The aggregation of the peptide hormone amylin in the pancreas is a pathological hallmark of type-2 diabetes. Additionally, amylin can form aggregates in the brain, promoting β-amyloid deposition and tau phosphorylation in Alzheimer's disease. The cross-seeding between amylin and tau exacerbates tau pathology spread and synaptic loss, leading to neurodegeneration and cognitive deficits. Given the link between lysosomal dysfunction and tauopathy in the brain and amylin aggregation in the pancreas, we hypothesized that amylin could potentially worsen tau pathology in diabetic mice. We administered streptozotocin and/or amylin peripherally to the PS19 model of tauopathy at 3 months and characterized them at 6 months of age. We found that streptozotocin diminished body weight gain, increased blood glucose levels, worsened motor performance, and improved fear-conditioned memory in PS19 mice. Both amylin and streptozotocin administration prompted the emergence of tau pathology in the pancreas, which coincided with a decrease in the number of lysosomes in pancreatic islets. Mice treated with amylin and streptozotocin also developed robust tau pathology concomitant with lowering lysosomal cathepsin D levels in the visual cortex. These findings suggest that in diabetic mice, amylin administration diminished pancreatic lysosomes, possibly increasing the number of amylin aggregates that reached the brain and contributing to the worsening of tau pathology due to lysosomal impairment in the visual cortex. The outcome of our research enhances the understanding of the cellular pathways by which amylin may serve as a link between the pancreas-brain axis during diabetes, influencing the risk of developing tau pathology.

BIN1 , the second strongest GWAS risk factor for late-onset Alzheimer’s disease (AD), encodes a nucleocytoplasmic adaptor protein that plays many roles in multiple tissue and cell types. It is known that BIN1 can directly bind to tau in vitro , and neuronal BIN1 expression decreases in patients with AD. Accumulation of intracellular hyperphosphorylated tau is a hallmark pathogenic feature of AD and related tauopathies. Neuronal BIN1 localizes to presynaptic terminals and influences excitatory synaptic transmission, however, the molecular events underpinning neuronal BIN1 function in disease progression i n vivo has remained unclear. To mimic the decrease in neuronal BIN1 expression in Alzheimer’s patients, we recently generated and characterized tau pathology in novel conditional Bin1 knock-out (tau P301S transgenic mice lacking BIN1 expression in the forebrain excitatory neurons; PS19: Bin1 -cKO) and examined tau pathogenesis. In order to establish a direct connection between neuronal BIN1 and the degree of neurofibrillary tangle pathology, we injected tau seeds into the brains of PS19: Bin1 -cKO mice and investigated tau propagation and spreading. We observed that the loss of excitatory neuronal BIN1 expression attenuates tau accumulation and neurodegeneration selectively in the hippocampus, entorhinal/piriform cortex, and amygdala. Furthermore, the knock-out mice had significantly reduced neuroinflammation, preservation of hippocampal synapses, and complex transcriptomic changes in the neurons and glial cells. In addition, we observed reduced brain-derived tau seed propagation from the site of injection in the hippocampus into connected cortical regions. Instead, there is an accumulation of Ser202/Thr205 phosphorylated tau and MC1 + tau in hippocampal CA1 pyramidal neurons. Thus, our findings reveal an interesting region-specificity in neuronal BIN1 regulation of tau pathogenesis and propagation. Overall, our findings reveal that excitatory neuronal BIN1 promotes region-specific tau pathogenesis and tau propagation through neuroanatomically connected brain regions. These findings add to our understanding of in vivo BIN1 function in the context of tau pathogenesis, revealing cell-autonomous and non-cell-autonomous mechanisms involved in BIN1 modulation of tau burden in AD.

