILC2 markers from least to most complex. ILC studies would benefit from more unbiased, holistic experiments including RNA-seq and mass spectroscopy to further define ILCs in steady state before adding more complex pathways like different disease states to enhance translational value and therapeutic targeting of these cells.

Astrocytes release functional mitochondria (Mt) that play regulatory and prosurvival functions on entering adjacent cells. We recently demonstrated that these released Mts could enter microglia to promote their reparative/prophagocytic phenotype that assists in hematoma cleanup and neurological recovery after intracerebral hemorrhage (ICH). However, the relevance of astrocytic Mt transfer into neurons in protecting brain after ICH is unclear. Here, we found that ICH causes a robust increase in superoxide generation and elevated oxidative damage that coincides with loss of the mitochondrial enzyme manganese superoxide dismutase (Mn-SOD). The damaging effect of ICH was reversed by intravenous transplantation of astrocytic Mt, which on entering the brain (and neurons), restored Mn-SOD levels and reduced neurological deficits in male mice subjected to ICH. Using an in vitro ICH-like injury model in cultured neurons, we established that astrocytic Mt on entering neurons prevented reactive oxygen species-induced oxidative stress and neuronal death by restoring neuronal Mn-SOD levels while at the same time promoted neurite extension and upregulation of synaptogenesis-related gene expression. Furthermore, we found that Mt genome-encoded small peptide humanin, which is normally abundant in Mt, could simulate Mt-transfer effect on neuronal Mn-SOD expression, oxidative stress, and neuroplasticity under ICH-like injury. This study demonstrates that adoptive astrocytic Mt transfer enhances neuronal Mn-SOD-mediated antioxidative defense and neuroplasticity in the brain, which potentiate functional recovery following ICH. SIGNIFICANCE STATEMENT Mitochondrial dysfunction and antioxidant defense play essential roles in brain damage after ICH. Astrocytes release functional Mt that enters adjacent cells to help brain homeostatic function. Here, we show that systemic transplantation of astrocytic Mt restores ICH-impaired neuronal antioxidative defense, enhances neurite outgrowth, and improves stroke recovery after ICH. Our study suggests that systemic transplantation of astrocytic Mt could be considered as a novel and potentially promising strategy for ICH treatment.

This meeting abstract was removed due to the OA licensing requirements of this journal. The full abstract is listed here : https://www.svin.org/files/SVIN_2021_Abstracts_for_Web.pdf

Cellular therapy is a promising investigational modality to enhance poststroke recovery. We conducted a single-arm, phase I clinical trial to determine the safety and feasibility of intravenous (IV) administration of autologous bone marrow mononuclear cells (MNCs) after acute ischemic stroke (AIS). Patients with moderate severity of AIS underwent bone marrow harvest followed by IV reinfusion of MNCs within 24–72 hours of onset. A target dose of 10 million cells per kilogram was chosen based on preclinical data. Patients were followed up daily during hospitalization and at 1, 3, 6, 12, and 24 months for incidence of adverse events using laboratory, clinical (12 months), and radiological (24 months) parameters. The trial was powered to detect severe adverse events (SAEs) with incidences of at least 10% and planned to enroll 30 patients. Primary outcomes were study-related SAEs and the proportion of patients successfully completing study intervention. A propensity score-based matched control group was used for the estimation of effect size (ES) for day-90 modified Rankin score (mRS). There were no study-related SAEs and, based on a futility analysis, enrolment was stopped after 25 patients. All patients successfully completed study intervention and most received the target dose. Secondary analysis estimated the ES to be a reduction of 1 point (95% confidence interval: 0.33–1.67) in median day-90 mRS for treated patients as compared with the matched control group. Bone marrow harvest and infusion of MNCs is safe and feasible in patients with AIS. The estimated ES is helpful in designing future randomized controlled trials. Stem Cells 2019;37:1481–1491

