Brain Infarct Size Research Articles

CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice

The studies on fractalkine and its unique receptor CX3CR1 in neurological disorders yielded contrasting results. We have explored the consequences of CX3CR1 deletion in ischemic (30' MCAo) mice on: (1) brain infarct size; (2) microglia dynamism and morphology; (3) expression of markers of microglia/macrophages (M/M) activation and polarization. We observed smaller infarcts in cx3cr1(-/-) (26.42 ± 7.41 mm(3) , mean ± sd) compared to wild type (36.29 ± 11.57) and cx3cr1(-/+) (34.49 ± 8.91) mice. We longitudinally analyzed microglia by in vivo two-photon microscopy before, 1 and 24 h after transient ischemia. Microglia were stationary in both cx3cr1(-/-) and cx3cr1(-/+) mice throughout the study. In cx3cr1(-/-) mice, they displayed a significantly higher number of ramifications >10 μm at baseline and at 24 h after ischemia compared to cx3cr1(-/+) mice, indicating that CX3CR1 deficiency impaired the development of microglia hypertrophic/amoeboid morphology. At 24 h after ischemia, we performed post mortem quantitative immunohistochemistry for different M/M markers. In cx3cr1(-/-) immunoreactivity for CD11b (M/M activation) and for CD68 (associated with phagocytosis) were decreased, while that for CD45(high) (macrophage and leukocyte recruitment) was increased. In addition, immunoreactivity for Ym1 (M2 polarization) was enhanced, while that for iNOS (M1) was decreased. Our data show that in cx3cr1(-/-) mice protection from ischemia at early time points after injury is associated with a protective inflammatory milieu, characterized by the promotion of M2 polarization markers.

Simvastatin Attenuates Stroke-induced Splenic Atrophy and Lung Susceptibility to Spontaneous Bacterial Infection in Mice

Statins are widely used in the primary and secondary prevention of ischemic stroke, but their effects on stroke-induced immunodepression and poststroke infections are elusive. We investigated the effects of simvastatin treatment on stroke-induced splenic atrophy and lung susceptibility to bacterial infection in acute experimental stroke in mice. Ischemic stroke was induced by transient middle cerebral artery occlusion, followed by reperfusion. In some experiments, splenectomies were performed 2 weeks before middle cerebral artery occlusion. Animals were randomly assigned to sham and middle cerebral artery occlusion groups treated subcutaneously with vehicle or simvastatin (20 mg/kg per day). Brain infarction, neurological function, brain interferon-γ expression, splenic atrophy and apoptosis, and lung infection were examined. Simvastatin reduced stroke-induced spleen atrophy and splenic apoptosis via increased mitochrondrial antiapoptotic Bcl-2 expression and decreased proapoptotic Bax translocation from cytosol into mitochondria. Splenectomy reduced brain interferon-γ (3 days) and infarct size (5 days) after stroke, and these effects were reversed by adoptive transfer of splenocytes. Simvastatin inhibited brain interferon-γ (3 days) and reduced infarct volume and neurological deficits (5 days) after stroke, and these protective effects were observed not only in naive stroke mice but also in splenectomied stroke mice adoptively transferred with splenocytes. Simvastatin also decreased the stroke-associated lung susceptibility to spontaneous bacterial infection. Results provide the first direct experimental evidence that simvastatin ameliorates stroke-induced peripheral immunodepression by attenuating spleen atrophy and lung bacterial infection. These findings contribute to a better understanding of the beneficial effects of statins in the treatment of stroke.

