Hours Of Middle Cerebral Artery Occlusion Research Articles

Abstract TMP109: Elovanoid Precursors Are Neuroprotective in Experimental Ischemic Stroke

Objective: Ischemic stroke triggers oxidative and biochemical responses that disrupt cell homeostasis. We previously demonstrated that Elovanoids (ELVs), a novel class of homeostatic lipid mediators, reduced infarct volumes and improved neurologic function when administered IV following middle cerebral artery occlusion (MCAo). The present study defines neuroprotection by very long-chain polyunsaturated fatty acid (VLC-PUFA) ELV precursors (C-32:6 and C-34:6) delivered intranasally following experimental ischemic stroke. Methods: Male SD rats underwent 2 hours of MCAo. Treatments with C-32:6, C-34:6 (1μg/μl, 20 μg per rat), or vehicle were administered at 1, 24, and 48h after onset of MCAo. Behavior testing was performed on days 1, 2, 3, and 7. Ischemic core and penumbra were computed from T2WI on day 7 followed by immunohistochemistry. RT-qPCR gene expression was investigated in a separate cohort of rats treated as above at 72 h. Results: C-32:6 and C-34:6 improved behavioral function on days three by (41% and 20%) and seven by (45% and 24%); T2WI total lesion volumes were reduced by (87% and 61%), respectively, compared to the vehicle. In the ischemic penumbra, C-32:6 and C-34:6 reduced TMEM119 + microglia by (38% and 29%), increased NeuN + neurons by (87% and 110%), reduced IgG staining by (54% and 25%), and increased the number of SMI-71 + vessels by (50% and 26%). In the core, C-32:6 and C-34:6 reduced TMEM119 + microglia by (12% and 15%), increased NeuN + neurons by (251% and 89%) and increased the number of SMI-71 + vessels by (31% and 70%). Anti-inflammatory marker genes for protective microglia and astrocyte phenotypes Tm4sf1, Thbs1, Ptx3, Gpc4, Fizz1, Cd14, Msr1, Manf , and Osmr were upregulated with C-32:6 and genes Fcrls, S1pr3, and Vim were downregulated with C-34:6. Conclusion: These data suggest that IN administered C-32:6 C-34:6 improved behavior, protected the ischemic penumbra, increased neurogenesis and angiogenesis, and reduced the number of microglia cells. The overall gene expression changes observed from free fatty acid C-32:6 indicate that its modulatory effects promote the biological activity of astrocytes, microglia, and cerebrovascular cells involved in plasticity.

Single-Cell Multiome Analysis Reveals Neuroprotection by Elovanoids in Experimental Ischemic Stroke

Ischemic stroke results in severe loss of neurological functions, and a large proportion of survivors display the onset of cognitive impairments. Administration of a novel class of very long-chain polyunsaturated fatty acids (VLC-PUFAs) termed elovanoids (ELV-N34) or its VLC-PUFA precursor (C34:6) are protective against both in vitro and in vivo models of uncompensated oxidative stress and ischemic stress. We found that ELV-N34 or C34:6 delivery attenuates sensorimotor deficits after ischemic stroke. Furthermore, we uncovered that ELV and its precursors protect against ischemic damage by modulating inflammatory signaling and increasing the expression of neuroprotective genes. We used 10X Single Cell Multiome (ATAC + Gene Expression) to compare genomic and epigenomic responses in naïve rats and after experimental ischemic treatment. Adult Sprague-Dawley rats underwent 2 hours of middle cerebral artery occlusion (MCAo) via intraluminal suture. Rats received vehicle, ELV-N34, or C34:6 delivered intranasally 1 hour, 1 day, and 2 days after 2 hours of MCAo. Brains were harvested on day 3 from the ipsilesional cortex (penumbra region) and subcortex. Nuclei were isolated and processed according to 10X Genomics Single Cell Multiome protocol. ELV-N34 or C34:6 treatment reduced neurological deficits after MCAo. Joint single-cell analysis revealed increases in microglia and leukocyte abundance as well as a loss of neuronal populations after MCAo. Differential gene expression revealed that ELV or its precursors protected against MCAo through complex mechanisms involving anti- inflammatory signaling and promoted neuroregeneration in microglia, astrocytes, neurons, and other neural cells. ELV/precursors exert their bioactivity by modulating conversion to neuroprotective cell phenotypes in neurodegenerative diseases such as stroke or Alzheimer's disease.

Leucine Reduced Blood-Brain Barrier Disruption and Infarct Size in Early Cerebral Ischemia-Reperfusion.