Bridging integrator 1 (BIN1) is the second most prevalent genetic risk factor identified by genome-wide association studies (GWAS) for late-onset Alzheimer's disease. BIN1 encodes an adaptor protein that regulates membrane dynamics in the context of endocytosis and neurotransmitter vesicle release. In vitro evidence suggests that BIN1 can directly bind to tau in the cytosol. In addition, BIN1's function limits extracellular tau seed uptake by endocytosis and subsequent propagation as well as influences tau release through exosomes. However, the in vivo roles of BIN1 in tau pathogenesis and tauopathy-mediated neurodegeneration remain uncharacterized. We generated conditional knockout mice with a selective loss of Bin1 expression in the forebrain excitatory neurons and oligodendrocytes in P301S human tau transgenic background (line PS19). PS19 mice develop age-dependent tau neuropathology and motor deficits and are commonly used to study Alzheimer's disease tau pathophysiology. The severity of motor deficits and neuropathology was compared between experimental and control mice that differ with respect to forebrain BIN1 expression. BIN1's involvement in tau pathology and neuroinflammation was quantified by biochemical methods and immunostaining. Transcriptome changes were profiled by RNA-sequencing analysis to gain molecular insights. The loss of forebrain BIN1 expression in PS19 mice exacerbated tau pathology in the somatosensory cortex, thalamus, spinal cord and sciatic nerve, accelerated disease progression and caused early death. Intriguingly, the loss of BIN1 also mitigated tau neuropathology in select regions, including the hippocampus, entorhinal/piriform cortex, and amygdala, thus attenuating hippocampal synapse loss, neuronal death, neuroinflammation and brain atrophy. At the molecular level, the loss of forebrain BIN1 elicited complex neuronal and non-neuronal transcriptomic changes, including altered neuroinflammatory gene expression, concomitant with an impaired microglial transition towards the disease-associated microglial phenotype. These results provide crucial new information on in vivo BIN1 function in the context of tau pathogenesis. We conclude that forebrain neuronal BIN1 expression promotes hippocampal tau pathogenesis and neuroinflammation. Our findings highlight an exciting region specificity in neuronal BIN1 regulation of tau pathogenesis and reveal cell-autonomous and non-cell-autonomous mechanisms involved in BIN1 modulation of tau neuropathology.

Background The BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth. Methods Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells. Bin1 expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches. Results Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing Bin1 expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specific Bin1 conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss of Bin1 impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene, Ifitm3 . Conclusions Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between Bin1 and Ifitm3 , two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3 upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases. Graphical Abstract

Introduction: Ischemic stroke remains one of the most debilitating diseases and is the fifth leading cause of death in the US. The ability to predict stroke outcomes within the acute period of stroke would be essential for care planning and rehabilitation. The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC; clinicaltrials.gov NCT03153683) study collects arterial blood immediately distal and proximal to the intracranial thrombus at the time of mechanical thrombectomy. These blood samples are an innovative resource in evaluating acute gene expression changes at the time of ischemic stroke. The purpose of this study was to identify inflammatory genes and important immune factors during mechanical thrombectomy for emergent large vessel occlusion (ELVO) and which patient demographics were predictors for stroke outcomes (infarct and/or edema volume) in acute ischemic stroke patients.Methods: The BACTRAC study is a non-probability sampling of male and female subjects (≥18 year old) treated with mechanical thrombectomy for ELVO. We evaluated 28 subjects (66 ± 15.48 years) relative concentrations of mRNA for gene expression in 84 inflammatory molecules in arterial blood distal and proximal to the intracranial thrombus who underwent thrombectomy. We used the machine learning method, Random Forest to predict which inflammatory genes and patient demographics were important features for infarct and edema volumes. To validate the overlapping genes with outcomes, we perform ordinary least squares regression analysis.Results: Machine learning analyses demonstrated that the genes and subject factors CCR4, IFNA2, IL-9, CXCL3, Age, T2DM, IL-7, CCL4, BMI, IL-5, CCR3, TNFα, and IL-27 predicted infarct volume. The genes and subject factor IFNA2, IL-5, CCL11, IL-17C, CCR4, IL-9, IL-7, CCR3, IL-27, T2DM, and CSF2 predicted edema volume. The overlap of genes CCR4, IFNA2, IL-9, IL-7, IL-5, CCR3, and IL-27 with T2DM predicted both infarct and edema volumes. These genes relate to a microenvironment for chemoattraction and proliferation of autoimmune cells, particularly Th2 cells and neutrophils.Conclusions: Machine learning algorithms can be employed to develop prognostic predictive biomarkers for stroke outcomes in ischemic stroke patients, particularly in regard to identifying acute gene expression changes that occur during stroke.