Abstract TP100: Meningeal Mast Cells Can Exacerbate Stroke Pathology In Mice

Inflammation critically contributes to post-stroke brain damage. Mast cells (MCs), perivascular cells best known as effector cells involved in the development of inflammatory processes, have been reported to exacerbate stroke pathology. Unlike other immune cells, mature MCs do not circulate but are resident in virtually all anatomical sites, including brain parenchyma and meninges. Thus a key question is which tissue-specific MCs are important after stroke. To address this, we used ‘mast cell knock-in’ mouse models whereby genetically MC-deficient mice were selectively repaired of their MC deficiency by engraftment of in vitro grown mast cells. Methods: Two different MC-deficient mouse models (KitW-sh/W-sh and KitW/W-v) were used. For each model, 3 groups were tested: wild-type, MC-deficient, and MC-engrafted. Mice were subjected to 30 min occlusion of the middle cerebral artery. Brain swelling and infarct size were assessed by T2-weighted MRI and histology. The immune response was quantified by flow cytometry. Results: MC-deficient mice had less brain swelling at 3d post-stroke, and smaller lesions at 3d and 2wk post-stroke than their corresponding wild-type or systemically MC-engrafted groups, implying that MCs exacerbate ischemic injury. MC-deficient mice also had fewer brain granulocytes at 3d post-stroke compared to the other two groups. However, no MC-dependent changes in granulocyte numbers in blood and spleen were observed at 3d, suggesting a role for central nervous system (CNS) MCs in modulating granulocyte trafficking to the brain and stroke pathology. Analysis of the CNS MC distribution in wild-type and MC-engrafted mice revealed equivalent numbers of MCs in meninges in both groups but almost no MCs in brain parenchyma of MC-engrafted groups. This suggests that meningeal MCs, rather than parenchymal MCs, are key effectors of stroke pathology. To test this, MCs were engrafted locally into the meninges. These meningeal MC-engrafted mice had significantly more brain swelling, larger infarcts, and more brain granulocytes after stroke than MC-deficient mice. Conclusions: Our results support the conclusion that meningeal MCs can exacerbate stroke pathology. Hence, targeting these cells may be a novel therapeutic strategy for stroke.

Abstract WP127: TAK-242, an Antagonist for Toll-like Receptor 4, Protects against Acute Cerebral Ischemia/Reperfusion Injury in Mice

Cerebral ischemia/reperfusion (I/R) induces in situ inflammatory responses which contribute to further brain injury. Toll-like receptors (TLRs) are a trans-membrane, pattern-recognition receptor family with important roles in the induction and regulation of immune/inflammatory responses. TLR4 is known to be involved in cerebral I/R injury and is considered a potential target for the treatment of ischemic stroke. This experiment evaluated the hypothesis that an exogenous TLR4 antagonist, TAK-242, protects brain from I/R injury. A mouse model of cerebral I/R was induced by transient middle cerebral artery occlusion (tMCAO). TAK-242 (3 mg/kg body weight) was intraperitoneally injected (i.p.) 1 hour after ischemia. Concentrations of TAK-242 in plasma and brain tissue were measured 3, 8, and 24 hrs after injection. Neurological scores were evaluated 24 hrs after cerebral I/R. Brain infarct areas were detected by TTC staining. Inflammatory cytokines were analyzed by antibody arrays 6 hrs after cerebral I/R. Group comparisons for infarct size were made with the t-test, and cytokine levels were analyzed by one-way ANOVA with post-hoc comparisons. Our results showed that the concentration of TAK-242 in plasma increased to 52.0 ng/ml 3 hrs after i.p, was maintained at 54.1 ng/ml 8 hrs after i.p., and decreased to 22.6 ng/ml 24 hrs after i.p. The concentration of TAK-242 in brain tissue increased to 26.1 ng/ml (ipsilateral) and 14.2 ng/ml (contralateral) 3 hrs after i.p, was maintained at 26.4 ng/ml (ipsilateral) and 15.1 ng/ml (contralateral) 8 hrs after i.p., and was still maintained at 25.0 ng.ml (ipsilateral) and 17.5 ng/ml (contralateral) 24 hrs after i.p. TAK-242 treatment significantly reduced brain infarct size (12.5%) compared to control mice (21.3%) (p<0.05). Cerebral I/R increased the levels of sTNF RI, sTNF RII, KC, GSCF, IL-6, MCP-1, MIP-1γ, and TIMP-1. TAK-242 treatment significantly reduced the levels of sTNF RII, MCP-1, MIP-1γ, and TIMP-1 (p<0.05). Our data demonstrate that TAK-242 can pass through the blood-brain barrier and that treatment with TAK-242 protects the brain from damage at the acute stage after cerebral I/R by mediating the expression of inflammatory cytokines.

