Hyperglycemic Stroke Research Articles

Abstract WMP115: Rapid Vascular Complement Activation: A Key Driver of Blood-Brain Barrier Disruption in Ischemic Stroke With Acute Hyperglycemia

Background: Acute hyperglycemia, which occurs in over 40% of ischemic stroke patients irrespective of pre-existing diabetes, exacerbates stroke injury. Understanding its mechanisms is vital for innovative treatments. Here we investigate the role of complement in hyperglycemia-exacerbated damage after stroke. Method: Male C57/BL6 mice (10-11 weeks) underwent 30-min suture-induced middle cerebral artery occlusion followed by reperfusion, resembling clinical thrombectomy. Acute hyperglycemia was induced 10 min pre-stroke via glucose injection. Mice were sacrificed at 4.5h and 24h post-stroke to analyze brain swelling, blood-brain barrier (BBB) leakage and hemorrhagic transformation (HT). Mortality, neurological deficits, and motor-sensory functions were assessed at 14 days post-stroke. Complement activation was visualized via C3d immunostaining. Complement C3's role was examined using knock-out mice and the targeted complement inhibitor CR2-Crry fusion protein. Result: Hyperglycemia rapidly worsens BBB leakage (p<0.0001), brain swelling (p<0.05), and HT (p<0.0001) at 4.5 hr after stroke. Hyperglycemic stroke mice exhibit higher mortality (100% vs 25%, p=0.0008), body weight loss, and behavior impairment at the sub-acute phase compared to normoglycemic mice. Notably, acute hyperglycemia rapidly increased plasma C3 levels at 1hr after stroke (p<0.01), accompanied by rapid and time-dependent activation of C3 in ischemic brain vessels, as marked by increased vascular C3d; and this co-localized with autoantibody (IgM/IgG) vessel buildup and vessel leakage. Additionally, C3d levels positively correlated with brain swelling and HT (p<0.01). Vascular C3d and leakage into parenchyma were markedly reduced in the absence of reperfusion. Blocking complement via C3 knock-out or post-reperfusion systemic injection of C3 inhibitor CR2-Crry mitigates hyperglycemia's detrimental effects, including BBB leakage. Conclusion: Rapid vascular activation of complement C3 is a significant driver of hyperglycemic-exacerbated pathology in experimental stroke. Inhibiting C3 activation could be a potential therapeutic approach to improve hyperglycemic stroke outcome.

Intra-arterial verapamil improves functional outcomes of thrombectomy in a preclinical model of extended hyperglycemic stroke.

The abrupt hyperglycemic reperfusion following thrombectomy has been shown to harm the efficacy of the intervention in stroke patients with large vessel occlusion. Studies of ours and others have shown thioredoxin-interacting protein (TXNIP) is critically involved in hyperglycemic stroke injury. We recently found verapamil ameliorates cerebrovascular toxicity of tissue plasminogen activators in hyperglycemic stroke. The present study aims to answer if verapamil exerts direct neuroprotective effects and alleviates glucose toxicity following thrombectomy in a preclinical model of hyperglycemic stroke. Primary cortical neural (PCN) cultures were exposed to hyperglycemic reperfusion following oxygen-glucose deprivation (OGD), with or without verapamil treatment. In a mouse model of intraluminal stroke, animals were subjected to 4h middle cerebral artery occlusion (MCAO) and intravenous glucose infusion. Glucose infusion lasted one more hour at reperfusion, along with intra-arterial (i.a.) verapamil infusion. Animals were subjected to sensorimotor function tests and histological analysis of microglial phenotype at 72h post-stroke. According to our findings, glucose concentrations (2.5-20mM) directly correlated with TXNIP expression in OGD-exposed PCN cultures. Verapamil (100nM) effectively improved PCN cell neurite growth and reduced TXNIP expression as well as interaction with NOD-like receptor pyrin domain-containing-3 (NLRP3) inflammasome, as determined by immunoblotting and immunoprecipitation. In our mouse model of extended hyperglycemic MCAO, i.a. verapamil (0.5mg/kg) could attenuate neurological deficits induced by hyperglycemic stroke. This was associated with reduced microglial pro-inflammatory transition. This finding encourages pertinent studies in hyperglycemic patients undergoing thrombectomy where the robust reperfusion may exacerbate glucose toxicity.