A disruption of the blood-brain barrier (BBB) is a crucial pathophysiological change that can impact the outcome of a stroke. Ribosomal protein S6 (S6) and protein kinase B (Akt) play significant roles in early cerebral ischemia-reperfusion injury. Studies have suggested that branched-chain amino acids (BCAAs) may have neuroprotective properties for spinal cord or brain injuries. Therefore, we conducted research to investigate if leucine, one of the BCAAs, could offer neuroprotection and alter BBB disruption, along with its effects on the phosphorylation of S6 and Akt during the early phase of cerebral ischemia-reperfusion, specifically within the thrombolytic therapy time window. In rats, ten min after left middle cerebral artery occlusion (MCAO), 5 µL of 20 mM L-leucine or normal saline was injected into the left lateral ventricle. After two hours of reperfusion following one hour of MCAO, we determined the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid to assess the BBB disruption, infarct size, and phosphorylation of S6 and Akt. Ischemia-reperfusion increased the Ki (+143%, p < 0.001) and the intra-cerebroventricular injection of leucine lowered the Ki in the ischemic-reperfused cortex (-34%, p < 0.001). Leucine reduced the percentage of cortical infarct (-42%, p < 0.0001) out of the total cortical area. Ischemia-reperfusion alone significantly increased the phosphorylation of both S6 and Akt (p < 0.05). However, the administration of leucine had no further effect on the phosphorylation of S6 or Akt in the ischemic-reperfused cortex. This study suggests that an acute increase in leucine levels in the brain during early ischemia-reperfusion within a few hours of stroke may offer neuroprotection, possibly due to reduced BBB disruption being one of the major contributing factors. Leucine did not further increase the already elevated phosphorylation of S6 or Akt by ischemia-reperfusion under the current experimental conditions. Our data warrant further studies on the effects of leucine on neuronal survival and its mechanisms in the later stages of cerebral ischemia-reperfusion.

Synergistic Neuroprotection by a PAF Antagonist Plus a Docosanoid in Experimental Ischemic Stroke: Dose-Response and Therapeutic Window

ObjectiveWe tested the hypothesis that blocking pro-inflammatory platelet-activating factor receptor (PAFR) with LAU-0901 (LAU) plus administering a selected docosanoid, aspirin-triggered neuroprotectin D1 (AT-NPD1), which activates cell-survival pathways after middle cerebral artery occlusion (MCAo), would lead to neurological recovery. Dose-response and therapeutic window were investigated. Materials and methodsMale SD rats were subjected to 2 hours of MCAo. Behavior testing (days 1-7) and ex vivo MRI on day 7 were conducted. In dose-response, rats were treated with LAU (45 and 60 mg/kg; IP), AT-NPD1 (111, 222, 333 µg/kg; IV), LAU+AT-NPD1 (LAU at 3 hours and AT-NPD1 at 3.15 hours) or vehicle. In the therapeutic window, vehicle, LAU (60 mg/kg), AT-NPD1 (222 µg/kg), and LAU+AT-NPD1 were administered at 3, 4, 5, and 6 hours after onset of MCAo. ResultsLAU and AT-NPD1 treatments alone improved behavior by 40-42% and 20-30%, respectively, and LAU+AT-NPD1 by 40% compared to the vehicle group. T2-weighted imaging (T2WI) volumes were reduced with all doses of LAU and AT-NPD1 by 73-90% and 67-83% and LAU+AT-NPD1 by 94% compared to vehicle. In the therapeutic window, LAU+AT-NPD1, when administered at 3, 4, 5, and 6 hours, improved behavior by 50, 56, 33, and 26% and reduced T2WI volumes by 93, 90, 82, and 84% compared to vehicle. ConclusionsWe have shown here for the first time that LAU plus AT-NPD1 treatment affords high-grade neuroprotection in MCAo, equaling or exceeding that afforded by LAU or AT-NPD1 alone at considerably moderate doses. It has a broad therapeutic window extending to 6 hours after stroke onset.

Inhibition of serum and glucocorticoid regulated kinases by GSK650394 reduced infarct size in early cerebral ischemia-reperfusion with decreased BBB disruption

Blood-brain barrier (BBB) disruption is one of the most important pathological changes following cerebral ischemia-reperfusion. We tested whether inhibition of the serum and glucocorticoid regulated kinase 1 (SGK1) would decrease BBB disruption and contribute to decreasing infarct size in the first few hours of cerebral ischemia-reperfusion within the thrombolysis therapy time window. After transient middle cerebral artery occlusion (MCAO), an SGK1 inhibitor GSK650394, or vehicle was administered into the lateral ventricle of rats. After one hour of MCAO and two hours of reperfusion, we determined BBB disruption using the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid, and also determined infarct size, phosphorylation of NDRG1, and MMP2 protein level. Ischemia-reperfusion increased (+34%, p < 0.05) and GSK650394 decreased (−25%, p < 0.05) the Ki in the ischemic-reperfused cortex. GSK650394 decreased the percentage of cortical infarct (−31%, p < 0.001). At the same time GSK650394 reduced NDRG1 phosphorylation and MMP2 protein level in the ischemic-reperfused cortex suggesting that SGK1 was inhibited by GSK650394 and that lower MMP2 could be one of the mechanisms of decreased BBB disruption. Collectively our data suggest that GSK650394 could be neuroprotective and one of the mechanisms of the neuroprotection could be decreased BBB disruption. SGK1 inhibition within the thrombolysis therapy time window might reduce cerebral ischemia-reperfusion injury.