Role of proinflammatory effects of nicotine on the brain microvasculature in stroke

Cigarette smoking is widely recognized as a major modifiable risk factor for stroke. Evidence suggests that nicotine adversely affects cerebral blood flow and the blood-brain barrier. Some recent findings have indicated that cigarette smoking, particularly nicotine, has a profound proinflammatory effect, causing a chronic inflammatory state of brain microvasculature. This was accompanied by enhanced leukocyte infiltration into brain during ischemia/reperfusion. The brain infarct size was closely associated with the exposed dose of nicotine, pinpointing that increased levels of nicotine directly aggravate brain injury. while the harmful effects of smoking on public health have been well demonstrated the underlying mechanisms of toxicity are not completely understood.

L‐theanine Administration Results in Neuroprotection and Prevents Glutamate Receptor Agonist‐Mediated Injury in the Rat Model of Cerebral Ischemia‐Reperfusion

While the neuroprotective effect of green tea (Camellia sinensis) might be explained by the presence of amino acid L-theanine in the tea leaves, it is not known whether postischemic administration of L-theanine could also provide neuroprotection. In the present study, we investigated the neuroprotective effect of L-theanine (1 and 4 mg/kg) administered at 3, 12, and 24 h after reperfusion in the rat model of stroke. We also studied the effect of L-theanine on brain injury caused by exogenous administration of N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate/kainate receptor agonists during reperfusion. Rats were subjected to 30-min middle cerebral artery occlusion followed by 48-h reperfusion. Neurological deficit and infarct size were determined at the end of reperfusion. At 3 and 12 h, but not at 24 h of reperfusion, L-theanine substantially reduced the size of brain infarct. Neurological status was improved when L-theanine was administered 3, 12, and 24 h after reperfusion. Repeated intrastriatal injections of L-theanine at a total dose of 800 µg/kg during reperfusion prevented brain injury caused by glutamate receptor agonists. In conclusion, L-theanine at reperfusion exerts neuroprotective effect in the in vivo rat model of stroke. Local treatment with L-theanine at reperfusion prevents glutamate receptor agonist-mediated brain injury.

Cytochrome P450 2J2 is protective against global cerebral ischemia in transgenic mice

Cytochrome P450 epoxygenase metabolites of arachidonic acid, EETs, have multiple cardiovascular effects, including reduction of blood pressure, protection against myocardial ischemia-reperfusion injury, and attenuation of endothelial apoptosis. This study investigated the hypothesis that transgenic mice with endothelial overexpression of CYP2J2 (Tie2-CYP2J2-Tr) would be protected against global cerebral ischemia induced by bilateral common carotid artery occlusion (BCCAO) and action mechanisms of EETs on cerebral ischemia in cultures of astrocytes exposed to oxygen–glucose deprivation (OGD). Tie2-CYP2J2-Tr mice had significantly increased CYP2J2 expression, increased 14,15-EET production, increases regional cerebral blood flow (rCBF) and microvascular density, decreased ROS production, decreased brain infarct size and apoptosis after ischemia compared to wild type mice, these were associated with increased activation of the PI3K/AKT and apoptosis-related protein in ischemic brain. Addition of exogenous EETs or CYP2J2 transfection attenuated OGD-induced apoptosis in astrocytes via activation of PI3K/AKT and anti-apoptosis pathways. However, these effects were reduced by pretreatments with inhibitor of the PI3K (LY294002) and 14,15-EET (14,15-EEZE), respectively. These results indicate that CYP2J2 overexpression exerts marked neuroprotective effects against ischemic injury by a mechanism linked to increased level of circulating EETs and increases CBF and reduction of apoptosis.

Protective effect of Danhong injection on cerebral ischemia–reperfusion injury in rats

Ethnopharmacological relevanceDanhong injection (DH), a Chinese medical product, is used extensively for the treatment of cerebrovascular diseases such as acutely cerebral infarction in clinic. Aim of the studyTo explore the protective effect and the relevant mechanisms of DH on cerebral ischemia–reperfusion (I/R) injury. Materials and methodsCerebral I/R injury was induced through four-vessel occlusion (4-VO) or middle cerebral artery occlusion (MCAO). Adult male SD rats were randomly divided into six kinds of groups: normal control group, sham-operated group, I/R injury group, DH-treated groups at doses of 0.5ml/kg, 1.0ml/kg and 2.0ml/kg. The effects of DH on murine neurological deficits and cerebral infarct volume, 6-keto-prostagladin F1α (6-keto-PGF1α) level, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in brain tissue, as well as the activities of plasma tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) after I/R were evaluated. Moreover, the expressions of Bcl-2 and Bax protein were detected by immunohistochemistry. ResultsThere was no significant difference between the control group and the sham-operated group based on the measurement indicators. Compared with the vehicle-treated group, rats treated with DH showed dose dependent reductions in brain infarction size, and improvement of neurological outcome. The level of 6-keto-PGF1α and the activities of SOD and plasma t-PA were enhanced significantly, whereas the level of MDA and the activity of plasma PAI were declined significantly. The immunohistochemical staining results also revealed that the expression of Bcl-2 protein was up-regulated and that of Bax protein was down-regulated when exposed to DH. ConclusionDH demonstrates a strong ameliorative effect on cerebral I/R damage in rats by its anticoagulant, antithrombotic, antifibrinolytic and antioxidant activities. Furthermore, suppressing apoptosis through regulating Bcl-2 and Bax protein expressions should be another potential mechanism by which DH exerts its neuroprotective function.