Introduction: Ischemic stroke is the one of the most severe and debilitating diseases, and despite animal models, there is much to learn about the neuropathology in humans in a way that could inform the development of therapies. We have developed a protocol to collect and evaluate arterial blood immediately distal and proximal from the removed intracranial thrombus during mechanical thrombectomy. These samples provide a unique resource in evaluating acute changes in acid/base and electrolyte concentrations at the time of ischemic stroke. The purpose of this study was to compare acid/base and electrolytes obtained proximal and distal to the occluded intracranial thrombi between male and female acute ischemic stroke subjects at the time of thrombectomy; and to determine whether arterial blood gas values predict outcomes in male and female subjects. Methods: We analyzed the first 49 subjects (age = 67 +/- 15.0, 21 males) in the BACTRAC registry. We compared arterial blood gas of blood distal versus proximal to the thrombus during thrombectomy which provided acid/base levels (pH, pCO(2), pO(2), BD, HCO3-) and electrolyte values (iCa(2+), K+, and Na+). Comparisons were evaluated by one-way repeated measures ANOVA (p < .05). Moderated multiple regression with an interaction term of sex determined predictors of infarct volume, edema volume, and infarct time. Results: In general, distal intracranial luminal blood sample showed a compensated metabolic acidosis with an elevated oxygen concentration in both blood samples. Analysis indicated several significant differences in the proximal blood samples between sexes (pH, pCO(2), and K+). Bicarbonate and base deficit were predictors of infarct time specifically in female subjects. Discussion and conclusion: Acid/base and electrolyte response to ischemic conditions differ between men and women, and these early changes could be used to predict local acid/base changes and how they develop differently in men and women during ischemia. These findings provide a novel insight into the pathology of large vessel stroke in humans, particularly potential variations based on sex.

Background and Purpose: The ability to predict ischemic stroke outcomes in the first day of admission could be vital for patient counseling, rehabilitation, and care planning. The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC; clinicaltrials.gov NCT03153683) collects blood samples distal and proximal to the intracranial thrombus during mechanical thrombectomy. These samples are a novel resource in evaluating acute gene expression changes at the time of ischemic stroke. The purpose of this study was to identify inflammatory genes and patient demographics that are predictive of stroke outcomes (infarct and/or edema volume) in acute ischemic stroke patients. Methods: The BACTRAC study is a non-probability, convenience sampling of subjects (≥ 18 year olds) treated with mechanical thrombectomy for emergent large vessel occlusion. We evaluated relative concentrations of mRNA for gene expression in 84 inflammatory molecules in static blood distal and proximal to the intracranial thrombus from adults who underwent thrombectomy. We employed a machine learning method, Random Forest, utilizing the first set of enrolled subjects, to predict which inflammatory genes and patient demographics were important features for infarct and edema volumes. Results: We analyzed the first 28 subjects (age = 66 ± 15.48, 11 males) in the BACTRAC registry. Results from machine learning analyses demonstrate that the genes CCR4, IFNA2, IL9, CXCL3, Age, DM, IL7, CCL4, BMI, IL5, CCR3, TNF, and IL27 predict infarct volume. The genes IFNA2, IL5, CCL11, IL17C, CCR4, IL9, IL7, CCR3, IL27, DM, and CSF2 predict edema volume. There is an intersection of genes CCR4, IFNA2, IL9, IL7, IL5, CCR3 to both infarct and edema volumes. Overall, these genes depicts a microenvironment for chemoattraction and proliferation of autoimmune cells, particularly Th2 cells and neutrophils. Conclusions: Machine learning algorithms can be employed to develop predictive biomarker signatures for stroke outcomes in ischemic stroke patients, particularly in regard to identifying acute gene expression changes that occur during stroke.