Adhesion molecule L1 inhibition increases infarct size in cerebral ischemia-reperfusion without change in blood-brain barrier disruption

ABSTRACT Objective Neural cell adhesion molecule L1CAM (L1) is involved in neuroprotection. To investigate a possible neuroprotective effect of L1 during ischemia, we determined whether blocking L1 with an antagonistic antibody would worsen the outcome of focal cerebral ischemia-reperfusion and increase blood-brain barrier (BBB) disruption. Methods Transient middle cerebral artery occlusion (MCAO) was performed in anesthetized rats. Five µg of antagonistic mouse IgG monoclonal L1 antibody 324 or non-immune control mouse IgG was applied on the ischemic-reperfused cortex during one hour of MCAO and two hours of reperfusion. At two hours of reperfusion, BBB permeability, size of infarct using tetrazolium staining, number of TUNEL-labeled apoptotic cells, and immunohistochemistry for expression of PTEN and p53 were studied. Results The antagonistic L1 antibody 324 increased the percentage of cortical infarct area (+36%), but did not affect BBB permeability in the ischemic-reperfused cortex. The antagonistic L1 antibody increased number of apoptotic neurons and p53 expression, but decreased PTEN expression. Conclusion Functional antagonism of L1 increases infarct size by increasing numbers of apoptotic neurons without affecting BBB permeability during the early stage of cerebral ischemia-reperfusion. Our data suggest that L1 affects primarily the brain parenchyma rather than BBB during early stages of cerebral ischemia-reperfusion and that endogenous brain L1 may be neuroprotective.

Novel Therapeutics for Treating Neuroinflammation Following Ischemic Stroke

Ischemic strokes are the fifth leading cause of death in the United States and are caused by the blockage of a major cerebral artery or one of its branches. This occlusion results in a lack of oxygen reaching the tissue, increases in intracellular calcium and glutamate release, and the activation of inflammatory processes, which can cause additional tissue loss and worsen neurological condition. The nuclear factor kappa B (NFkB) cascade is an inflammatory pathway activated by many cytokines and inflammatory signals that are elevated following ischemic stroke, making it a central hub for post-stroke inflammation. We have designed a novel inhibitor of the NFkB cascade attached to drug carrier elastin-like polypeptide (ELP). ELP is based on the sequence of human elastin, has a long plasma half-life, is thermally responsive, and has a tunable size. Our hypothesis is that ELPs can be used to deliver NFkB inhibitory peptide SynB1-ELP-p50i to the infarct region after ischemic stroke, blocking inflammation and ultimately sparing brain tissue. To determine if SynB1-ELP-p50i is an effective inhibitor of the NFkB cascade, we assessed its efficacy in in vitro experiments. RAW 264.7 cells were treated with 1-50 mM SynB1-ELP-p50i then stimulated with lipopolysaccharide (LPS). TNF-a produced in response to LPS stimulation was measured using a TNF-a ELISA with the cell media. SynB1-ELP-p50i treatment completely blocked LPS-stimulated TNF-a release at a concentration of 10 mM. SynB1-ELP-p50i was determined to be an effective inhibitor for translation to in vivo efficacy studies using the middle cerebral artery occlusion (MCAO) model of ischemic stroke. Biodistribution and pharmacokinetic studies were conducted comparing two routes of delivery (intravenous, via the femoral vein, or intraarterial, via a catheter in the external carotid artery) of 50 mg/kg fluorescently labeled SynB1-ELP-p50i after two hours of MCAO. Our results show significant accumulation of SynB1-ELP-p50i in the ischemic hemisphere following MCAO (two-way ANOVA F(1,12) = 10.34; Sidak's p = 0.045). However, there was no difference in intrabrain deposition between intravenous and intraarterial routes of delivery. Future studies will assess the effects of SynB1-ELP-p50i treatment on infarct size, inflammatory cytokine production, and neurological deficits following MCAO in Wistar rats.