Inhibitory effects of Qushuanling Capsule (祛栓灵胶囊) on thrombus formation and platelet aggregation in rats

To investigate the effects of Qushuanling Capsule ( QSLC) on thrombus formation and platelet aggregation in rats. Arteriovenous bypass, venous thrombosis, and middle cerebral artery thrombosis models were used in rats to investigate the anti-thrombotic effects of QSLC, a compound of nine Chinese herbs. The platelet aggregation induced by adenosine diphosphate (ADP), thrombin or arachidonic acid (AA), as well as the contents of thromboxane B(2) (TXB(2)) and 6-keto-prostaglandin F1α (6-keto-PGF1α) in rat plasma and aortic walls, were determined to investigate the possible mechanisms of the anti-thrombotic effects of QSLC. After oral administration with QSLC for 7 days, arteriovenous bypass thrombosis was obviously suppressed compared with the model group, venous thrombosis was also obviously suppressed, rat behaviors were obviously improved, and brain infarct size as well as water content were also reduced. The platelet aggregation induced by ADP or thrombin was inhibited by QSLC, but the drug had no effect on AA-induced platelet aggregation and content of TXB(2) and 6-keto-PGF1α in plasma and the aortic wall. These results suggest that QSLC can be used in the prevention and treatment of thrombotic diseases, and that its mechanism of action may be related to inhibition of platelet aggregation.

Perinatal nicotine exposure increases vulnerability of hypoxic-ischemic brain injury in neonatal rats: role of angiotensin II receptors.

Maternal cigarette smoking increases the risk of neonatal morbidity. We tested the hypothesis that perinatal nicotine exposure causes heightened brain vulnerability to hypoxic-ischemic (HI) injury in neonatal rats through aberrant expression patterns of angiotensin II type 1 (AT(1)R) and type 2 (AT(2)R) receptors in the developing brain. Nicotine was administered to pregnant rats through subcutaneous osmotic minipumps. HI brain injury was determined in 10-day-old pups. AT(1)R and AT(2)R expression patterns were assessed through Western blotting, quantitative polymerase chain reaction, immunofluorescence, and confocal imaging. Perinatal nicotine exposure significantly increased HI brain infarct size in male, but not female, pups. In fetal brains, nicotine caused a decrease in mRNA and protein abundance of AT(2)R but not AT(1)R. The downregulation of AT(2)R persisted in brains of male pups, and nicotine treatment resulted in a significant increase in methylation of CpG locus 3 bases upstream of TATA-box at the AT(2)R gene promoter. In female brains, there was an increase in AT(2)R but a decrease in AT(1)R expression. Both AT(1)R and AT(2)R expressed in neurons but not in astrocytes in the cortex and hippocampus. Central application of AT(1)R antagonist losartan or AT(2)R antagonist PD123319 increased HI brain infarct size in both male and female pups. In male pups, AT(2)R agonist CGP42112 abrogated nicotine-induced increase in HI brain infarction. In females, PD123319 uncovered the nicotine's effect on HI brain infarction. Perinatal nicotine exposure causes epigenetic repression of the AT(2)R gene in the developing brain resulting in heightened brain vulnerability to HI injury in neonatal male rats in a sex-dependent manner.