Excitotoxicity and microglia/macrophage over-activation are the important pathogenic steps in brain damage caused by ischemic stroke. Recent studies from our group suggest that the neurons in ischemic penumbra generate an anti-inflammatory cytokine, interleukin-4 (IL-4). This neuron-produced IL-4 could subsequently convert surrounding microglia/macrophages to a reparative (M2)-phenotype. The present study was designed to establish the mechanisms by which neurons under transient ischemic condition produce/secrete IL-4. We employed primary rat cortical neurons and a validated in vitro ischemic injury model involving transient oxygen–glucose deprivation (OGD). We discovered that only sublethal OGD induces IL-4 production/secretion by neurons. We then showed that excitotoxic stimulus (an integral component of OGD-mediated damage) involving N-methyl-D-aspartate (NMDA), and not kainate receptor, triggers neuronal IL-4 production/release. Of note, oxidative stress or pro-apoptotic stimuli did not induce IL-4 production by neurons. Next, using the calcineurin inhibitor FK506, we implicated this phosphatase in activation of the nuclear factor of activated T-cells (NFAT; a transcription factor activated through calcineurin-mediated dephosphorylation) and propose that this pathway is involved in transcriptional upregulation of the IL-4 synthesis in NMDA-treated neurons. Finally, using a transfer of culture medium from NMDA-conditioned neuron to microglia, we showed that the neuronal IL-4 can polarize microglia toward a restorative, phagocytic phenotype.

Aging affects immunologic responses by a global suppression of the immune system, including dysregulation of cytokine mediators, leading to increased inflammation throughout all systems, termed inflammaging. However, understanding mechanisms of healthy aging can bypass this effect. Inflammaging also leads to poor outcomes during brain injury, making immune-targeting therapeutics tantamount to overall brain health and longevity. Two candidate immune cells microglia and group 2 innate lymphoid cells (ILC2s) may control immune responses in the aged brain Therefore, we hypothesized that ILC2s are the brains’ gatekeepers of microglia polarization to a reparative, M2-like phenotype under: (1) homeostatic conditions and (2) with aging. To address this, we used flow cytometry to quantify ILC2s between young and aged male and female C57Bl6/J mice, showing an age-related increase in aged males. Moreover, when we stimulated ILC2s, the data indicated an age-related increase in cytokine production in males. We also utilized conditioned media transfer experiments from stimulated young and aged ILC2s to quiescent microglia and assessed their morphologic, genomic, proteomic, and functional changes using microscopy, qRT-PCR, Western immunoblotting, and phagocytosis assays. We showed more complex microglia morphology (indicating microglial priming) and an increase in reparative mRNA, proteins, and phagocytic function in microglia, which was augmented by aged ILC2s. Taken together, we have defined the capacity of ILC2 soluble factors to polarize microglia to a reparative phenotype and maintain this in aging. Supported by the NIH NINDS F31NS118983

Background and Purpose: Marrow stromal cells (MSCs) are being tested in clinical trials for stroke patients. MSCs appear to promote recovery through secretomes that promote modulation of immune cells, including myeloid phagocytes. Many stroke patients have comorbidities such as metabolic syndrome, hypertension, hypercholesterolemia, and diabetes for which they are prescribed medications that might affect the function of MSCs and monocytes (Mo) when they are administered in stroke patients. We studied the effects of the two most commonly prescribed stroke medications, statin and statin plus aspirin, on the secretomes of MSCs and their modulation of Mo derived from stroke patients.Methods: Human MSCs, Mo and their co-cultures were exposed to atorvastatin or atorvastatin plus aspirin followed by secretome analysis at 24 h. Monocytes were isolated from healthy controls as well as stroke patients with NIHSS ranging from 11 to 20. Secretome composition was measured using multiplex immunoassay. We used MTT assay to measure proliferation of monocytes. The mixed model was used to analyze experimental data. p-values less than 0.05 were considered significant.Results: Atorvastatin and aspirin combination increased the release of IL-1RA from stroke Mo. In MSCs, atorvastatin and aspirin combination reduced the release of pro-inflammatory cytokines such as IL-6, IL-8, MCP-1 and IFN-γ. Atorvastatin alone reduced the release of IL-6, IL-8 and MCP-1 from co-cultures of stroke monocytes and MSCs. Combination of atorvastatin and aspirin had additive effect on reducing the secretion of IL-6 from co-cultures of stroke Mo and MSCs.Conclusion: Atorvastatin, alone and in combination with aspirin can promote anti-inflammatory effect by modulating the secretome profile of Mo and MSCs. Our results suggest that stroke trials involving the use of intravenous MSCs should consider the effect of aspirin and atorvastatin, both of which are administered to the majority of hospitalized ischemic stroke patients.