Lysophosphatidic acid increased infarct size in the early stage of cerebral ischemia-reperfusion with increased BBB permeability

BackgroundWe investigated whether exogenous lysophosphatidic acid (LPA), a phospholipid extracellular signaling molecule, would increase infarct size and blood-brain barrier (BBB) disruption during the early stage of cerebral ischemia-reperfusion, and whether it works through Akt-mTOR-S6K1 intracellular signaling. Material and methodsRats were given either vehicle or LPA 1 mg/kg iv three times during reperfusion after one hour of middle cerebral artery (MCA) occlusion. In another group, prior to administration of LPA, 30 mg/kg of PF-4708671, an S6K1 inhibitor, was injected. After one hour of MCA occlusion and two hours of reperfusion the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid and the volume of 3H-dextran distribution were determined to measure the degree of BBB disruption. At the same time, the size of infarct was determined and western blot analysis was performed to determine the levels of phosphorylated Akt (p-Akt) and phosphorylated S6 (pS6). ResultsLPA increased the Ki in the ischemic-reperfused cortex (+43%) when compared with Control rats and PF-4708671 pretreatment prevented the increase of Ki by LPA. LPA increased the percentage of cortical infarct out of total cortical area (+36%) and PF-4708671 pretreatment prevented the increase of the infarct size. Exogenous LPA did not significantly change the levels of p-Akt as well as pS6 in the ischemic-reperfused cortex. ConclusionOur data demonstrate that the increase in BBB disruption could be one of the reasons of the increased infarct size by LPA. S6K1 may not be the major target of LPA. A decrease of LPA during early cerebral ischemia-reperfusion might be beneficial for neuronal survival.

ENT1 inhibition attenuates apoptosis by activation of cAMP/pCREB/Bcl2 pathway after MCAO in rats

Background and purposeThe present study was designed to investigate the potential role and the mechanism of equilibrative nucleoside transporter 1 (ENT1) on neuronal apoptosis and neurological deficits after middle cerebral artery occlusion (MCAO) in rats. MethodsOne hundred and thirty-four male Sprague-Dawley rats were subjected to two hours of MCAO followed by reperfusion. The time course of the expression level of ENT1 and phosphorylation of CREB were detected by western blot and immunofluorescence staining. Another set of animals were administrated with NBTI, the ENT1 inhibitor, by daily intraperitoneal injection starting at 0.5 h post-MCAO, infarction volume and neurological deficits were measured both at 24 h and 72 h post MCAO. We further explored the neuroprotection machenism by using H89, cAMP dependent protein kinase inhibitor, the expression of Bcl-2, Bax, phosphorylated CREB and Cleaved caspase-3 were quantified by Western blot, neuronal apoptosis were analyed by TUNEL staining. ResultsThe endogenous expression of ENT1 were significantly increased and peaked at 12 h after MCAO. High-dose of NBTI (15 mg/kg) reduced brain infarction volume and improved neurologic deficits both at 24 h and 72 h post MCAO. Moreover, NBTI significantly increased the level of CREB phosphorylation and extracellular adenosine concentration, and decreased the neuronal apoptosis 24 h after MCAO. NBTI treatment reduced the expression of Bax and cleaved caspase-3, while up-regulated Bcl-2 compared with vehicle group. These effects were abolished by H89 pretreatment. ConclusionsENT1 inhibition prevented neuronal apoptosis and improves neurological deficits through cAMP/PKA/CREB/Bcl-2 signaling pathway after MCAO in rats. ENT1 might be an effective target in the treatment strategy for ischemic stroke.

A Genome-Wide Analysis of the Penumbral Volume in Inbred Mice following Middle Cerebral Artery Occlusion

Following ischemic stroke, the penumbra, at-risk neural tissue surrounding the core infarct, survives for a variable period of time before progressing to infarction. We investigated genetic determinants of the size of penumbra in mice subjected to middle cerebral artery occlusion (MCAO) using a genome-wide approach. 449 male mice from 33 inbred strains underwent MCAO for 6 hours (215 mice) or 24 hours (234 mice). A genome-wide association study using genetic data from the Mouse HapMap project was performed to examine the effects of genetic variants on the penumbra ratio, defined as the ratio of the infarct volume after 6 hours to the infarct volume after 24 hours of MCAO. Efficient mixed model analysis was used to account for strain interrelatedness. Penumbra ratio differed significantly by strain (F = 2.7, P < 0.001) and was associated with 18 significant SNPs, including 6 protein coding genes. We have identified 6 candidate genes for penumbra ratio: Clint1, Nbea, Smtnl2, Rin3, Dclk1, and Slc24a4.

Activation of Akt by SC79 decreased cerebral infarct in early cerebral ischemia-reperfusion despite increased BBB disruption