BACKGROUND:The goal of this study was to determine whether leukemia inhibitory factor (LIF) promotes anti-inflammatory activity after stroke in a sex-dependent manner.METHODS:Aged (18-month-old) Sprague-Dawley rats of both sexes underwent sham surgery or permanent middle cerebral artery occlusion (MCAO). Animals received three doses of intravenous LIF (125 μg/kg) or PBS at 6, 24, and 48 h before euthanization at 72 h. Spleen weights were measured immediately following euthanization. Western blot was used to measure protein levels of CCL8, CD11b, CXCL9, CXCL10, IL-12 p40, IL-3, and the LIF receptor (LIFR) in spleen tissue. ELISA was used to measure IL-1β, IL-6, TNFα, and IFNγ in spleen tissue. A Griess Assay was used to indirectly quantify NO levels via measurement of nitrite. Levels of cellular markers and inflammatory mediators were normalized to the baseline (sham) group from each sex. Statistical analysis was performed using two-way ANOVA and followed by Fisher's LSD post hoc test.RESULTS:Aged female rats showed a significantly lower spleen weight after MCAO, but showed a significant increase in spleen size after LIF treatment. This effect was observed in aged male rats, but not to as great of an extent. CD11b levels were significantly higher in the spleens of MCAO+PBS males compared to their female counterparts, but there was no significant difference in CD11b levels between MCAO+LIF males and females. LIF significantly increased CXCL9 after LIF treatment in aged male and female rats. LIFR and IL-3 were upregulated after LIF treatment in aged females. Splenic nitrate increased after MCAO but decreased after LIF treatment in aged females. Splenic nitrate levels did not increase after MCAO but did increase after LIF treatment in aged males. The following cytokines/chemokines were not altered by sex or treatment: TNFα, IL-6, IL-12 p40, CCL8, IFNγ, and CXCL10.CONCLUSIONS:LIF treatment after permanent MCAO induces sex-dependent effects on the poststroke splenic response and the production of proinflammatory cytokines among aged rats.