Abstract WP225: Rapid Complement Activation Induced By Acute Hyperglycemia Worsens Ischemic Stroke Outcome

Background: Acute hyperglycemia, which occurs in over 40% of ischemic stroke patients regardless of pre-existing diabetes, increases brain edema, hemorrhagic transformation (HT) and worsens stroke outcome. Understanding the mechanisms of hyperglycemia-exacerbated stroke injury will be vital for developing novel treatments. Here we identify systemic complement activation as a novel contributor to hyperglycemic-exacerbated damage in rodent stroke. Method: Male C57/BL6 mice (10-11 weeks) were subjected to middle cerebral artery occlusion (MCAO) for 30 min, followed by reperfusion to mimic ischemic stroke. Acute hyperglycemia was induced by glucose injection 10 min before MCAO. To assess the effect of hyperglycemia on stroke outcomes, mice were sacrificed at 4.5 and 24 hr post-stroke to analyze brain edema, blood-brain barrier (BBB) leakage, and HT; or allowed to survive 14 days to examine mortality rate, neurological deficit, and motor-sensory dysfunction using the horizontal rotating beam test. Complement activation was evaluated at 1, 2, 4.5 hr post-stroke. To inhibit C3 activation, the targeted complement inhibitor CR2-Crry fusion protein, or PBS control, was injected via femoral vein 15 min after reperfusion. Result: Hyperglycemia worsens stroke outcomes in our mouse model, with rapid increased BBB leakage (p<0.0001), brain swelling (p<0.001) and HT at (p<0.0001) 4.5 and 24 hr after stroke compared to normoglycemia; and poorer prognosis including higher mortality (100% VS 25%, p=0.0008), body weight loss, and impaired behavioral performance by day 14. Interestingly, acute hyperglycemia rapidly increased plasma complement C3 levels at 1 and 2 hr after stroke (p<0.01 compared to normoglycemic mice), accompanied by activation of C3 in ischemic brain vessels as indicated by elevated levels of vascular C3d. Vascular C3d colocalized with IgM/IgG, suggesting the involvement of classical pathway in C3 activation. Notably, activated C3 levels positively correlated with brain swelling and HT (p<0.01). Inhibition of C3 by CR2-Crry markedly reduced brain swelling (p<0.05) and HT in hyperglycemic stroke. Conclusion: Rapid activation of complement C3 by acute hyperglycemia may be an important contributor to BBB leakage, brain swelling and HT.

Abstract 38: Admission Hyperglycemia In Stroke Patients Increases Oxidative Stress In Red Blood Cells

Background: Our recent study has shown that admission hyperglycemia in stroke patients is associated with red blood cell (RBC) dysregulation, however, the underlying mechanism is still unclear. In addition to transporting oxygen via hemoglobin, RBCs also carry a robust antioxidant system to remove the reactive oxygen species (ROS) continuously produced from hemoglobin autoxidation (Fig A). It has been reported that in vitro hyperglycemia can increase oxidative stress in RBCs. We thus hypothesize that acute hyperglycemia in stroke patients may also affect the antioxidant system in RBCs. Method: Stroke patients with hyperglycemia and normoglycemia (n=12/group) were prospectively recruited in accordance with IRB protocol. Blood was collected at 2 hr, 24 hr, 72 hr and 3 mon post stroke. RBCs were precipitated from whole blood, and leucocytes were removed with depletion filter. The expression and glycosylation of RBC antioxidants, such as catalase (CAT), superoxide dismutase type 1 (SOD1), glutathione peroxidase 1 (GPX1) and peroxiredoxin 2 (PRX2), were quantified using mass spectrometry-based selected reaction monitoring (SRM) method. Result: CAT, SOD1 and GPX1 showed gradually decreased expression in hyperglycemic stroke patients, which may impair the antioxidant capacity of RBCs (Fig B). The glycosylation level of PRX2 was increased in stroke-related hyperglycemia (Fig C), and increased glycosylation was associated with decreased PRX2 activity (Fig D). Consistent with the expression and glycosylation changes in antioxidants, total RBC antioxidant capacity was reduced in hyperglycemic patients, and ROS was significantly accumulated in RBCs (Fig E). Conclusion: Acute hyperglycemia in stroke patients intensifies RBC oxidative stress by affecting the expression and post-translational modification of RBC antioxidants. Larger studies are underway to verify these findings, and explore how hyperglycemia and RBC oxidative stress influence stroke outcome.