Activation of Akt has been suggested to produce neuronal protection in cerebral ischemia. Decreasing blood-brain barrier (BBB) disruption has been associated with a better neuronal outcome in cerebral ischemia. We hypothesized that activation of Akt would decrease BBB disruption and contribute to decreasing the size of infarct in the early stage of cerebral ischemia-reperfusion within the therapeutic window. Transient middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. Rats were treated with SC79 (a selective Akt activator which is cell and BBB permeable) 0.05 mg/kg × 3 i.p. or vehicle i.p. perioperatively. After one hour of MCAO and two hours of reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (14C-AIB, molecular weight 104 Da) and the volume of 3H-dextran (molecular weight 70,000 Da) distribution were determined to measure the degree of BBB disruption. At the same time point, the size of infarction was determined using tetrazolium staining. In an additional group of rats, a higher dose of SC79 (0.5 mg/kg × 3) was administered to determine the size of infarct. Administration of SC79 increased the Ki in the ischemic-reperfused cortex (IR-C, +32%, p < 0.05) as well as in the contralateral cortex (CC, +35%, p < 0.05) when compared with the untreated animals with MCAO/reperfusion. The volume of dextran distribution was not significantly changed by SC79. SC79 treatment significantly produced a decrease in the percentage of cortical infarct out of total cortical area (12.7 ± 1.7% vs 6.9 ± 0.9%, p < 0.001). Increasing the dose of SC79 by ten times did not significantly affect the size of cortical infarct. Contrary to our hypothesis, our data demonstrated that SC79 decreased the size of the infarct in the ischemic-reperfused cortex despite an increase in BBB disruption. Our data suggest the importance of activation of Akt for neuronal survival in the early stage of cerebral ischemia-reperfusion within the therapeutic window and that the mechanism of neuroprotection may not be related to the BBB effects of SC79.

Rapamycin decreased blood-brain barrier permeability in control but not in diabetic rats in early cerebral ischemia.

Diabetes causes functional and structural changes in blood-brain barrier (BBB). The mammalian target of rapamycin (mTOR) has been associated with glucose metabolism, diabetes, and altering BBB permeability. Since there is only a narrow therapeutic window (3h) for stroke victims, it is important to investigate BBB disruption in the early stage of cerebral ischemia. We compared the degree of BBB disruption in diabetic and in control rats at two hours of reperfusion after one hour of middle cerebral artery (MCA) occlusion with or without inhibition of mTOR. Two weeks after streptozotocin ip to induce diabetes, MCA occlusion was performed. In half of the rats, an mTOR inhibitor, rapamycin was given for 2days before MCA occlusion. After one hour of MCA occlusion and two hours of the reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid was determined to quantify degree of BBB disruption. Ischemia-reperfusion increased the Ki in the control animals. Streptozotocin increased the Ki in the ischemic-reperfused (IR-C, +22%) as well as in the contralateral cortex (CC, +40%). Rapamycin decreased the Ki in the IR-C (-32%) as well as in the CC (-26%) in the control rats. However, rapamycin did not affect Ki in the IR-C or in the CC in the diabetic rats. Our data demonstrated a greater BBB disruption in diabetes in the ischemic as well as non-ischemic cortex even in the early stage of cerebral ischemia-reperfusion and that acute administration of rapamycin did not significantly affect BBB permeability in diabetes. From our quantitative analysis of BBB disruption, the vulnerability of BBB in diabetes has been emphasized in the early stage of cerebral ischemia-reperfusion and a less important role of the mTOR pathway is suggested in altering BBB permeability in diabetes.

The effect of high dose albumin on permeability of blood-brain barrier in brain of rats after ischemic-reperfusion

Objective To determine the effect of high dose albumin on permeability of blood brain barrier (BBB) in brain of rats after ischemic-reperfusion (IR) in order to explore its possible mechanism. Methods Establishment of brain ischemic reperfusion rat model by using middle cerebral artery occlusion (MCAO). Medicine treatment was given by caudal vein injection after 2 hours of MCAO. Thirty-six healthy male SD rats were then randomly (random number)divided into 6 groups (n=6 in each): 6 h and 24 h sham-operation groups (Group Sham: operation without ischemia), 6 h and 24 h normal saline groups (Group NS: NS injection 5 ml/kg) and 6 h and 24 h albumin group (Group Alb: 25 % Alb injection 1.25 g/kg). Six hours and 24 hours after the end of reperfusion, rats were measured by Zea-Longa score (neural function deficit) separately. Serum concentration of S100B was examined by the ELISA kit and Evans blue in brain tissue was detected by spectrophotometer. The level of AQP4 was examined by Western blot and immunohistochemistry. All data were analyzed by one-way analysis of variance (ANOVA), The intergroup comparisons were analyzed by the least-significant-difference (LSD) test by using SPSS version 17.0 software. Differences were considered statistically significant if P<0.05. Results Zea-Longa score significantly increased in both group NS and group Alb at 6 h and 24 h(P=0.000). However, there was no significant difference in ZEA-LONGA score of 6 h and 24 h between group Alb and group NS (P=1.000). The serum concentration of S100B in group NS 6 h was significantly lower than that in group Alb at 6 h(196.67±20.11 vs 160.04±14.00, P=0.000), and at 24 h(2.45±0.07 vs. 2.23±0.07, P=0.000). Furthermore, concentration of Evans blue in brain tissue in group Alb was significantly higher than that in group NS at both 6 h(0.97±0.08 vs. 0.74±0.06, P=0.000) and 24 h (2.45±0.07 vs. 2.23±0.07, P=0.000). The expression of AQP4 in brain tissue was higher in group Alb than that in group NS at both 6 h (0.72±0.11 vs. 0.57±0.06, P<0.01)and 24 h (0.80±0.03 vs 0.61±0.02, P<0.01). Conclusions High dose albumin contribute slightly in improvement of neural deficit in rats after IR. On the contrary, it can also aggravate the IR injury, which increases brain edema then increase the permeability of BBB. The mechanism may be associated with over-expression of AQP4 in brain tissue. Key words: Albumin; BBB; IR; Brain edema; Nuro-defect; S100B; AQP4