BackgroundThe goal of this study was to determine whether LIF differentially affects pro‐inflammatory signaling in aged male and female animals and determine whether changes in inflammatory signaling after LIF treatment were linked to the mitochondrial function in splenocytes.Methods18‐month‐old male/female Sprague‐Dawley rats underwent middle cerebral artery occlusion (MCAO). Animals were treated with PBS or LIF at 6, 24, and 48 h after MCAO (125 μg/kg). Protein levels of the LlF receptor (LIFR), TNFα, IL‐1β, IL‐3, IFNγ, CXCL8, CXCL9, and CXCL10 were measured in splenic tissue using immunoblotting and ELISA. A Griess Assay kit was used to measure nitrite in spleen tissue. The oxygen consumption rate in electron transport chain States III‐V was measured to assess mitochondrial function. Statistical analyses were performed using one or two‐way ANOVA followed by Fisher's LSD post hoc test.ResultsAged male rats (*p<0.05) and aged female rats (***p<0.001) showed significant decreases in spleen size after MCAO + PBS treatment. LIF treatment significantly increased the spleen weight in aged males (**p<0.01). Among female rats, LIF significantly increased spleen weight compared to PBS‐treated (****p<0.001) and sham‐operated groups (**p<0.01) among aged females. LIF treatment resulted in a significant increase in splenic LIFR and CXCL9 expression regardless of sex (**p<0.01). Aged male rats showed significantly higher levels of IL‐1β in the spleen compared to their female counterparts (**p<0.01). MCAO + LIF treatment significantly altered levels of IFNγ compared to sham animals (*p<0.05) in a sex‐dependent manner (*p<0.05). IL‐3 levels were significantly increased in the spleens of female rats after MCAO + LIF treatment compared to sham females (****p<0.0001), MCAO + PBS females (**p<0.01), and MCAO + LIF males (***p<0.001). However, drug treatment did not significantly alter splenic IL‐3 levels among aged male rats. Levels of nitrite significantly increased in the spleens of aged female rats after MCAO + PBS treatment (*p<0.05), but returned to sham levels after LIF treatment (**p<0.01). Aged males showed significantly higher levels of nitrite after MCAO + LIF treatment (**p<0.01). There was no significant change in TNFα, CXCL8, or CXCL10 levels between treatment groups among aged male and female animals. The OCR was significantly increased in State III and State V (Complex II) after MCAO+ LIF treatment compared to MCAO + PBS treatment (***p<0.01), but not State IV or State V (Complex I).ConclusionsLIF treatment promotes alterations in mitochondrial activity and cytokine expression in the spleen at 72 h after MCAO, with some cytokines exhibiting sex‐specific differences in their expression. These data demonstrate that rats show sex differences in the post‐stroke inflammatory response after stroke and differential responses to LIF.Support or Funding InformationExperiments were supported by the following awards: VA Award # 5I01BX003405‐02 (Sullivan) and NINDS Award # 5R01NS091146‐04 (Pennypacker)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background and Purpose: In the setting of mechanical thrombectomy for emergent large vessel occlusion (ELVO), we have developed a protocol to collect and evaluate blood immediately distal and proximal from the removed intracranial thrombus. These samples provide a unique resource in evaluating acute changes in acid/base and electrolyte concentrations at the time of ischemic stroke. The purpose of this study is to compare acid/base and electrolyte differences obtained proximal and distal to the occluded intracranial thrombus in acute ischemic stroke patients. Methods: We developed the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol: an IRB-approved tissue banking strategy for ELVO (clinicaltrials.gov NCT03153683). We compared arterial blood gases (ABG) of blood distal versus proximal to the thrombus during thrombectomy. Comparisons were evaluated by Paired Samples T-Tests (p < 0.05). Results: We analyzed the first 46 subjects (age = 67 ± 14.23, 20 males) in the BACTRAC registry. Preliminary results demonstrate that, while pH is nonsignificant (p = 0.513), distal blood in relation to proximal blood showed significantly lower oxygen (p < 0.001), carbon dioxide (p < 0.001), bicarbonate (p < 0.001), ionized calcium (p < 0.001), and potassium (p < 0.001). Sodium concentration was significantly higher (p < 0.001) in distal blood. These results suggest alterations occurring intravascularly during ischemia. Conclusions: These preliminary findings provide a novel insight into the pathology of large vessel stroke in humans, particularly in regard to identifying acute changes in acid/base balance and electrolyte concentrations that occur during stroke.

The aim of this study was to determine whether leukemia inhibitory factor (LIF) exerts its neuroprotective effects through signal transduction of the transcription factor myeloid zinc finger-1 (MZF-1). According to the hypothesis of this study, MZF-1 mediates LIF-induced neuroprotective signaling during ELVO through increased expression and transcriptional activity. To determine the in vivo role of MZF-1 in LIF-induced neuroprotection, we used Genomatix software was used to MZF-1 sites in the promoter region of the rat superoxide dismutase 3 (SOD3) gene. Stroke was induced via middle cerebral artery occlusion, and animals were administered PBS or 125 μg/kg LIF at 6, 24, and 48 h after the injury. MZF-1 binding activity was measured using electrophoretic mobility shift assay (EMSA) and its expression/localization were determined using western blot and immunohistochemical analysis. To determine whether MZF-1 relays LIF-induced neuroprotection in vitro, primary cultured neurons were subjected to oxygen-glucose deprivation (OGD) after treatment with PBS or LIF. MZF-1 expression was measured in vitro using real time PCR and immunohistochemical staining. Transfection with siRNA was used to determine whether LIF protected cultured neurons against OGD after silencing MZF-1 expression. Four MZF-1 binding sites were identified by Genomatix, and EMSA confirmed in vivo binding activity in brain after MCAO. LIF significantly increased MZF-1 protein levels compared to PBS treatment at 72 h post-MCAO. In vivo nuclear localization of MZF-1 as well as co-localization of SOD3 and MZF-1 was observed in the cortical neurons of LIF-treated rats. Primary cultured neurons treated with LIF had significantly higher levels of MZF-1 mRNA and protein after LIF treatment compared to neurons treated with PBS. Finally, knockdown MZF-1 using siRNA counteracted the neuroprotective effects of LIF in vitro. These data demonstrate that LIF-mediated neuroprotection is dependent upon MZF-1 activity. Furthermore, these findings identify a novel neuroprotective pathway that employs MZF-1, a transcription factor associated with hematopoietic gene expression.