Abstract WP253: Increased Hydrogen Sulfide As A New Mechanism For Hyperglycemic Worsening Of Stroke Outcome

Introduction: Hyperglycemia at stroke onset worsens outcome and reduces the effectiveness of reperfusion therapies. Increased oxidative and mitochondrial injury likely contribute. To treat this mechanism, we synthesized (PEG)-ylated carbon nanoparticles (CNPs) from acid oxidation of activated charcoal (OAC) generating 3 nm discs, catalytic superoxide dismutase mimetics that protect mitochondrial complexes. Cellular uptake is rapid and delayed I.V. PEG-CNPs are highly protective in reversible MCAO in hyperglycemic rats. Hydrogen sulfide (H 2 S) is an essential gaseous transmitter with a narrow therapeutic index associated with many disorders e.g., diabetes. Its synthesis is influenced by radicals. Excess H 2 S is toxic to mitochondrial complex IV. H 2 S is endogenously oxidized to polysulfides (PS), potent antioxidants also needed for protein persulfidation. We hypothesized that OAC’s favorable redox potential will catalyze H 2 S oxidization to PS, acute hyperglycemia will increase H 2 S and PEG-OACs will blunt the increase. Methods: b.End3 brain endothelial and HEK293 cultured cells were employed. SSP4 fluorescence measured PS levels with increasing concentrations of PEG-OACs. AzMC fluorescence detected H 2 S levels in cells incubated in 100 mg/dL glucose media followed by glucose 500 mg/dL with or without PEG-OACs, first in normoxia followed by anoxia/normoxia. Results: PEG-OACs dose dependently increased cellular PS levels (Fig 1a). High glucose increased H 2 S levels especially during anoxia/normoxia (Fig 1b). PEG-OACs completely eliminated the glucose-induced increase in H 2 S (Fig 1c). Conclusions: Acute hyperglycemia increased H 2 S production especially under conditions mimicking ischemia/reperfusion, an effect eliminated by PEG-OACs. Because of mitochondrial toxicity, an H 2 S increase may contribute to worsened outcome in hyperglycemic stroke. These results suggest a new therapeutic target for this important cause of poor stroke outcome.

Application of hyperglycemia/diabetes-derived polygenic risk scores on the risk of poor outcomes after an ischemic stroke.

Unfavorable prognoses are often accompanied for hyperglycemic stroke patients. This study aimed to construct a hyperglycemia/diabetes-derived polygenic risk score (PRS) to improve the predictive performance for poor outcome risks after a stroke and to evaluate its potential clinical application. A hospital-based cohort study was conducted including 1320 first-ever acute ischemic stroke (AIS) patients and 1210 patients who completed the follow-up at 3 months. PRSs were calculated for hyperglycemia/diabetes mellitus using results from genome-wide association studies in Asians. An unfavorable functional outcome was defined as a modified Rankin Scale score of ≥3 at 3, 6, and 12 months of follow-up. The prediction of a poor prognosis was evaluated using measures of model discrimination, calibration, and net reclassification improvement (NRI). The second to fourth PRS quartiles (≥Q2) were significantly associated with higher risks of unfavorable outcomes at 3 months compared with the first quartile as the reference group after adjusting for age, baseline stroke severity, hypertension, diabetes, dyslipidemia, smoking, heart disease, and ischemic stroke subtype (p for trend <0.0001). The addition of the PRS to traditional risk predictors of poor outcomes after an AIS significantly improved the model fit (likelihood ratio test p < 0.0001) and enhanced measures of reclassification (NRI, 0.245; 95% confidence interval [CI], 0.195-0.596). The corrected C-index for the PRS combining traditional risk factors at 3 months after a stroke was 0.899 (95% CI, 0.878-0.980). Among hyperglycemic AIS patients, those who did not take an antidiabetic drug and whose PRS was ≥Q2 had higher risks of an unfavorable outcome at 3 months compared with patients who took the medicine. The hyperglycemia/diabetes-derived PRS was associated with poor outcomes after an AIS, but further studies are needed to validate its use for clinical applications.

Effect of Intensive Glucose Control on Outcomes of Hyperglycemic Stroke Patients Receiving Mechanical Thrombectomy: Secondary Analysis of the SHINE Trial.