Blood -brain barrier disruption was less under isoflurane than pentobarbital anesthesia via a PI3K/Akt pathway in early cerebral ischemia.

One of the important factors altering the degree of blood-brain barrier (BBB) disruption in cerebral ischemia is the anesthetic used. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been reported to be involved in modulating BBB permeability and in isoflurane induced neuroprotection. This study was performed to compare the degree of BBB disruption in focal cerebral ischemia under isoflurane vs pentobarbital anesthesia and to determine whether inhibition of PI3K/Akt would affect the disruption in the early stage of focal cerebral ischemia. Permanent middle cerebral artery (MCA) occlusion was performed in rats under 1.4% isoflurane or pentobarbital (50mg/kg i.p.) anesthesia with controlled ventilation. In half of each group LY294002, which is a PI3K/Akt inhibitor, was applied on the ischemic cortex immediately after MCA occlusion. After one hour of MCA occlusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (14C-AIB) was determined to quantify the degree of BBB disruption. MCA occlusion increased the Ki both in the isoflurane and pentobarbital anesthetized rats. However, the value of Ki was lower under isoflurane (11.5±6.0μL/g/min) than under pentobarbital (18.3±7.1μL/g/min) anesthesia. The Ki of the contralateral cortex of the pentobarbital group was higher (+74%) than that of the isoflurane group. Application of LY294002 on the ischemic cortex increased the Ki (+99%) only in the isoflurane group. The degree of BBB disruption by MCA occlusion was significantly lower under isoflurane than pentobarbital anesthesia in the early stage of cerebral ischemia. Our data demonstrated the importance of choice of anesthetics and suggest that PI3K/Akt signaling pathway plays a significant role in altering BBB disruption in cerebral ischemia during isoflurane but not during pentobarbital anesthesia.

The effect of hypertonic saline on notch signaling pathway in experimentally induced cerebral ischemic rats

Objective To explore whether hypertonic saline would partake in regulating Notch signaling in microglia in experimentally induced cerebral ischemic rats. Methods Male SD rats were randomly divided into sham group, cerebral ischemia group, normal saline group (NS group), 10% hypertonic saline group (10%HS group), the model of cerebral ischemia were established in all rats except the sham group by using middle cerebral artery occlusion (MCAO). After 2 hours of MCAO, the rats were through reperfusion for 24 h. In addition, rats in the normal saline group and 10% HS group were respectively treated with a continuous intravenous injection of normal saline (0.3 mL/h) and 10% HS (0.3 mL/h) by tail vein for 24 h. Immunofluorescence methods, RT-PCR and Western blot were used to detect the expression of Notch1 and intracellular Notch receptor domain (NICD). All data was analyzed by one-way analysis of variance (ANOVA), The intergroup comparisons were analyzed by the least-significant-difference (LSD) tests. Differences were considered statistically significant if P<0.05. Results Immunofluorescence showed that the expression of Notch1 and NICD were significantly increased in the microglia around peri-ischemia area in cerebral ischemia group and normal saline group compared to sham group; the expression of Notch1 and NICD in the microglia around peri-ischemia area were significantly reduced in 10% HS group compared to ischemia group and NS group. RT-PCR showed that the mRNA expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 1.000±0.076; ischemia group: 2.203±0.283; NS group: 1.616±0.185; P<0.01); however, it was significantly reduced in 10% HS group compared to ischemia group and NS group ( ischemia group: 2.203±0.283; NS group: 1.616±0.185; 10%HS group: 1.202±0.177; P<0.05). Western blot showed that the protein expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.290±0.079; ischemia group: 0.750±0.029; NS group: 0.765±0.182; P<0.01); but was significantly reduced in 10% HS group compared to ischemia group and NS group (ischemia group: 0.750±0.029; NS group: 0.765±0.182; 10%HS group: 0.390±0.195; P<0.05). The protein expression of NICD was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.401±0.196; ischemia group: 0.906±0.359; NS group: 0.847±0.153; P<0.01); but was significantly reduced in 10% HS group compared to ischemia group and NS group ( ischemia group: 0.906±0.359; NS group: 0.847±0.153; 10%HS group: 0.561±0.165; P<0.05). Conclusion Our results suggest that HS markedly suppresses Notch signaling in microglia around the ischemia tissue area in experimental induced cerebral ischemic rats. Key words: Hypertonic saline; Normal saline; Cerebral ischemia; Middle cerebral artery occlusion (MCAO); Microglia; Notch signaling; Notch1; Notch receptor domain (NICD)