Background and purpose Mechanical thrombectomy (MT) is the standard of care for emergent large vessel occlusion (ELVO), one of the most severe subtypes of ischemic stroke, which accounts for 30–40% of all cases. Through MT, we can isolate distal blood within the artery immediately downstream from the clot and compare it to systemic arterial blood to provide insight into local intraluminal changes during ischemia. CXCL9 is in interferon gamma-inducible chemokine that binds CXCR3, degrading endothelial tight junctions, attracting T cells, and facilitating immune cell extravasation into brain parenchyma. We aimed to study local CXCL9 expression distal to the intracranial thrombus during large vessel occlusion in human patients. Methods Tissue samples of distal and proximal blood were collected as part of the BACTRAC tissue bank (www.clinicaltrials.gov NCT03153683). Adult subjects with ELVO were prospectively enrolled, and arterial blood distal (intracranial) and proximal (cervical) were collected and processed to optimize RNA quality. RNA were isolated and used to evaluate gene expression in both samples for each subject; proximal systemic blood was used as an internal control for each subject. Results 22 subjects were included in this preliminary analysis. 15 (68.2%) were female. 54.5% of subjects had a CTA collateral score of 1 (18.2% had a score of 0). 4.5% (1 subject) did not attain TICI 2B or 3 recanalization. Infarct time (last known normal to thrombectomy recanalization) was 491 ± 243 minutes. Mean change in NIHSS from admission to discharge was -8 ± 8. CXCL9 expression in distal blood was upregulated an average of 106-fold with a maximum upregulation of 805-fold in one subject. In plotting CXCL9 expression against infarct time, there was a clear negative correlation (Spearman coefficient -0.43, p=0.05).Abstract O-004 Figure 1 Graph demonstrating time-dependent differential gene expression of CXCL9 in distal vs proximal blood sample (n=21). The y-axis shows fold-changes in distal expression over proximal expression. Infarct time is defined as Last Known Normal to thrombectomy recanalization moment. The panel below shows these changes mapped to a published timeline of BBB permeability Conclusion For the first time, we evaluate chemokine alterations in human stroke patients in distal stagnant blood during ELVO. There is a significant variance in CXCL9 expression in distal blood in relationship to infarct time, which mirrors the known timing of blood-brain barrier disruption. Disclosures J. Fraser: None. S. Martha: None. L. Collier: None. S. Davis: None. A. Alhajeri: None. S. Grupke: None. K. Pennypacker: None.