Ischaemic Postconditioning Rescues Brain Injury Caused by Focal Ischaemia/Reperfusion via Attenuation of Protein Oxidization

To investigate the effects of ischaemic postconditioning on brain injury and protein oxidization in focal ischaemia/reperfusion. Adult male Wistar rats (n = 30) were randomly divided into sham-operated, ischaemia, and ischaemic postconditioning groups. Ischaemia was produced by middle cerebral artery occlusion and ischaemic postconditioning was performed using three cycles of 30-s/30-s reperfusion/reocclusion after 2 h of ischaemia. Brain infarction size, hydrogen peroxide concentration, superoxide dismutase (SOD), catalase (CAT) and proteasome activities, protein carbonyl derivatives and advanced oxidized protein products (AOPPs) were evaluated. The size of brain infarction after ischaemic postconditioning was significantly smaller compared with the ischaemia group, and was concomitant with significant reduction in protein carbonyl derivatives and AOPPs. The activities of SOD, CAT and proteasomes were elevated by ischaemic postconditioning compared with the ischaemia group. Ischaemic postconditioning is an effective way of reducing the size and effects of brain infarction caused by focal ischaemia/reperfusion, possibly due to a decrease in oxidized protein levels. Decreasing protein oxidization may, therefore, be a useful target for preventing cerebral injury.

Antithrombotic Potential of Blockers of Store-Operated Calcium Channels in Platelets

Platelet Orai1 channels mediate store-operated Ca(2+) entry (SOCE), which is required for procoagulant activity and arterial thrombus formation. Pharmacological blockage of these channels may provide a novel way of antithrombotic therapy. Therefore, the thromboprotective effect of SOCE blockers directed against platelet Orai1 is determined. Candidate inhibitors were screened for their effects on SOCE in washed human platelets. Tested antagonists included the known compounds, SKF96365, 2-aminoethyl diphenylborate, and MRS1845 and the novel compounds, Synta66 and GSK-7975A. The potency of SOCE inhibition was in the order of Synta66>2-aminoethyl diphenylborate>GSK-7975A>SKF96365>MRS1845. The specificity of the first 3 compounds was verified with platelets from Orai1-deficient mice. Inhibitory activity on procoagulant activity and high-shear thrombus formation was assessed in plasma and whole blood. In the presence of plasma, all 3 compounds suppressed platelet responses and restrained thrombus formation under flow. Using a murine stroke model, arterial thrombus formation was provoked in vivo by transient middle cerebral artery occlusion. Postoperative administration of 2-aminoethyl diphenylborate markedly diminished brain infarct size. Plasma-soluble SOCE blockers such as 2-aminoethyl diphenylborate suppress platelet-dependent coagulation and thrombus formation. The platelet Orai1 channel is a novel target for preventing thrombotic events causing brain infarction.

Protective effect of celastrol in rat cerebral ischemia model: Down-regulating p-JNK, p-c-Jun and NF-κB

Oxidative stress and inflammatory damage play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Celastrol has been proved to elicit a vanity of biological effects through its anti-oxidant, anti-inflammatory properties in the treatment of Alzheimer's disease, systemic lupus erythematosus, and rheumatoid arthritis. However, little is known regarding the effect of celastrol in the acute phase of ischemic stroke. This study investigated the potential protective effects of celastrol and underlying mechanisms in cerebral ischemia. We used a permanent middle cerebral artery occlusion (pMCAO) model and administered celastrol intraperitoneally immediately after stroke. At 24h after stroke, we found that celastrol dramatically reduced neurological deficit, brain water content and infarct sizes, and downregulated the expression of p-JNK, p-c-Jun and NF-κB. The results indicated that celastrol may have the possibility of protective effect against ischemic injury, and this effect may be through downregulation of the expression of p-JNK, p-c-Jun and NF-κB.

Unfavorable Neurological Outcome among Patients Admitted during On-Call Hours (P06.251)

