Transient Focal Brain Ischemia Research Articles

Impact of inflammatory preconditioning on murine microglial proteome response induced by focal ischemic brain injury.

Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).

Gut microbiota of old mice worsens neurological outcome after brain ischemia via increased valeric acid and IL-17 in the blood

BackgroundAging is a significant risk factor for ischemic stroke and worsens its outcome. However, the mechanisms for this worsened neurological outcome with aging are not clearly defined.ResultsOld C57BL/6J male mice (18 to 20 months old) had a poorer neurological outcome and more severe inflammation after transient focal brain ischemia than 8-week-old C57BL/6J male mice (young mice). Young mice with transplantation of old mouse gut microbiota had a worse neurological outcome, poorer survival curve, and more severe inflammation than young mice receiving young mouse gut microbiota transplantation. Old mice and young mice transplanted with old mouse gut microbiota had an increased level of blood valeric acid. Valeric acid worsened neurological outcome and heightened inflammatory response including blood interleukin-17 levels after brain ischemia. The increase of interleukin-17 caused by valeric acid was inhibited by a free fatty acid receptor 2 antagonist. Neutralizing interleukin-17 in the blood by its antibody improved neurological outcome and attenuated inflammatory response in mice with brain ischemia and receiving valeric acid. Old mice transplanted with young mouse feces had less body weight loss and better survival curve after brain ischemia than old mice transplanted with old mouse feces or old mice without fecal transplantation.ConclusionsThese results suggest that the gut microbiota-valeric acid-interleukin-17 pathway contributes to the aging-related changes in the outcome after focal brain ischemia and response to stimulus. Valeric acid may activate free fatty acid receptor 2 to increase interleukin-17.

Critical role of FPR1 in splenocyte migration into brain to worsen inflammation and ischemic brain injury in mice.

Background: Splenocyte contribution to ischemic brain injury has been suggested. It is not known whether this effect is due to systemic action or direct influence in ischemic brain tissues. It is also not known how splenocytes migrate into the brain and worsen neurological outcome after brain ischemia. We determined the role of formyl peptide receptor 1 (FPR1), a receptor expressed in monocytes, in the migration of splenocytes into ischemic brain tissues and the contribution of these splenocytes to ischemic brain injury.Methods: Mice with or without fpr1 knockout were subjected to transient focal brain ischemia. The migration of splenocytes was assessed under in vivo and in vitro conditions.Results: cFLFLF, a FPR1 antagonist, inhibited splenocyte migration into the brain and neuroinflammation after ischemic stroke. cFLFLF improved neurological outcome assessed 24 hours or 28 days after stroke. cFLFLF did not alter blood-brain barrier permeability in the ischemic brain. fpr1-/- mice had an attenuated peripheral monocyte and neutrophil infiltration into the brain, a reduced proinflammatory cytokine level and an improved neurological outcome compared with wild-type mice after brain ischemia. cFLFLF did not affect the proinflammatory cytokine levels in the spleen and brain of fpr1-/- mice after ischemic stroke.Conclusions: These results suggest that FPR1 facilitates splenocyte migration into the brain and proinflammatory cytokine production to worsen neurological outcome after brain ischemia, indicating a direct effect of splenocytes on ischemic brain tissues. Our results support the notion that cFLFLF via blocking FPR1 signaling inhibits those pathological processes and is a potential agent for neuroprotection.

Could Galectin-3 be a key player in the etiology of neuromyelitis optica spectrum disorder?