Background and Purpose: The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) tissue bank collects blood proximal and distal to the intracranial thrombus during the process of mechanical thrombectomy in human stroke patients. These samples provide a unique resource in evaluating acute gene expression changes at the time of ischemic stroke. The purpose of this study was to evaluate gene expression changes occurring within the thrombus and across the occlusion in the intravascular space in acute ischemic stroke patients. Methods: We developed the BACTRAC protocol: an IRB-approved tissue banking approach for ELVO (clinicaltrials.gov NCT03153683). We evaluated relative concentrations of gene expression in 84 inflammatory molecules in thrombi removed from adults who received thrombectomy for ischemic stroke, in static blood distal to thrombus, and in peripheral circulation. Results: We analyzed the first 46 subjects (age = 67 ± 14.23, 20 males) in the BACTRAC registry. Results from qPCR gene expression analyses demonstrate that 21 genes (CCL1, CCL11, CCL13, CCL17, CCL26, CCL8, CSF3, CX3CL1, CXCL1, CXCL9, IFNA2, IL13, IL17C, IL17F, IL1A, IL27, IL3, IL33, LTA, TNFSF11, and TNFSF13) had at least 25 mean fold change in the distal blood compared to the peripheral blood. Fourteen genes (AIMP1, CCL11, CCL13, CCL15, CCL16, CCL23, CCR2, CCR4, CCR8, CD40LG, CXCL10, IL10RA, IL15, and TNFSF13B) had at least 10 mean fold change in the thrombus compared to the peripheral blood. Overall, these genes are associated with chemo-attraction and cell proliferation of monocytes, neutrophils, and T cells. Conclusions: These findings provide a novel insight into the initial pathology of large vessel stroke in humans, particularly in regard to identifying acute gene expression changes that occur during stroke.

BACKGROUND:Early changes in acid/base and electrolyte concentrations could provide insights into the development of neuropathology at the onset of stroke. We evaluated associations between acid/base and electrolyte concentrations, and outcomes in permanent middle cerebral artery occlusion (pMCAO) model. METHODS:18-month-old male and female Sprague-Dawley rats underwent pMCAO. Pre-, post- (7 min after occlusion), and at 72 hr of pMCAO venous blood samples provided pH, carbon dioxide, oxygen, glucose, hematocrit, hemoglobin, and electrolyte values of ionized calcium, potassium, and sodium. Multiple linear regression determined predictors of infarct and edema volumes from these values, Kaplan-Meier curve analyzed morality between males and females at 72 hr, and a Cox regression model was used to determine predictors for mortality. RESULTS:Analysis indicated significant differences in acid/base balance and electrolyte levels in aged rats not dependent on sex between the three time points in the pMCAO model. Changes in pH (from pre- to post and post- to 72 hr) and changes in sodium and ionized calcium (from post- to 72 hr) were predictors of infarct volume and edema volume, respectively. Cox Regression revealed there is a 3.25 times increased risk for mortality based on changes in bicarbonate (pre- to post-MCAO). CONCLUSIONS:These early venous blood changes in acid/base balance and electrolytes can be used to predict stroke outcomes in our rat model of stroke. This study provides potential biomarkers to be examined in the human condition that could provide profound prognostic tools for stroke patients.

Background and Purpose: Monitoring for hypotension, hyperglycemia, hypoxia, and dehydration is key to reducing early death in stroke patients. Evaluation of physiological predictors of infarct volume and mortality may provide opportunities for effective interventions to improve outcomes. The purpose of this study is: 1) to describe and compare the predictive effects of venous blood gas (VBG) on infarct volume and mortality in acute stroke in rats; 2) and we have begun to collect arterial blood gas (ABG) to compare differences obtained proximal and distal to the occluded intracranial thrombi in acute ischemic stroke patients. Methods: 3-month old Sprague-Dawey rats (n = 9) underwent permanent or transient middle cerebral artery occlusion (MCAO). Pre- and post-MCAO venous samples provided pH, pCO 2 , pO 2 , and electrolyte values (iCa 2+ , K + , and Na + ). Linear regression determined predictors of infarct volume from these values, and Cox regression analyzed VBG changes between tMCAO (n = 28) and pMCAO (n = 29) to determine predictors of mortality. We compared mean proximal and distal pH, pCO 2 , pO 2 , and electrolytes (iCa 2+ , K + , and Na + ) in stroke patients (n = 7) arterial samples using Wilcoxon Signed Ranks test. Results: Animal studies demonstrated pH and iCa 2+ are predictors of infarct volume, but not mortality. After pMCAO (n = 9), change in pH or iCa 2+ significantly predicted infarct volume [F(1,7) = 7.351, β = -0.716, p = 0.03] and [F(1, 7) = 6.782, β = -0.701, p = 0.035]; as pH and calcium decreased, infarct volume increased. These variables explained 44% and 42% of the total variance in these models. In human patients (n = 7), there were significant differences in blood samples proximal and distal to the intracranial thrombus for pCO 2 (p = 0.018), HCO 3- (p = 0.028), iCa 2+ (p = 0.043), K + (p = 0.028), and Na + (p = 0.044). Conclusions: In conclusion, there are acute changes in acid/base balance and electrolytes during stroke in rodent models and humans. In cross-species comparison ionized calcium changes were significant in both, with iCa 2+ changes predicting stroke volume in the rat model. These preliminary findings are novel, and warrant further exploration in human patients.