Hyperglycemia is common among patients presenting with acute ischemic stroke (AIS) and is associated with poor clinical outcomes. We studied the effects of intensive blood glucose control among AIS patients presenting with hyperglycemia treated with mechanical thrombectomy (MT). We analyzed publicly available data from the Stroke Hyperglycemia Insulin Network Effort trial. In this nonpreplanned secondary analysis, we compared hyperglycemic AIS patients treated with MT who received intensive blood glucose control (80 to 130 mg/dL) with those who received standard blood glucose control (80 to 179 mg/dL). Outcomes included rates of favorable 90-day outcome (modified Rankin Scale score ≤2) and death. This analysis included 146 patients (74 in the intensive treatment group and 72 in the standard treatment group). Intensive blood glucose was not associated with higher rates of 90-day favorable outcomes (intensive 31.1% vs. standard 30.6%, P =1.0; odds ratio 1.025, 95% confidence interval 0.51 to 2.07) or a decrease in rates of death (intensive 20.3% vs. standard 22.2%, P =0.84; odds ratio 0.98, 95% confidence interval 0.40 to 1.97). Intensive blood glucose control among AIS patients presenting with hyperglycemia and treated with MT was not associated with lower rates of death or higher rates of long-term favorable outcomes when compared with standard treatment.

A randomized trial to investigate the efficacy and safety of insulin glargine in hyperglycemic acute stroke patients receiving intensive care

This pilot, randomized, open-label controlled study compared the basal–bolus regimens of insulin glargine (IG) and neutral protamine Hagedorn (NPH) insulin in stroke patients with hyperglycemia receiving intensive care. The study recruited acute stroke patients requiring intensive care within 72 h (h) of onset and had blood glucose > 200 mg/dL. 50 patients received IG (n = 26) or NPH (n = 24) with added short-acting prandial regular insulin over a 72-h period. The primary end point was the percentage of glucose within 80–180 mg/dL assessed through continuous glucose monitoring. The baseline characteristics were comparable, except the IG had higher glucose pre-randomization than the NPH (290.69 ± 82.31 vs. 246.04 ± 41.76 mg/dL, P = 0.021). The percentage of time with glucose between 80 and 180 mg/dL was 45.88 ± 27.04% in the IG and 53.56 ± 22.89% in the NPH (P = 0.341) and the percentage of glucose reduction was 31.47 ± 17.52% in the IG and 27.28 ± 14.56% in the NPH (P = 0.374). The percentage of time with glucose < 60 mg/dL was 0.14 ± 0.49% in the IG and 0.47 ± 1.74% in the NPH. Poststroke outcomes were not significantly different. In conclusion, IG is safe and equally effective as an NPH-based basal-bolus regimen for acute stroke patients with hyperglycemia receiving intensive care.Trial registration ClinicalTrials.gov, NCT02607943. Registered 18/11/2015, https://clinicaltrials.gov/ct2/show/NCT02607943.

Verapamil as an Adjunct Therapy to Reduce tPA Toxicity in Hyperglycemic Stroke: Implication of TXNIP/NLRP3 Inflammasome.

Thrombolytic therapy has remained quite challenging in hyperglycemic patients for its association with poor prognosis and increased hemorrhagic conversions. We recently showed that tissue plasminogen activator (tPA)-induced cerebrovascular damage is associated with thioredoxin-interacting protein (TXNIP) upregulation, which has an established role in the detrimental effects of hyperglycemia. In the present work, we investigated whether verapamil, an established TXNIP inhibitor, may provide protection against hyperglycemic stroke and tPA-induced blood-brain barrier (BBB) disruption. Acute hyperglycemia was induced by intraperitoneal administration of 20% glucose, 15 min prior to transient middle cerebral artery occlusion (tMCAO). Verapamil (0.15 mg/kg) or saline was intravenously infused with tPA at hyperglycemic reperfusion, 1 h post tMCAO. After 24 h of ischemia/reperfusion (I/R), mice were assessed for neurobehavioral deficits followed by sacrifice and evaluation of brain infarct volume, edema, and microbleeding. Alterations in TXNIP, inflammatory mediators, and BBB markers were further analyzed using immunoblotting or immunostaining techniques. As adjunctive therapy, verapamil significantly reduced tPA-induced BBB leakage, matrix metalloproteinase 9 (MMP-9) upregulation, and tight junction protein deregulation, which resulted in lesser hemorrhagic conversions. Importantly, verapamil strongly reversed tPA-induced TXNIP/NLRP3 (NOD-like receptor pyrin domain-containing-3) inflammasome activation and reduced infarct volume. This concurred with a remarkable decrease in high-mobility group box protein 1 (HMGB-1) and nuclear factor kappa B (NF-κB) stimulation, leading to less priming of NLRP3 inflammasome. This preclinical study supports verapamil as a safe adjuvant that may complement thrombolytic therapy by inhibiting TXNIP's detrimental role in hyperglycemic stroke.