Aneurysmal subarachnoid hemorrhage (aSAH) causes a robust inflammatory response which leads worse brain injury and poor outcomes. We investigated if stimulation of nicotinic acetylcholine α(7) receptors (α(7)-AChR) (receptors shown to have anti-inflammatory effects) would reduce inflammation and improve outcomes. To investigate the level of peripheral inflammation after aSAH, inflammatory markers were measured in plasma samples collected in a cohort of aSAH patients. To study the effect of α(7)-AChR stimulation, SAH was induced in adult mice which were then treated with a α(7)-AChR agonist, galantamine, or vehicle. A battery of motor and cognitive tests were performed 24 h after subarachnoid hemorrhage. Mice were euthanized and tissue collected for analysis of markers of inflammation or activation of α(7)-AChR-mediated transduction cascades. A separate cohort of mice was allowed to survive for 28 days to assess long-term neurological deficits and histological outcome. Microglia cell culture subjected to hemoglobin toxicity was used to assess the effects of α(7)-AChR agonism. Analysis of eighty-two patient plasma samples confirmed enhanced systemic inflammation after aSAH. α(7)-AChR agonism reduced neuroinflammation at 24 h after SAH in male and female mice, which was associated with improved outcomes. This coincided with JAK2/STAT3 and IRAK-M activity modulations and a robust improvement in neurological/cognitive status that was effectively reversed by interfering with various components of these signaling pathways. Pharmacologic inhibition partially reversed the α(7)-AChR agonist’s benefits, supporting α(7)-AChR as a target of the agonist’s therapeutic effect. The cell culture experiment showed that α(7)-AChR agonism is directly beneficial to microglia. Our results demonstrate that activation of α(7)-AChR represents an attractive target for treatment of SAH. Our findings suggest that α(7)-AChR agonists, and specifically galantamine, might provide therapeutic benefit to aSAH patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-021-01052-3.

Background: Systemic administration of bone marrow derived stromal cells (MSCs) releases a broad range of trophic factors mediating stroke recovery. How MSCs release these factors may depend on the state of the circulation after systemic administration. We assessed whether the secretomes of MSCs, upon exposure to peripheral blood monocytes (Mo), differs depending on the temporal course when the Mo were isolated after stroke. Methods: Peripheral blood mononuclear cells (PBMCs) from stroke patients was isolated at 24h (N=10) and day 5-7 (N=7) after stroke, and from healthy controls (N=5) using Ficoll gradient. CD14+ Mo were isolated using indirect magnetic beads labelling. Contact and Trans-well co-culture assays were established using 100,000 CD14+ Mo and MSCs each in a 0.8um transwell system. After 24h, media was collected and analyzed for secretome using multiplex cytokine assay. Subtypes of Mo were evaluated using flow cytometry. Results: There was a significant increase in non-classical (CD14+CD16++) Mo and a significant decrease in classical (CD14++CD16-) Mo at Day5-7 after stroke as compared to 24h after stroke (Fig). When Day5-7 CD14+ Mo were co-cultured with MSCs, we found a significant reduction in inflammatory IL-1β and TNF-α, and a significant increase in anti-inflammatory IL-4 as compared to co-cultures of MSCs with healthy Mo. This effect was more pronounced in transwell (vs contact) co-cultures. Co-cultures of MSCs and Mo from controls or from patients 24h after stroke led to reductions in TNF-α and upregulation in IL-4 compared with Mo alone. There were no differences in effect of MSCs co-cultures with healthy vs 24h stroke Mo. Conclusion: Our study indicates that the immunomodulatory effect of MSCs may be dependent on the phenotype of Mo in the peripheral circulation of stroke patients. Hence, depending on the temporal course after stroke, the systemic circulation of stroke patients may differentially regulate MSCs to release soluble factors.