Neuromyelitis Optica Spectrum Disorder (NMOSD) is a chronic, inflammatory, demyelinating disorder of the central nervous system (CNS) characterized primarily by transverse myelitis (TM) and optic neuritis (ON). Serum antibodies of the IgG class to the water channel protein aquaporin-4 (AQP4) are associated with NMOSD in most cases. These antibodies are thought to cause functional abnormality or changed expressional pattern of AQP4 channel proteins in the CNS lesions.Activation of microglia is one of the chief antibody-mediated effects in NMOSD and it has opposing detrimental and protective effects in NMOSD. On the one hand, it promotes neuroinflammation, demyelination and BBB breakdown. On the other, it aids in remyelination. What controls the switch between these effects is unknown.Recently, Galectin- 3, a lectin, has been identified as a key player in several neurodegenerative diseases. In transient focal brain ischemia, alzheimer’s disease (AD), huntington disease (HD), and experimental autoimmune encephalitis (EAE), Galectin-3 promotes microglia-mediated inflammation.Conversely, in amyotrophic lateral sclerosis (ALS), Galectin-3 reduces inflammation. It also suppresses Th17 cytokines, which play a crucial role in NMOSD pathogenesis. Being devoid of a leader signal, Gal-3 localizes in different cellular compartments and is subject to various post-translational modifications. These reasons explain why Galectin-3 expression has opposing effects under different physiological conditions. Microglia-mediated inflammation in NMOSD has not been extensively studied. The factors that regulate microglia-mediated inflammation in NMOSD are unknown. Here, I hypothesize that Galectin-3 might be an etiological factor in NMOSD that regulates microglia-mediated inflammation. Analysing the role of Gal-3 in NMOSD could help in the development of novel therapies to treat NMOSD.

Hypertonic saline mediates the NLRP3/IL-1β signaling axis in microglia to alleviate ischemic blood-brain barrier permeability by downregulating astrocyte-derived VEGF in rats.

IntroductionThe aim of this study was to explore whether the antibrain edema of hypertonic saline (HS) is associated with alleviating ischemic blood‐brain barrier (BBB) permeability by downregulating astrocyte‐derived vascular endothelial growth factor (VEGF), which is mediated by microglia‐derived NOD‐like receptor protein 3 (NLRP3) inflammasome.MethodsThe infarct volume and BBB permeability were detected. The protein expression level of VEGF in astrocytes in a transient focal brain ischemia model of rats was evaluated after 10% HS treatment. Changes in the NLRP3 inflammasome, IL‐1β protein expression, and the interleukin‐1 receptor (IL1R1)/pNF‐кBp65/VEGF signaling pathway were determined in astrocytes.ResultsHS alleviated the BBB permeability, reduced the infarct volume, and downregulated the expression of VEGF in astrocytes. HS downregulates IL‐1β expression by inhibiting the activation of the NLRP3 inflammasome in microglia and then downregulates VEGF expression by inhibiting the phosphorylation of NF‐кBp65 mediated by IL‐1β in astrocytes.ConclusionsHS alleviated the BBB permeability, reduced the infarct volume, and downregulated the expression of VEGF in astrocytes. HS downregulated IL‐1β expression via inhibiting the activation of the NLRP3 inflammasome in microglia and then downregulated VEGF expression through inhibiting the phosphorylation of NF‐кBp65 mediated by IL‐1β in astrocytes.

N,N-dimethyltryptamine reduces infarct size and improves functional recovery following transient focal brain ischemia in rats