Abstract 4: Glucose Control and Risk of Tpa-Related Symptomatic Intracerebral Hemorrhage in Patients With Hyperglycemic Acute Ischemic Stroke: Preplanned Analysis From the SHINE Trial

Introduction: In acute ischemic stroke (AIS), hyperglycemia promotes enhanced blood brain barrier permeability, tissue acidosis, and oxygen free radicals, and may increase risk of post-tPA symptomatic intracerebral hemorrhage (sICH). We performed a pre-planned analysis from the SHINE trial (NCT01369069) to examine the effects of blood glucose (BG) control on post-thrombolysis ICH. Hypothesis: In AIS, (1) post-tPA BG measures are associated with sICH, and (2) intensive insulin therapy can reduce the risk of sICH. Methods: Hyperglycemic AIS patients &lt;12 hours onset were randomized to intensive insulin (target range 80-130 mg/dL) vs standard BG control (80-179 mg/dL) over a 72-hour period. Randomization was stratified by tPA treatment. Three independent vascular neurologists reviewed all sICH events occurring within 7 days, defined by neurologic deterioration of ≥4 points on the NIHSS. Associations between BG control and sICH were analyzed using a logistic regression model accounting for NIHSS, age, systolic blood pressure, onset to tPA time, and endovascular therapy. Associations were reported as odds ratios (95% CI). Categorical variables and outcomes were compared using the chi-square test (p &lt; 0.05). Results: Of the 1151 SHINE participants, 725 (63%) received IV tPA (median age 65, 46% women, 29% Black, 18% Hispanic). Median NIHSS was 7, baseline BG 187 (IQR 153-247) mg/dL, and onset to tPA was 2.2 hrs (1.6-2.9). Post-tPA sICH occurred in 3.6% (3% intensive vs. 4.3% standard, OR 1.10, 95% CI 0.60-2.01, p=0.697). There was a consistent association between post-tPA BG measures and sICH. In the first 12 hours, every 10 mg/dL increase in median BG increased odds of sICH by ~8% (OR 1.08, 95% CI 1.03-1.14, p=0.004), and a greater percentage of BG measures 80-130 mg/dL decreased odds of sICH by ~11% (0.89, 95% CI 0.80-0.99, p=0.030). Conclusion: In this pre-planned analysis, intensive insulin therapy was not associated with a reduced risk of post-tPA sICH. However, post-tPA hyperglycemia was associated with a higher risk of sICH overall, particularly in the early post-treatment period. These data provide class IIa, level B-R evidence that post-tPA glucose levels between 80-130 mg/dL are associated with decreased risk of sICH. Acknowledgments: NIH-NINDS U01 NS069498.

High glucose-induced effects on Na+-K+-2Cl- cotransport and Na+/H+ exchange of blood-brain barrier endothelial cells: involvement of SGK1, PKCβII, and SPAK/OSR1.

Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCβII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCβII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCβII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCβII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCβII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

The peripheral immune response in hyperglycemic stroke

ObjectivesHyperglycemia is common in acute ischemic stroke patients and is associated with poor clinical outcome. However, aggressive reduction of post-stroke hyperglycemia did not improve clinical outcome, suggesting that other mechanisms are playing a detrimental role in hyperglycemic stroke. We hypothesize that the acute post-stroke immune response is altered in the hyperglycemic state leading to higher mortality and morbidity. The objective of this study was to characterize temporal changes in circulating immune cells after stroke and their association with clinical outcomes in hyperglycemic compared to euglycemic patients. Patients and methodsThis retrospective study included 97 (58 % euglycemic, 42 % hyperglycemic) patients presenting within 12 h of symptom onset of stroke. Blood neutrophil, monocyte and lymphocyte concentrations were measured sequentially for 96 h post stroke. Primary clinical outcome was the difference in the NIH stroke scale at admission compared to discharge. Secondary outcome measures included discharge disposition and the modified Rankin Scale (mRS) at 90 days. ResultsCirculating neutrophils were significantly higher in hyperglycemic than in euglycemic patients within the first 48 h post stroke, while lymphocyte counts trended to be lower. Hyperglycemic patients had higher mortality rates, less favorable discharge disposition and worse neurological function at 90 days. In both groups, the neutrophil to lymphocytes ratio ((NLR) remained strongly associated with neurological function at discharge within the first 24 h (p < 0.001), and remained significant in hyperglycemic patients up to 48 h (p < 0.001). Multivariate regression analysis showed no confounding by other factors and a significant correlation with differences in NIHSS score (CI; - 9.287 to -1.46, p = 0.0077**) and NLR (CL; 0.6058–6.901, p = 0.0203*) in hyperglycemic patients. ConclusionsOur data suggests that circulating immune cells play an important role in mediating poor clinical outcome in hyperglycemic patients following stroke. The NLR is a strong predictor of neurological outcomes in hyperglycemic patients. Thus, the modulation of immune cells may be a viable therapeutic approach to improve outcomes for this high risk group.