Background: Multipotent adult progenitor cells (MAPC) are an adherent adult stem cell being evaluated as a treatment for ischemic stroke in humans under the name MultiStem®. However, the efficacy of MAPC cells for the treatment of intracerebral hemorrhage (ICH), the most devastating form of stroke for which there is no effective treatment, is not clear Method: The therapeutic efficacy of MAPC administration was evaluated in both autologous blood injection (ABI) and collagenase (COL) rat ICH models. We treated rats intravenously with 1.2x10 6 cells (sub-optimal dose based on MAPC efficacy in ischemic stroke) and 1.2x10 7 cells (optimal dose) at either 2 or 24h after ICH, and used 2 different doses of collagenase to better understand the dose responses. Outcome measurements included 4 sensorimotor tests (up to 28d), ventricular hypertrophy, spleen size, and body weight (N=128 rats tested across 4 separate experiments). Results: MAPC offered a robust benefit in both ICH models in a dose-dependent fashion. (1) ABI model: at the sub-optimal dose MAPCs had no significant effect on behavioral performance, but effectively reduced ventricular hypertrophy. At an optimal dose, MAPCs at 2h or 24h after ICH, robustly reduced deficits in all 4 behavioral tests, and reduced ventricular hypertrophy by 59% and 35% in 2h and 24h post-treatment groups, respectively. No difference in body weight and spleen size was observed. (2) COL model: MAPC administered 2h after high collagenase dose, reduced hematoma volume (hemispheric hemoglobin level), as measured at 48h after collagenase injection. In addition, MAPC administration significantly reduced neurological deficit in the COL model. Conclusions: MAPC provide a uniquely robust therapeutic effect on clinically relevant neurological and morphological outcomes in two different ICH models. MAPC also reduced bleeding in the COL model, suggesting the potential for MAPC as a safe acute therapeutic treatment after ICH. In addition to having beneficial effects on recovery processes, MAPC could be further evaluated as a candidate to limit the hematoma enlargement during the initial postictal period. We are currently investigating the mechanism of MAPC-induced post-ICH recovery as well as hemostasis using tissue microarray analysis.

Background: Intracerebral hemorrhage (ICH) constitutes upto 40% mortality in first 30 days. Early identification of predictors of hematoma expansion (HE) may improve efforts to prevent its occurrence and improve clinical outcome. Methods: We identified patients with ICH and follow-up imaging. HE was defined as a combination of absolute volume increase of 6cc, new IVH, or proportional increase of 33% in our dataset on 72h follow up scan. Presence of IVH was also included in hematoma expansion. We evaluated the predictive ability of 3 machine learning classifiers, Random Forest, Support Vector Machine (with RBF kernel) and Logistic regression (with L1 regularization). The evaluation was done using a K-fold stratified cross validation to avoid overfitting. K was selected to be the number of subjects with HE. The features employed by classifiers were entirely based on the baseline imaging: Hematoma volume, Systolic BP, Diastolic BP, Black hole signs, Island signs, Blend signs, Fluid level, Swirl signs, Spot signs. Results: Our dataset comprised of 91 patients (n=21 HE, n=70 no HE). According to the area under the ROC (AUC), the two top performing classifiers were Support Vector Machine (AUC=0.66 CI 0.50-0.79) and Logistic Regression (AUC=0.64 CI 0.49-0.80). The statistical significance of the prediction is confirmed by the Mann-Whitney U test, p=0.01 and p=0.04 respectively. Random Forest did not reach statistical significance. Finally, we evaluated what were the highest and lowest weighted features across the cross-validation with Logistic Regression. The 3 top features were: presence of black hole and island signs and the systolic blood pressure. The 3 least useful features were: presence of spot and swirl signs and hematoma volume. Conclusion: Using our cohort, we developed a machine learning algorithm that predicts hematoma expansion using imaging features and blood pressure. MBL provided better sensitivity of these imaging markers compared with previous studies.