Effects of rapamycin pretreatment on blood-brain barrier disruption in cerebral ischemia-reperfusion

The mammalian target of rapamycin (mTOR) pathway is essential in neuronal survival and repair in cerebral ischemia. Decreases in blood-brain barrier (BBB) disruption are associated with a decrease in neuronal damage in cerebral ischemia. This study was performed to investigate how pre-inhibition of the mTOR pathway with rapamycin would affect BBB disruption and the size of the infarcted cortical area in the early stage of focal cerebral ischemia-reperfusion using quantitative analysis of BBB disruption. Rats were treated with 20mg/kg of rapamycin i.p. once a day for 2days (Rapamycin Group) or vehicle (Control Group) before transient middle cerebral artery (MCA) occlusion. After one hour of MCA occlusion and two hours of reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (14C-AIB) to measure the degree of BBB disruption and the size of the cortical infarct were determined. Ischemia-reperfusion increased the Ki in the Rapamycin treated (+15%) as well as in the untreated control group (+13%). However, rapamycin pretreatment moderately decreased Ki in the contralateral (−30%) as well as in the ischemic-reperfused (−29%) cortex when compared with the untreated control group. Rapamycin pretreatment substantially increased the percentage of cortical infarct compared with the control group (+56%). Our data suggest that activation of mTOR pathway is necessary for neuronal survival in the early stage of cerebral ischemia-perfusion and that the reason for the enlarged cortical infarct by rapamycin pretreatment may be related to its non-BBB effects on the mTOR pathway.

Abstract WP275: Hemorrhagic Transformation in Large Cerebral Infarction of Rats Pretreated With Dabigatran or Warfarin

Introduction: Dabigatran is effective in preventing stroke and reduces the risk of intracerebral hemorrhage when comparing with warfarin in patients with atrial fibrillation. However, it is uncertain whether hemorrhagic transformation (HT) after large cerebral infarction is also less frequent in dabigatran users than warfarin users. Hypothesis: We hypothesized that intracerebral HT would occur less frequently following large cerebral infarction in rats pretreated with dabigatran than those with warfarin. We also compared the change of matrix metalloproteinases (MMPs), which play a key role in HT. Methods: We performed randomized, double-blind experiments comparing warfarin and dabigatran in rats. After treatment with warfarin (0.2 mg/kg), dabigatran (20 mg/kg), or normal saline for 7 days, male wistar rats were subjected to permanent middle cerebral artery occlusion (MCAO) using a nylon thread. After 22 hours of MCAO, magnetic resonance imaging (MRI) was undergone (9.4T MR scanner, slice thickness = 0.5 mm). Operative procedures and assessment of MRI findings were performed by investigators who were blind to the group information. The presence of HT was assessed on gradient recalled-echo and infarction volume was measured on diffusion-weighted image. The MMP-2 and MMP-9 activities in brain tissues (obtained 24 hours after MCAO) were investigated using gelatin zymography. Results and Conclusions: Of the 33 rats with MRI, HT was observed less frequently in the dabigatran group than the warfarin group (placebo 14% [2/14], dabigatran 0% [0/10], and warfarin 100% [9/9], p &lt; 0.001 [dabigatran vs warfarin]). The volume of infarction was similar between the groups (placebo 432.45 ± 78.35 mm3, dabigatran 432.42 ± 60.27 mm3, and warfarin 446.55 ± 73.55 mm3). MMP-2 and MMP-9 activities were not different between the groups. In the placebo, dabigatran, and warfarin groups, mean international normalized ratio were 1.38 ± 0.13, 1.73 ± 0.20, and 4.72 ± 2.92, respectively; mean activated partial thromboplastin times were 30.20 ± 7.49, 31.14 ± 7.02, and 55.06 ± 24.05, respectively. The risk of HT after large cerebral infarction was remarkably reduced in rats pretreated with dabigatran. MMPs did not seem to play a major role in reduced risk of HT.