Objective: We assessed the relation between hospital admission during on-call hours and two outcome measures: in hospital mortality (IHM) and neurological deterioration (ND) during hospital stay. Background Acute ischemic stroke admissions during weekends are associated with an increased mortality. It is unclear whether adverse outcome is also more frequent among patients admitted during on-call hours. Design/Methods: We assessed a consecutive cohort of ischemic stroke patients admitted between 01/01/2007 and 12/31/2010. Admission during on-call hours was defined as that occurring during weekends and between 8pm and 8am on weekdays. We defined ND as a difference of ≥ 4 points between the National Institutes of Health Stroke Scale (NIHSS) score on admission and upon discharge. We compared risk factors, comorbidities, neuroimaging results, and outcome measures in patients admitted during on-call and non-on-call hours. We assessed the effect of arriving during on-call hours on IHM and ND by using multiple logistic regression analysis (MLRA), adjusted for age, sex, brain infarct size, and severity of ischemic stroke. Results: We included 229 patients, 82 admitted during on-call hours (35.8%, 95% CI 29.9-42.2). Twenty patients experienced ND (8.7%, 95% CI 5.7-13.1) and 16 died during hospital stay (7.0% CI 95% 4.4-11.1). Patients arriving during on-call hours had more severe strokes (NIHSS 7.7 ± 7.0 vs. 5.3 ± 5.7, p=.008) and a higher proportion of new onset atrial fibrillation (13.4 vs. 5.4%, p=.036), and ND (15.9 vs. 4.8%, p=.004). In the MLRA, admission during on-call hours was associated with ND (OR 3.1, 95% CI 1.1-8.6, p=.024), but not with IHM. Conclusions: Stroke patients admitted during on-call hours had more severe strokes and higher frequency of new onset atrial fibrillation and ND. Admission during on-call hours was associated with ND, regardless the severity of the ischemic stroke, age, sex, and brain infarct size. Admission during on-call hours was not associated with IHM. Disclosure: Dr. Riccio has nothing to disclose. Dr. Sposato has nothing to disclose. Dr. Sposato has nothing to disclose. Dr. Sposato has nothing to disclose. Dr. Sposato has nothing to disclose. Dr. Sposato has nothing to disclose. Dr. Sposato has nothing to disclose. Dr. Sposato has nothing to disclose.

Neuroprotection of early and short-time applying berberine in the acute phase of cerebral ischemia: Up-regulated pAkt, pGSK and pCREB, down-regulated NF-κB expression, ameliorated BBB permeability

BackgroundBerberine (BBR) has gained attention for its vast beneficial biological effects through immunomodulation, anti-inflammatory and anti-apoptosis properties. Inflammatory and apoptosis damage play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. The aim of this study was to explore BBR's effect in ischemic injury and the role of the Akt/GSK (glycogen synthase kinase) signaling cascade in mediating the anti-apoptosis and anti-inflammatory effects in the rat brain of permanent middle cerebral artery occlusion (pMCAO). Male Sprague–Dawley rats were subjected to pMCAO and randomly assigned into four groups: Sham (sham-operated) group, pMCAO (pMCAO+0.9% saline) group, BBR-L (pMCAO+BBR 10mg/kg) and BBR-H (pMCAO+BBR 40mg/kg) group. BBR was administered immediately after pMCAO and the neuroprotection was detected. Phospho-Akt (pAkt), phospho-glycogen synthase kinase 3-β (pGSK3β), phospho-cAMP response element binding protein (pCREB), nuclear factor-kappa B (NF-κB) and claudin-5 in ischemic cerebral cortex were detected by immunohistochemistry, reverse transcription-polymerase chain reaction and western blotting. Compared with pMCAO group, BBR dramatically lessened neurological deficits scores, brain water contents and infarct sizes, upregulated the expression of pAkt, pGSK3β, pCREB and claudin-5, and decreased the nuclear accumulation of NF-κB (P<0.05) in ischemic brain. The results showed that BBR reduced ischemic brain injury after pMACO, and this effect may be via the increasing the activation of Akt/GSK signaling and claudin-5, and decreasing NF-κB expression.

Perinatal Nicotine Exposure Increases Hypoxic‐Ischemic Brain Injury in Neonatal Rats

Maternal cigarette smoking increases the risk of neonatal morbidity. This study determined whether perinatal nicotine exposure causes heightened brain vulnerability to hypoxic‐ischemic (HI) injury in neonatal rats via altered expression patterns of angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors. Nicotine was administered via subcutaneous osmotic minipumps from gestational day 4 to postnatal day 10. HI brain injury was determined in 10‐day‐old pups. Nicotine treatment significantly enhanced HI brain infarct size in male pups. In fetal brains, nicotine caused downregulation of AT2R, but not AT1R, expression. The downregulation of AT2R persisted in male pup brains. In female brains, there was an increase in AT2R, but a decrease in AT1R. Intracerebroventricular (ICV) administration of AT1R antagonist losartan or AT2R antagonist PD123319 exaggerated HI brain infarction in both male and female pups. AT2R agonist CGP42112 (ICV) abrogated nicotine‐induced increase in HI brain infarction in male pups. PD123319 (ICV) uncovered nicotine's effect on HI brain infarction in females. The results suggest that perinatal nicotine exposure causes aberrant AT2R gene expression in the developing brain, resulting in heightened brain vulnerability to HI injury in neonatal male rats in a sex‐dependent manner. (Supported in part by NIH grants HL82779 and HL83966).