Background and purposeN,N-dimethyltryptamine (DMT) is an endogenous ligand of the Sigma 1 receptor (Sig-1R) with documented in vitro cytoprotective properties against hypoxia. Our aim was to demonstrate the in vivo neuroprotective effect of DMT following ischemia-reperfusion injury in the rat brain. MethodsTransient middle cerebral occlusion (MCAO) was induced for 60 min in male Wistar rats using the filament occlusion model under general anaesthesia. Before the removal of the filament the treatment group (n = 10) received an intra-peritoneal (IP) bolus of 1 mg/kg-body weight (bw) DMT dissolved in 1 ml 7% ethanol/saline vehicle, followed by a maintenance dose of 2 mg/Kg-bw/h delivered over 24 h via osmotic minipumps. Controls (n = 10) received a vehicle bolus only. A third group (n = 10) received a Sig-1R antagonist (BD1063, 1 mg/kg-bw bolus +2 mg/kg-bw/h maintenance) in parallel with the DMT. Lesion volume was measured by MRI 24 h following the MCAO. Shortly after imaging the animals were terminated, and the native brains and sera were removed. Four rats were perfusion fixed. Functional recovery was studied in two separate group of pre-trained animals (n = 8–8) using the staircase method for 30 days. The expression levels of proteins involved in apoptosis, neuroplasticity and inflammatory regulation were assessed by real-time qPCR and ELISA. ResultsDMT treated rats were characterized by lower ischemic lesion volume (p = .0373), and better functional recovery (p = .0084) compared to the controls. Sig-1R was expressed both in neurons and in microglia in the peri-infarct cortex, and the DMT induced change in the lesion volume was hindered by BD1063. Lower APAF1 expression (mRNA and protein) and higher BNDF levels were documented on DTM, while decreased TNF-α, IL1-β, IL-6 and increased IL-10 expressions indicated the compound's anti-inflammatory potential. ConclusionOur results indicate a Sig-1R dependent reduction of the ischemic brain injury following exogenous DMT administration in rats, presumably through a combined anti-apoptotic, pro-neurotrophic and anti-inflammatory treatment effect.

Study of the association between gait variability and gene expressions in a mouse model of transient focal ischemic stroke

Purpose: Gait variability analysis has been clinically adopted to characterize the presentation of various neurological diseases. However, literature and practice lack a comprehensive murine model assessment of the gait deficits that result from transient focal ischemic stroke. Further, correlations between gait parameters and the gene expression profiles associated with brain ischemia have yet to be identified. This study quantitatively assesses gait deficits through a murine model of transient focal cerebral ischemia on day 7 to determine associations between gait deficits and ischemia-related gene expressions.Methods: A total of 182 dynamic and static gait parameters from the transient middle cerebral artery occlusion (MCAO) murine model for simulating human transient focal ischemic stroke on day 7 were measured using the CatWalk system. Pearson’s correlation analysis and genes associated with ischemia were identified from the existing literature to aid the investigation of the relationship between gait variability and gene expression profiles.Results: Thirty-nine gait parameters and the mRNA expression levels of four of the eight ischemia-associated genes exhibited more significant change in the MCAO models (p < 0.005) on day 7. Twenty-six gait parameters exhibited strong correlations with four ischemia-associated genes.Conclusion: This examination of gait variability and the strong correlation to the gene expression profiles associated with transient focal brain ischemia on day 7 provides a quantitative and reliable assessment of the MCAO model’s motor performance. This research provides valuable insights into the study of disease progression and offers novel therapeutic interventions in the murine modeling of ischemic stroke.

Post-stroke gastrodin treatment ameliorates ischemic injury and increases neurogenesis and restores the Wnt/β-Catenin signaling in focal cerebral ischemia in mice

Cerebral ischemic stroke is one of the leading causes of death and disability worldwide, and the only available drug treatment is limited to a short window following the ischemic event. Gastrodin is the major bioactive constituent extracted from thetuberGastrodia elata, and is currently used to treat dizziness in the clinic. “Early” application of gastrodin (before modeling or immediately after ischemic injury) has shown antioxidative and neuroprotective effects in a transient focal brain ischemia model in rodents; however, it is not known whether the delayed administration of gastrodin after permanent focal cerebral ischemia ameliorates neural injury and increases neurogenesis. In this study, we performed a permanent middle cerebral artery occlusion (MCAO) model for the study of cerebral ischemic stroke in adult male mice to examine the effects of gastrodin. Gastrodin treatment that was started “late” (one day after the ischemic injury) significantly improved neural function, reduced infarct volume and apoptosis, and increased the number of DCX/BrdU double-positive cells in permanent MCAO mice. Moreover, gastrodin treatment markedly preserved the Wnt/β-Catenin signaling pathway, which could promote neurogenesis and provide neuroprotection brain injury. Our findings suggest that gastrodin treatment following ischemic injury can induce neuroprotection, promote neurogenesis and restored the Wnt /β-Catenin signaling pathway.