Abstract W P240: S1177-eNOS Phosphorylation Protects Against Stroke Injury in Akt1 Deficient Mice

Background: Phosphorylation of eNOS at serine 1177 (S1177) by Akt1 increases NO production and vascular reactivity. The goal of this study is to test the hypothesis that Akt1 deficiency increases susceptibility to stroke because of insufficient eNOS phosphorylation. Thus, constitutively phosphorylated eNOS will overcome Akt1 deficiency and protect against stroke. We generated knock-in mice (S1177D) carrying phosphomimetic mutation (serine to aspartate substitutions) in the eNOS gene. We bred S1177D mice with Akt1 knockout (Akt1KO) mice to test whether constitutively active eNOS could improve stroke outcome in Akt1KO mice. Methods: Adult male mice were anesthetized by 30% oxygen, 70% nitrous oxide, and 1.5% isoflurane. Body temperature was maintained at 36-37°C. The common carotid artery was catheterized to measure systemic blood pressure using a blood pressure transducer. For stroke studies we used the middle cerebral artery occlusion (MCAO) by filament with subsequent reperfusion with Laser Doppler cortical blood flow monitoring. Mice were examined for neurologic deficits by 5 point scale 24 hours after stroke. Infarct sizes were determined by the indirect method by 2,3,5-triphenyltetrazolium chloride staining. Results: Mean blood pressure was higher in Akt1KO (95±7 mmHg, Mean±SD, n=9) and WT mice (93±3 mmHg, n=8) than in S1177D/Akt1KO mice (80±11 mmHg, n=6, P&lt;0.05). One hour of MCAO with 23 hours of reperfusion produced increased cerebral infarct volume in Akt1KO mice (106±21 mm3, n=9) as compared with WT (70±33, n=9, p&lt; 0.05) mice. S1177D phosphomimetic mutation decreased cerebral infarct volume in Akt1KO mice (64±20 mm3, n=9, P&lt;0.05). This was associated with a functional impairment in the neurologic deficit, as quantities by neurologic scoring (2.7 point deficit for Akt1KO; 1.9 for WT and 1.3 for S1177D/Akt1KO mice). Conclusion: Stroke injury was more pronounced in Akt1KO mice than in WT mice. Introducing S1177D mutation in Akt1KO mice resulted in decreased infarct volume, neurological deficit and blood pressure. These results demonstrated that S1177D eNOS-derived NO is able to protect against stroke in the absence of Akt1.

Follistatin-like 1 attenuates apoptosis via disco-interacting protein 2 homolog A/Akt pathway after middle cerebral artery occlusion in rats.

Follistatin-like 1 (FSTL1), an extracellular glycoprotein, has been reported to decrease apoptosis in ischemic cardiac diseases, but its effect in ischemic stroke has not been examined. We hypothesized that recombinant FSTL1 attenuates neuronal apoptosis through its receptor disco-interacting protein 2 homolog A (DIP2A) and the Akt pathway after middle cerebral artery occlusion (MCAO) in rats. One hundred forty male Sprague-Dawley rats were subjected to 2 hours of MCAO followed by reperfusion. In a subset of animals, the time course and location of FSTL1 and DIP2A were detected by Western blot and immunofluorescence double staining. Another set of animals were intracerebroventricularly given either recombinant FSTL1 1 hour after reperfusion or FSTL1-small interfering RNA (siRNA) 48 hours before reperfusion. Additionally, DIP2A was knockdown by siRNA in some animals. Infarction volume and neurological deficits were measured, and the expression of FSTL1, DIP2A, phosphorylated Akt, cleaved caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling were quantified using Western blot. The expression of FSTL1 and DIP2A was increased in neurons and peaked 24 hours after MCAO. Recombinant FSTL1 reduced brain infarction and improved neurological deficits 24 and 72 hours after MCAO via activation of its receptor DIP2A and downstream phosphorylation of Akt. These effects were reversed by DIP2A-siRNA and FSTL1-siRNA. Recombinant FSTL1 decreases neuronal apoptosis and improves neurological deficits through phosphorylation of Akt by activation of its receptor DIP2A after MCAO in rats. Thus, FSTL1 may have potentials as a treatment for patients with ischemic stroke.

Exercise Therapy Augments the Ischemia-Induced Proangiogenic State and Results in Sustained Improvement after Stroke

The induction of angiogenesis will stimulate endogenous recovery mechanisms, which are involved in the long-term repair and restoration process of the brain after an ischemic event. Here, we tested whether exercise influences the pro-angiogenic factors and outcomes after cerebral infarction in rats. Wistar rats were exposed to two hours of middle-cerebral artery occlusion and reperfusion. Different durations of treadmill training were performed on the rats. The expression of matrix metalloproteinase 2 (MMP2) and vascular endothelial growth factor (VEGF)-related genes and proteins were higher over time post-ischemia, and exercise enhanced their expression. Sixteen days post-ischemia, the regional cerebral blood flow in the ischemic striatum was significantly increased in the running group over the sedentary. Although no difference was seen in infarct size between the running and sedentary groups, running evidently improved the neurobehavioral score. The effects of running on MMP2 expression, regional cerebral blood flow and outcome were abolished when animals were treated with bevacizumab (BEV), a VEGF-targeting antibody. Exercise therapy improves long-term stroke outcome by MMP2-VEGF-dependent mechanisms related to improved cerebral blood flow.