Tissue Plasminogen Activator Promotes TXNIP-NLRP3 Inflammasome Activation after Hyperglycemic Stroke in Mice

Hyperglycemia has been shown to counterbalance the beneficial effects of tissue plasminogen activator (tPA) and increase the risk of intracerebral hemorrhage in ischemic stroke. Thioredoxin interacting protein (TXNIP) mediates hyperglycemia-induced oxidative damage and inflammation in the brain and reduces cerebral glucose uptake/utilization. We have recently reported that TXNIP-induced NLRP3 (NOD-like receptor pyrin domain-containing-3) inflammasome activation contributes to neuronal damage after ischemic stroke. Here, we tested the hypothesis that tPA induces TXNIP-NLRP3 inflammasome activation after ischemic stroke, in hyperglycemic mice. Acute hyperglycemia was induced in mice by intraperitoneal (IP) administration of a 20% glucose solution. This was followed by transient middle cerebral artery occlusion (t-MCAO), with or without intravenous (IV) tPA administered at reperfusion. The IV-tPA exacerbated hyperglycemia-induced neurological deficits, ipsilateral edema and hemorrhagic transformation, and accentuated peroxisome proliferator activated receptor-γ (PPAR-γ) upregulation and TXNIP/NLRP3 inflammasome activation after ischemic stroke. Higher expression of TXNIP in hyperglycemic t-MCAO animals augmented glucose transporter 1 (GLUT-1) downregulation and increased vascular endothelial growth factor-A (VEGF-A) expression/matrix metallopeptidase 9 (MMP-9) signaling, all of which result in blood brain barrier (BBB) disruption and increased permeability to endogenous immunoglobulin G (IgG). It was also associated with a discernible buildup of nitrotyrosine and accumulation of dysfunctional tight junction proteins: zonula occludens-1 (ZO-1), occludin and claudin-5. Moreover, tPA administration triggered activation of high mobility group box protein 1 (HMGB-1), nuclear factor kappa B (NF-κB), and tumor necrosis factor-α (TNF-α) expression in the ischemic penumbra of hyperglycemic animals. All of these observations suggest a powerful role for TXNIP-NLRP3 inflammasome activation in the tPA-induced toxicity seen with hyperglycemic stroke.

The Stress-Immune Response in Acute Hyperglycemic Stroke

Diabetic and hyperglycemic stroke patients have worse clinical outcome, higher mortality rates, larger infarcts, and more severe neurological disability than euglycemic individuals. The reasons for the worse outcome in diabetes are poorly understood, but may not be fully explained by hyperglycemia alone. The objective of this prospective clinical study was to explore whether there is an association between the stress-immune response and clinical outcomes in the hyperglycemic state. Circulating immune cells including neutrophils, monocytes, B-cells and subpopulations of T-lymphocytes were measured over 96 hours post stroke in both hyperglycemic and euglycemic patients. Cortisol and C-reactive protein (CRP) were measured daily over 4 days in the acute post-stroke phase. Short- and long-term neurological outcomes were assessed by the NIH stroke scale, modified Ranking scale and discharge disposition. Discharge dispositions were captured at discharge and 90 days post stroke. Data on infection rates and all course mortality were collected during the hospital stay and at 3 months follow up. Hyperglycemic stroke patients had worse clinical outcomes, more in-hospital infections, less favorable discharge dispositions and higher mortality rates. Hyperglycemic individuals had higher cortisol and CRP levels, higher peripheral neutrophil concentrations and suppressed lymphocyte levels compared to their euglycemic counterparts. Our data suggests an altered stress-immune response in the hyperglycemic state following acute ischemic stroke which could contribute to the poor clinical outcome in this high risk group. Key words Key words: Diabetes, hyperglycemia, ischemic stroke, lymphocytes, neutrophils

Pioglitazone Treatment for Hyperglycemic Acute Ischemic Stroke

Pioglitazone Treatment for Hyperglycemic Acute Ischemic Stroke

Prehospital exenatide in hyperglycemic stroke-A randomized trial.