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from ± 25 min to ± 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (± 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (± 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia.

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from ± 25 min to ± 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (± 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (± 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

Accumulation of corticosterone and interleukin-1β in the hippocampus after focal ischemic damage of the neocortex: Selective vulnerability of the ventral hippocampus

Most ischemic strokes are caused by the occlusion of the middle cerebral artery (MCAO), which results in focal brain lesions in different areas of the neocortex. Secondary damage develops in brain regions located out of the infarct area, including the hippocampus. Hippocampal lesion may lead to cognitive impairments and post-stroke depression. Here, we studied the time course of changes in the levels of corticosterone and proinflammatory cytokine interleukine-1β (IL-1β) in the blood and hippocampus of rats after transient focal brain ischemia. Activation of the hypothalamo–pituitary–adrenal axis, which causes a release of corticosterone into blood, was observed at the early stage after MCAO and was accompanied by the presence of the stress hormone in the hippocampi of both the ischemic and contralateral hemispheres. We show for the first time that this effect was observed only in the ventral hippocampus (VH) but not in the dorsal hippocampus (DH). MCAO induced accumulation of the proinflammatory cytokine IL-1β, which coexisted with the elevated level of corticosterone at the early and delayed stages after reperfusion and was also observed in the VH of both hemispheres. Our data show that the VH is more vulnerable to remote damage induced by MCAO compared to the DH and corticosteroid response and neuroinflammation may be detected in the VH of both ischemic and contralateral hemispheres.

Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity

Astrocytes are believed to bridge interactions between infiltrating lymphocytes and neurons during brain ischemia, but the mechanisms for this action are poorly understood. Here we found that interleukin-15 (IL-15) is dramatically up-regulated in astrocytes of postmortem brain tissues from patients with ischemic stroke and in a mouse model of transient focal brain ischemia. We generated a glial fibrillary acidic protein (GFAP) promoter-controlled IL-15-expressing transgenic mouse (GFAP-IL-15tg) line and found enlarged brain infarcts, exacerbated neurodeficits after the induction of brain ischemia. In addition, knockdown of IL-15 in astrocytes attenuated ischemic brain injury. Interestingly, the accumulation of CD8+ T and natural killer (NK) cells was augmented in these GFAP-IL-15tg mice after brain ischemia. Of note, depletion of CD8+ T or NK cells attenuated ischemic brain injury in GFAP-IL-15tg mice. Furthermore, knockdown of the IL-15 receptor α or blockade of cell-to-cell contact diminished the activation and effector function of CD8+ T and NK cells in GFAP-IL-15tg mice, suggesting that astrocytic IL-15 is delivered in trans to target cells. Collectively, these findings indicate that astrocytic IL-15 could aggravate postischemic brain damage via propagation of CD8+ T and NK cell-mediated immunity.

Dimethyl Fumarate and Monomethyl Fumarate Promote Post-Ischemic Recovery in Mice.

Oxidative stress plays an important role in cerebral ischemia–reperfusion injury. Dimethyl fumarate (DMF) and its primary metabolite monomethyl fumarate (MMF) are antioxidant agents that can activate the nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway and induce the expression of antioxidant proteins. Here, we evaluated the impact of DMF and MMF on ischemia-induced brain injury and whether the Nrf2 pathway mediates the effects provided by DMF and MMF in cerebral ischemia–reperfusion injury. Using a mouse model of transient focal brain ischemia, we show that DMF and MMF significantly reduce neurological deficits, infarct volume, brain edema, and cell death. Further, DMF and MMF suppress glial activation following brain ischemia. Importantly, the protection of DMF and MMF was mostly evident during the subacute stage and was abolished in Nrf2−/− mice, indicating that the Nrf2 pathway is required for the beneficial effects of DMF and MMF. Together, our data indicate that DMF and MMF have therapeutic potential in cerebral ischemia–reperfusion injury and their protective role is likely mediated by the Nrf2 pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s12975-016-0496-0) contains supplementary material, which is available to authorized users.