The migration of peripheral immune cells and splenocytes to the ischemic brain is one of the major causes of delayed neuroinflammation after permanent large vessel stroke. Other groups have demonstrated that leukemia inhibitory factor (LIF), a cytokine that promotes neural cell survival through upregulation of antioxidant enzymes, promotes an anti-inflammatory phenotype in several types of immune cells. The goal of this study was to determine whether LIF treatment modulates the peripheral immune response after stroke.

Preclinical studies using rodent models of stroke have had difficulty in translating their results to human patients. One possible factor behind this inability is the lack of studies utilizing aged rodents of both sexes. Previously, this lab showed that leukemia inhibitory factor (LIF) promoted recovery after stroke through antioxidant enzyme upregulation. This study examined whether LIF promotes neuroprotection in aged rats of both sexes. LIF did not reduce tissue damage in aged animals, but LIF-treated female rats showed partial motor skill recovery. The LIF receptor (LIFR) showed membrane localization in young male and aged rats of both sexes after stroke. Although LIF increased neuronal LIFR expression in vitro, it did not increase LIFR in the aged brain. Levels of LIFR protein in brain tissue were significantly downregulated between young males and aged males/females at 72 h after stroke. These results demonstrated that low LIFR expression reduces the neuroprotective efficacy of LIF in aged rodents of both sexes. Furthermore, the ability of LIF to promote motor improvement is dependent upon sex in aged rodents.

BACKGROUND AND PURPOSE:Acid/base and electrolytes could provide clinically valuable information about cerebral infarct core and penumbra. We evaluated associations between acid/base and electrolyte changes and outcomes in 2 rat models of stroke, permanent, and transient middle cerebral artery occlusion. METHODS:Three-month old Sprague-Dawley rats underwent permanent or transient middle cerebral artery occlusion. Pre- and post-middle cerebral artery occlusion venous samples for permanent and transient models provided pH, carbon dioxide, oxygen, glucose, and electrolyte values of ionized calcium, potassium, and sodium. Multiple regression determined predictors of infarct volume from these values, and Kaplan-Meier curve analyzed morality between permanent and transient middle cerebral artery occlusion models. RESULTS:Analysis indicated significant differences in the blood gas and electrolytes between pre- to post-middle cerebral artery occlusion. A decrease in pH and sodium with increases in carbon dioxide, potassium, ionized calcium, and glucose changes were found in both middle cerebral artery occlusion models; while hematocrit and hemoglobin were significant in the transient model. pH and ionized calcium were predictors of infarct volume in the permanent model, as changes in pH and ionized calcium decreased, infarct volume increased. CONCLUSIONS:There are acute changes in acid/base balance and electrolytes during stroke in transient and permanent rodent models. Additionally, we found pH and ionized calcium changes predicted stroke volume in the permanent middle cerebral artery occlusion model. These preliminary findings are novel, and warrant further exploration in human conditions.