Hyperglycemia is a predictor for poor stroke outcome. Hyperglycemic stroke patients treated with thrombolysis have an increased risk of intracranial hemorrhage. Insulin is the gold standard for treating hyperglycemia but comes with a risk of hypoglycemia. Glucagon-like peptide-1 receptor agonists (GLP-1RA) are drugs used in type 2 diabetes that have a low risk of hypoglycemia and have been shown to exert neuroprotective effects. The primary objective was to determine whether prehospital administration of the GLP-1RA exenatide could lower plasma glucose in stroke patients. Secondary objective was to study tolerability and safety. Randomized controlled trial comparing exenatide administrated prehospitally with a control group receiving standard care for hyperglycemia. Patients with Face Arm Speech Test ≥1 and glucose ≥8mmol/L were randomized. Glucose was monitored for 24hours. All adverse events were recorded. Nineteen patients were randomized, eight received exenatide. An interim recruitment failure analysis with subsequent changes of the protocol was made. The study was stopped prematurely due to slow inclusion. No difference was observed in the main outcome of plasma glucose at 4hours, control vs exenatide (mean, SD); 7.0±1.9 vs 7.6±1.6; P=.56). No major adverse events were reported. We found no evidence that prehospital exenatide had effect on hyperglycemia. However, it was given without adverse events in this study with limited sample size that was prematurely stopped due to slow inclusion.

Intravenous delivery of adipose tissue-derived mesenchymal stem cells improves brain repair in hyperglycemic stroke rats

BackgroundOver 50% of acute stroke patients have hyperglycemia, which is associated with a poorer prognosis and outcome. Our aim was to investigate the impact of hyperglycemia on behavioral recovery and brain repair of delivered human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) in a rat model of permanent middle cerebral artery occlusion (pMCAO).MethodsHyperglycemia was induced in rats by the administration of nicotinamide and streptozotocin. The rats were then subjected to stroke by a pMCAO model. At 48 h post-stroke, 1 × 106 hAD-MSCs or saline were intravenously administered. We evaluated behavioral outcome, infarct size by MRI, and brain plasticity markers by immunohistochemistry (glial fibrillary acidic protein [GFAP], Iba-1, synaptophysin, doublecortin, CD-31, collagen-IV, and α-smooth muscle actin [α-SMA]).ResultsThe hyperglycemic group exhibited more severe neurological deficits; lesion size and diffusion coefficient were larger compared with the non-hyperglycemic rats. GFAP, Iba-1, and α-SMA were increased in the hyperglycemic group. The hyperglycemic rats administered hAD-MSCs at 48 h after pMCAO had improved neurological impairment. Although T2-MRI did not show differences in lesion size between groups, the rADC values were lower in the treated group. Finally, the levels of GFAP, Iba-1, and arterial wall thickness were lower in the treated hyperglycemic group than in the nontreated hyperglycemic group at 6 weeks post-stroke.ConclusionsOur data suggest that rats with hyperglycemic ischemic stroke exhibit increased lesion size and impaired brain repair processes, which lead to impairments in behavioral recovery after pMCAO. More importantly, hAD-MSC administration induced better anatomical tissue preservation, associated with a good behavioral outcome.

Abstract WP151: Ischemic Injury in Diabetes Augments Cerebral Microvascular Matrix Metalloproteinase Activation in Female Rats