Intracerebral hemorrhage (ICH) is the deadliest form of stroke for which there is no effective treatment, despite an endless number of pre-clinical studies and clinical trials. The obvious therapeutic target is the neutralization of toxic products of red blood cell (RBC) lysis that lead to cytotoxicity, inflammation, and oxidative damage. We used rigorous approaches and translationally relevant experimental ICH models to show that lactoferrin-(LTF)-based monotherapy is uniquely robust in reducing brain damage after ICH. Specifically, we designed, produced, and pharmacokinetically/toxicologically characterized an optimized LTF, a fusion of human LTF and the Fc domain of human IgG (FcLTF) that has a 5.8-fold longer half-life in the circulation than native LTF. Following dose-optimization studies, we showed that FcLTF reduces neurological injury caused by ICH in aged male/female mice, and in young male Sprague Dawley (SD) and spontaneously hypertensive rats (SHR). FcLTF showed a remarkably long 24-h therapeutic window. In tissue culture systems, FcLTF protected neurons from the toxic effects of RBCs and promoted microglia toward phagocytosis of RBCs and dead neurons, documenting its pleotropic effect. Our findings indicate that FcLTF is safe and effective in reducing ICH-induced damage in animal models used in this study.

Background: Inflammatory responses after stroke consists of central and peripheral immune responses. The role of the spleen after stroke is well-known, however the role of the lungs has not been studied in detail. We explored the relation between stroke severity and immunomodulatory changes in lung endothelial cells. Methods: Human pulmonary endothelial cells (hPECs, Cell Biologics) were cultured at passage 3. Serum from stroke patients with NIH Stroke Scale (NIHSS) severity ranging from 0 to 20 was collected at 24 hours after stroke. hPECs were exposed to media with 1) 10% FBS alone (N=6), 2) 10% serum from stroke patients (N=72), or 3) 10% serum from stroke mimic patients (N=6). After 3 hour of exposure, fresh media was added and secretomes from hPECs were measured after 24 hours. We isolated RNA from hPECs after 3 hour of serum exposure and measured gene expression (N=6 for each group). Secretome and gene changes in hPECs were analyzed based on stroke severity, tPA treatment, and co-morbidities. Results: Serum from stroke patients reduced the secretion of IL-8, MCP-1 and Fractalkine (p Conclusion: Exposure of hPECs to serum from stroke patients alters their immunomodulatory properties. Higher severity of stroke leads to more protective response from hPECs by reducing the secretion of pro-inflammatory factors, while increasing the secretion of anti-inflammatory factors.

Introduction: White matter tract (WMT) injury occurs in patients with acute cerebrovascular disorders. In this study, we elucidate longitudinal differences in mechanism of injury and repair between ischemic stroke (ISC) and intracerebral hemorrhage (ICH). Methods: Twenty patients (10 ISC and ICH) were prospectively imaged at 1, 3, and 12 months of onset on a 3T MRI. 3D anatomical and DTI images were obtained and integrity of the corticospinal tract (CST) assessed at the ipsi and contralesional posterior limb of internal capsule (PLIC). Fractional anisotropy (FA), mean diffusivity (MD) and pixel volume were recorded. A linear regression model was applied for statistical analysis. Results: ISC group had 4 men, 6 women whereas ICH group had 7 men, 3 women, both with average age 52. Baseline NIHSS in ISC was 11 (IQR=4.5-20) and ICH 6 (IQR=2-13). All lesions were unilateral, hemispheric, completely subcortical or with a significant subcortical component. The average lesion and hematoma volume at 1 month was 37 and 39 cc in ISC and ICH, respectively. The MD in the PLIC of the ISC increased from 1 to 3m (P Conclusions: ISC and ICH display unique patterns of WMT changes over one year in which ICH injury reflects a compression of the CST that resolves over time, while in ISC our data show degeneration and microstructural injury. These changes reflect different mechanisms of injury and remodeling on a cellular level. A better understanding of these changes could improve recovery therapies. Larger studies are needed to better characterize long term WMT changes in IS and ICH.