Neuroprotective effect of hydroxypropyl-β-cyclodextrin in hypoxia-ischemia

Neonatal cerebral ischemic injury is a common and debilitating pathology for which there is currently no known purely pharmacological treatments that are effective when delivered immediately after injury. Cyclodextrins are cyclic oligosaccharides that can remove cholesterol from cell membranes and thereby affect receptor function. Cyclodextrins have previously been shown to be neuroprotective in vitro. We showed that hydroxypropyl-β-cyclodextrin is neuroprotective in rats in vivo when delivered by intraperitoneal injection 30 min following hypoxia-ischemia, when assessed 15 days after surgery. A single dose of 1 g/kg hydroxypropyl-β-cyclodextrin reduced brain infarction size by 28.57% compared with control (P<0.001). We also report that the same compound reduces neuronal excitability in hippocampal slices and propose that hydroxypropyl-β-cyclodextrin is neuroprotective by reducing excitotoxicity in the delayed phase of brain damage.

Abstract 3676: MyD88-Dependent Pathway Has a Dual Effect on Brain Injury after Cerebral Ischemia/Reperfusion in Mouse

Myeloid differentiation primary-response protein-88 (MyD88) is one of the intracellular adaptors in Toll-like receptor (TLR) signaling pathways, which regulates the activation of nuclear factor kappa B (NF-kappa-B) and transcription of other proinflammatory cytokines. We assessed the hypothesis that MyD88-dependent signaling mediates the inflammatory responses in ischemic brain and is implicated in the process of brain injury. A mouse model of cerebral ischemia/reperfusion (I/R) was induced by transient middle cerebral artery occlusion (tMCAO). MyD88 knockout (KO) mice and wild type control mice (WT, C57BL/6J) were assigned to I/R or sham-operated groups. The mice were killed at 4 hrs or 24 hrs after reperfusion following 60 minutes of MCAO. Using Nissl staining, brain infarct areas were evaluated and expressed as a percentage of the whole brain. Brain edema was evaluated and presented as the percentage of water content in the total weight of the brain tissue. The expression of mRNA was detected by real-time PCR, and the levels of inflammatory cytokines were measured by ELISA and Western Blots. The infarct size was analyzed by t-test. Brain edema and inflammatory response were analyzed by one way ANOVA with post hoc comparisons. Our results showed that the brain infarct size was not significantly different in MyD88KO mice compared with WT mice 24 hrs after cerebral I/R. However, brain edema (water content) significantly increased in MyD88KO mice compared with WT 24 hrs after ischemia (p&lt;0.05). The data also showed that the levels of mRNA of CD14, Pellino-1, cyclooxygenase-2 (COX-2), and Interferon beta-1 (INFb1) increased significantly 4 hrs after cerebral I/R in WT mice compared to sham-operated controls (p&lt;0.05). MyD88 deficiency inhibited the increased expression of Pellino-1 (p&lt;0.05) and COX-2 (p&lt;0.05), but not CD14. Twenty-four hrs after cerebral I/R, the protein levels of Interleukin - 1-beta, Interleukin - 6 (IL-6), p-IkappaB alpha, and the activity of NFkappaB, significantly increased in the ischemic brain in WT mice, and were inhibited in MyD88KO mice. Interestingly, VEGF significantly increased in MyD88KO mice compared with WT mice (p&lt;0.05). In addition, ZO-1, a tight-junction protein on the blood-brain barrier (BBB) decreased in MyD88KO mice compared with WT mice subjected to cerebral I/R. Our data demonstrates that MyD88 contributes to the activation of inflammatory responses after cerebral I/R. However, MyD88 reduces brain edema by mediating the expression of VEGF, and tight-junction proteins, which are important for maintaining BBB integrity. We concluded that the MyD88-dependent pathway has a dual effect on brain injury induced by cerebral I/R. Interpreting the results for the role of MyD88 dependent pathway based on brain injury from whole animal experiments is cautioned.