Introduction: Diabetic female patients are at greater risk of stroke and worsened recovery. We have previously shown that matrix metalloprotease 3 (MMP3) exacerbates hemorrhagic transformation (HT) in hyperglycemic stroke in male rats while high glucose and hypoxia increases MMP3 activity in brain microvascular endothelial cells (BMVECs). Furthermore, diabetic female rats develop greater HT. Current study was designed to test the hypothesis that ischemic stroke increases the cerebral microvascular MMP3 activity in diabetic female animals. Methods: Female rats after 7-8 weeks of diabetes (high fat diet and low dose of streptozotocin) were subjected to 60 min MCAO. After 3 days, adhesive removal time (ART) and behavioral composite score were measured and cerebral microvessels were isolated. Human female BMVECs were grown under normal (5.5mM glucose) or diabetic condition (25mM glucose plus 100μM sodium palmitate) for 48 hours, followed by hypoxia insult for 12 hours (200μM cobalt chloride). Cell lysate, media and microvascular homogenates were analyzed for MMP profiling by immunoblotting, zymography and MMP3 enzyme activity by fluorescence resonance energy transfer (FRET) assay. Results: MMP3 protein expression and activity were significantly increased in the ischemic microvasculature and this was accompanied by a worse neurobehavioral score (Table 1). In vitro, expression of MMP3, 2 and 9 proteins in cell lysate were not different between the groups. However, MMP3 and MMP9 enzyme activities were significantly increased in the diabetes condition while, there was no additional increase in enzyme activity with hypoxia. MMP2 activity was significantly increased with hypoxia in diabetes condition (Table 1). Conclusions: In female rats, diabetes and hypoxia increase MMP activity in the microvasculature and endothelial cells. These observations provide initial evidence to further explore the role of MMPs in the increased HT seen in females with diabetes.

Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood–brain barrier Na–K–Cl cotransport and Na/H exchange

Cerebral edema is exacerbated in diabetic ischemic stroke through poorly understood mechanisms. We showed previously that blood–brain barrier (BBB) Na–K–Cl cotransport (NKCC) and Na/H exchange (NHE) are major contributors to edema formation in normoglycemic ischemic stroke. Here, we investigated whether hyperglycemia-exacerbated edema involves changes in BBB NKCC and NHE expression and/or activity and whether inhibition of NKCC or NHE effectively reduces edema and injury in a type I diabetic model of hyperglycemic stroke. Cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundances and activities were determined by Western blot, radioisotopic flux and microspectrofluorometric methods. Cerebral edema and Na in rats subjected to middle cerebral artery occlusion (MCAO) were assessed by nuclear magnetic resonance methods. Hyperglycemia exposures of 1-7d significantly increased CMEC NKCC and NHE abundance and activity. Subsequent exposure to ischemic factors caused more robust increases in NKCC and NHE activities than in normoglycemic CMEC. MCAO-induced edema and brain Na uptake were greater in hyperglycemic rats. Intravenous bumetanide and HOE-642 significantly attenuated edema, brain Na uptake and ischemic injury. Our findings provide evidence that BBB NKCC and NHE contribute to increased edema in hyperglycemic stroke, suggesting that these Na transporters are promising therapeutic targets for reducing damage in diabetic stroke.

Peroxynitrite-Induced Tyrosine Nitration Contributes to Matrix Metalloprotease-3 Activation: Relevance to Hyperglycemic Ischemic Brain Injury and Tissue Plasminogen Activator.

Matrix metalloprotease-3 (MMP3) activation mediates the tissue plasminogen activator (tPA)-induced hemorrhagic transformation after stroke. Hyperglycemia (HG) further exacerbates this outcome. We have recently shown that HG increases MMP3 activity in the brain after stroke. However, the combined HG-tPA effect on MMP3 activation, and the mechanisms through which MMP3 is activated were not previously reported. Accordingly, this study tested the hypothesis that tPA and HG increases MMP3 activity in the brain after stroke through peroxynitrite induced tyrosine nitration. Normoglycemic and mildly hyperglycemic male Wistar rats were subjected to middle cerebral artery suture occlusion for 90min or thromboembolic occlusion, and up to 24h reperfusion, with and without tPA. MMP3 activity and tyrosine nitration were evaluated in brain homogenates at 24h. Brain microvascular endothelial cells (BMVEC) were subjected to either 3h hypoxia or 6h OGD under either normal or high glucose conditions with or without tPA, with or without peroxynitrite scavenger, FeTPPs. MMP3 activity and MMP3 tyrosine nitration were assessed at 24h. HG and tPA significantly increased activity and tyrosine nitration of MMP3 in the brain. In BMVECs, tPA but not HG increased MMP3 activity. Treating BMVEC with FeTPPs significantly reduced the tPA-induced increase in MMP3 activity and nitration. Augmented oxidative and nitrative stress may be potential mechanisms contributing to MMP3 activation in hyperglycemic stroke, especially with tPA administration. Peroxynitrite may be playing a critical role in mediating MMP3 activation through tyrosine nitration in hyperglycemic stroke.