Background and Purpose— Phagocytic cells, such as microglia and blood-derived macrophages, are a key biological modality responsible for phagocytosis-mediated clearance of damaged, dead, or displaced cells that are compromised during senescence or pathological processes, including after stroke. This process of clearance is essential to eliminate the source of inflammation and to allow for optimal brain repair and functional recovery. Transcription factor, RXR (retinoic-X-receptor) is strongly implicated in phagocytic functions regulation, and as such could represent a novel target for brain recovery after stroke. Methods— Primary cultured microglia and bone marrow macrophages were used for phagocytic study. Mice with deleted RXR-α in myeloid phagocytes (Mac-RXR-α −/− ) were subjected to transient middle cerebral artery occlusion to mimic ischemic stroke and then treated with RXR agonist bexarotene. RNA-sequencing and long-term recovery were evaluated. Results— Using cultured microglia, we demonstrated that the RXR-α promotes the phagocytic functions of microglia toward apoptotic neurons. Using mice with deleted RXR-α in myeloid phagocytes (Mac-RXR-α −/− ), we have shown that despite behaving similarly to the control at early time points (up to 3 days, damage established histologically and behaviorally), these Mac-RXR-α −/− mice demonstrated worsened late functional recovery and developed brain atrophy that was larger in size than that seen in control mice. The RXR-α deficiency was associated with reduced expression of genes known to be under control of the prominent transcriptional RXR partner, PPAR (peroxisome proliferator-activated receptor)-γ, as well as genes encoding for scavenger receptors and genes that signify microglia/macrophages polarization to a reparative phenotype. Finally, we demonstrated that the RXR agonist, bexarotene, administered as late as 1 day after middle cerebral artery occlusion, improved neurological recovery, and reduced the atrophy volume as assessed 28 days after stroke. Bexarotene did not improve outcome in Mac-RXR-α −/− mice. Conclusions— Altogether, these data suggest that phagocytic cells control poststroke recovery and that RXR in these cells represents an attractive target with exceptionally long therapeutic window.

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20925667 for Optimized lactoferrin as a highly promising treatment for intracerebral hemorrhage: Pre-clinical experience by Xiurong Zhao, Marian Kruzel, Shun-Ming Ting, Guanghua Sun, Sean I Savitz and Jaroslaw Aronowski in Journal of Cerebral Blood Flow & Metabolism

Marrow stromal cells (MSCs) are in different stages of clinical trials for stroke patients. MSCs are proposed to promote recovery through the release of secretomes that modulate the function of beneficial immune cells. The majority of stroke patients have comorbidities including hypertension, for which they are prescribed antihypertensive medications that might affect the function of MSCs, when they are administered in stroke patients. Here, we studied the effects of common antihypertensive medications on the secretomes of human MSCs and their modulation of human monocytes (Mo) derived from stroke patients. MTT assay was used to assess the proliferation of MSCs after they were exposed to increased levels of antihypertensive medications. MSCs were exposed to the following medications: atenolol, captopril, and losartan. Monocytes were isolated from stroke patients with NIHSS ranging from 11 to 20 and from healthy controls. MSC-Mo cocultures were established, and a secretome profile was analyzed using the Magpix Multiplex cytokine array from Luminex technology. The linear mixed-effect model was used for statistical analysis. All analyses were performed using SAS 9.4, and p values less than 0.05 were considered significant. At clinically relevant levels, there was no change in MSC proliferation after exposure to atenolol, captopril, or losartan. Atenolol increased IL-1RA in stroke-Mo and decreased IL-8 secretion from MSCs indicating an anti-inflammatory effect of atenolol on secretomes of these cells. Captopril increased IL-8 from stroke-Mo and increased IL-6, IL-8, and MCP-1 secretions from MSCs. Captopril also increased IL-6 secretion from cocultures of stroke-Mo and MSCs indicating a strong proinflammatory effect on MSCs and their interaction with Mo. Atenolol increased the secretion of IL-8 and MCP-1 while captopril increased the secretion of IL-6 and MCP-1 from MSCs. Losartan decreased the release of IL-6 from MSCs. Losartan reduced MCP-1 and TNF-α from stroke-Mo and reduced IL-8 from cocultures of stroke-Mo and MSCs. Our results show that antihypertensive medications such as atenolol, captopril, and losartan, at concentrations comparable to doses prescribed for patients hospitalized for acute stroke, modulate the secretome profile of MSCs and their modulatory effects on target immune cells. Our results suggest that stroke trials involving the use of intravenous MSCs should consider the effect of these antihypertensive drugs administered to stroke patients.