Probucol and atorvastatin in combination protect rat brains in MCAO model: Upregulating Peroxiredoxin2, Foxo3a and Nrf2 expression

Inflammation and oxidative stress play an important role in cerebral ischemic pathogenesis. It has been well established that atorvastatin and probucol could elicit a variety of biological effects through its anti-inflammatory and anti-oxidant properties respectively. This study was to examine whether probucol and atorvastatin in combination had the enhanced protective effect against cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO). Experiment 1 was used to evaluate the time course expression of Peroxiredoxin2 (Prx2) and Foxo3a after cerebral ischemia. Experiment 2 was used to detect neuroprotective effect of atorvastatin and probucol in cerebral ischemia. At 24h or 72h, neurologic deficit, brain water content and infarct size were measured. Immunohistochemistry, RT-PCR, Western blot and confocal microscope were used to analyze the expressions of Prx2, Foxo3a and nuclear factor erythroid 2-related factor 2 (Nrf2). Compared with the normal-control group, the expressions of Prx2 and Foxo3a were down-regulated in ischemic brain. Compared with the use of probucol or atorvastatin alone, the combined treatment dramatically reduced the brain water content and the infarct volume (P<0.05). Meanwhile, the decrease of Prx2, Foxo3a and Nrf2 was significantly alleviated in combined treatment group. Probucol combined with atorvastatin can get the augmented neuroprotection from the damage caused by MCAO, this effect may be through up-regulation of Prx2, Foxo3a and Nrf2.

Tetrahydrocurcumin Ameliorates Homocysteinylated Cytochrome-c Mediated Autophagy in Hyperhomocysteinemia Mice after Cerebral Ischemia

High levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy), contribute to autophagy and ischemia/reperfusion injury (I/R). Previous studies have shown that I/R injury and HHcy cause increased cerebrovascular permeability; however, the associated mechanism remains obscure. Interestingly, during HHcy, cytochome-c becomes homocysteinylated (Hcy-cyto-c). Cytochrome-c (cyto-c) transports electrons and facilitates bioenergetics in the system. However, its role in autophagy during ischemia/reperfusion injury is unclear. Tetrahydrocurcumin (THC) is a major herbal antioxidant and anti-inflammatory agent. Therefore, the objective of this study was to determine whether THC ameliorates autophagy during ischemia/reperfusion injury by reducing homocysteinylation of cyto-c in hyperhomocysteinemia pathological condition. To test this hypothesis, we employed 8-10-week-old male cystathionine-beta-synthase heterozygote knockout (CBS⁺/⁻) mice (genetically hyperhomocystemic mice). Experimental group was: CBS⁺/⁻, CBS⁺/⁻ + THC (25 mg/kg in 0.1% DMSO dose); CBS ⁺/⁻/I/R, and CBS⁺/⁻/I/R + THC (25 mg/kg in 0.1% DMSO dose). Ischemia was performed for 30 min and reperfusion for 72 h. THC was injected intra-peritoneally (I.P.) once daily for a period of 3 days after 30 min of ischemia. The infarct area was measured using 2,3,5-triphenyltetrazolium chloride staining. Permeability was determined by brain edema and Evans Blue extravasation. The brain tissues were analyzed for oxidative stress, matrix metalloproteinase-9 (MMP-9), damage-regulated autophagy modulator (DRAM), and microtubule-associated protein 1 light chain 3 (LC3) by Western blot. The mRNA levels of S-adenosyl-L-homocysteine hydrolases (SAHH) and methylenetetrahydrofolate reductase (MTHFR) genes were measured by quantitative real-time polymerase chain reaction. Co-immunoprecipitation was used to determine the homocysteinylation of cyto-c. We found that brain edema and Evans Blue leakage were reduced in I/R + THC-treated groups as compared to sham-operated groups along with reduced brain infarct size. THC also decreased oxidative damage and ameliorated the homocysteinylation of cyto-c in-part by MMP-9 activation which leads to autophagy in I/R groups as compared to sham-operated groups. This study suggests a potential therapeutic role of dietary THC in cerebral ischemia.