Xenon (Xe), a noble gas, has promising neuroprotective properties with no proven adverse side-effects. We evaluated neuroprotective effects of Xe delivered by Xe-containing echogenic liposomes (Xe-ELIP) via ultrasound-controlled cerebral drug release on early brain injury following subarachnoid hemorrhage (SAH). The Xe-ELIP structure was evaluated by ultrasound imaging, electron microscopy and gas chromatography-mass spectroscopy. Animals were randomly divided into five groups: Sham, SAH, SAH treated with Xe-ELIP, empty ELIP, or Xe-saturated saline. Treatments were administrated intravenously in combination with ultrasound application over the common carotid artery to trigger Xe release from circulating Xe-ELIP. Hematoma development was graded by SAH scaling and quantitated by a colorimetric method. Neurological evaluation and motor behavioral tests were conducted for three days following SAH injury. Ultrasound imaging and electron microscopy demonstrated that Xe-ELIP have a unique two-compartment structure, which allows a two-stage Xe release profile. Xe-ELIP treatment effectively reduced bleeding, improved general neurological function, and alleviated motor function damage in association with reduced apoptotic neuronal death and decreased mortality. Xe-ELIP alleviated early SAH brain injury by inhibiting neuronal death and bleeding. This novel approach provides a noninvasive strategy of therapeutic gas delivery for SAH treatment.

BACKGROUND: Animal models have demonstrated the deleterious contribution of splenic immunocytes on secondary brain injury after stroke. While previous work has demonstrated splenic contraction (SC) in patients with acute ischemic stroke (AIS) and intracranial hemorrhage (ICH), no clinical studies have examined the relationship between the systemic inflammatory response syndrome (SIRS) with SC in stroke patients. METHODS: This is a retrospective analysis of a previous prospective observational study where daily spleen sizes were evaluated in 178 acute stroke patients. Spleen contraction was based on previously established normograms of healthy volunteers from the same study. SC from the first 24 hrs of stroke onset was evaluated against criteria for SIRS for the first 5 days of admission after AIS. RESULTS: 91patients had verified AIS without concurrent infection at admission. SIRS was not associated with SC at admission. African American patients with early SIRS had higher odds of having SC. Older patients with persistent SIRS at 72 hrs had lower odds of SC. At 48 hrs, there was significantly higher lymphocytosis and lower neutrophils present in patients with SC. Patients with SIRS at 72 hrs were more likely to have worse discharge mRS. CONCLUSION: This study provides evidence for an association among SC and SIRS in African American patients suggesting that spleen changes could be a biomarker for detecting SIRS in this population. Our data also indicate a counter association between SC and a lack of SIRS in patients older than 75. Further studies are needed to ascertain how age affects this association.

Subarachnoid hemorrhage (SAH) results in severe neuronal dysfunction and degeneration. Since the nicotinic acetylcholine α(7) receptors (α(7)-AChR) are involved in neuronal function and survival, we investigated if stimulation of α(7)-AChR would promote neuronal survival and improve behavioral outcome following SAH in mice. Male mice subjected to SAH were treated with either galantamine (α(7)-AChR agonist) or vehicle. Neurobehavioral testing was performed 24 hours after SAH, and mice were euthanized for analysis of neuronal cell death or a cell survival (PI3K/Akt) signaling pathway. Neuron cell cultures were subjected to hemoglobin toxicity to assess the direct effects of α(7)-AChR agonism independent of other cells. Treatment with the α(7)-AChR agonist promoted neuronal survival and improved functional outcomes 24 hours post-SAH. The improved outcomes corresponded with increased PI3K/Akt activity. Antagonism of α(7)-AChR or PI3K effectively reversed galantamine’s beneficial effects. Tissue from α(7)-AChR knockout mice confirmed α(7)-AChR’s role in neuronal survival after SAH. Data from the neuronal cell culture experiment supported a direct effect of α(7)-AChR agonism in promoting cell survival. Our findings indicate that α(7)-AChR is a therapeutic target following SAH which can promote neuronal survival, thereby improving neurobehavioral outcome. Thus, the clinically relevant α(7)-AChR agonist, galantamine, might be a potential candidate for human use to improve outcome after SAH.