Intravenous Treatment With Coenzyme Q10 Improves Neurological Outcome and Reduces Infarct Volume After Transient Focal Brain Ischemia in Rats

Coenzyme Q10 (CoQ10) crosses the blood-brain barrier when administered intravenously and accumulates in the brain. In this study, we investigated whether CoQ10 protects against ischemia-reperfusion injury by measuring neurological function and brain infarct volumes in a rat model of transient focal cerebral ischemia. In male Wistar rats, we performed transient middle cerebral artery occlusion (tMCAO) for 60 minutes, followed by reperfusion for 24 hours or 7 days. Forty-five minutes after the onset of occlusion (or 15 minutes before reperfusion), rats received a single intravenous injection of solubilized CoQ10 (30 mg·mL(-1)·kg(-1)) or saline (2 mL/kg). Sensory and motor function scores and body weights were obtained before the rats were killed by decapitation, and brain infarct volumes were calculated using tetrazolium chloride staining. CoQ10 brain levels were measured by high-performance liquid chromatography with electrochemical detection. CoQ10 significantly improved neurological behavior and reduced weight loss up to 7 days after tMCAO (P < 0.05). Furthermore, CoQ10 reduced cerebral infarct volumes by 67% at 24 hours after tMCAO and 35% at 7 days (P < 0.05). Cerebral ischemia resulted in a significant reduction in endogenous CoQ10 in both hemispheres (P < 0.05). However, intravenous injection of solubilized CoQ10 resulted in its increase in both hemispheres at 24 hours and in the contralateral hemisphere at 7 days (P < 0.05). Our results demonstrate that CoQ10 is a robust neuroprotective agent against ischemia-reperfusion brain injury in rats, improving both functional and morphological indices of brain damage.

Abstract 144: Astrocyte-Derived Interleukin-15 Accelarates Ischemic Brain Injury

Background and Purpose: Astrocytes play a pivotal role in post-ischemic brain inflammation, but the relevant astrocyte-derived mediators of ischemic brain injury remain to be defined. This study aims to investigate the impact of astrocyte-derived factors such as IL-15 on ischemic brain injury. Methods and Results: We show that IL-15 is a prominent factor relased by astrocytes after brain ischemia in a mouse model of transient focal brain ischemia. To further eluciate the biological functions of astrcoyte-derived IL-15 in ischemic stroke, we generated a glial fibrillary acidic protein (GFAP) promoter-controlled IL-15-expressing transgenic mouse line (GFAP-IL-15 tg ). We demonstrate that astrocyte-specific overproduction of IL-15 leads to larger brain infarcts, worse neurodeficits and enhanced lymphocyte infiltration. In GFAP-IL-15 tg mice, we found increased accumulation and activation of CD8 + T and natural killer (NK) cells after cerebral ischemia. Importantly, depletion of either CD8 + T or NK cells in GFAP-IL-15 tg mice prior to cerebral ischemia attenuates the accelerated brain infarction and neurodeficits. Of note, CD8 + T and NK cells are within the proximity of astrocytes in the post-ischemic brain and either knockdown of IL-15 receptor α or blockade of cell-to-cell contact diminishes the activation and effector function of CD8 + T and NK cells caused by astrocytic overproduction of IL-15, suggesting that astrocytic IL-15 is delivered in trans to target cells. Additionally, CD8 + T and NK cells are seen closely adjacent to IL-15-producing astrocytes in acute ischemic lesions of postmortem human brain tissues, implying that astrocytic IL-15-mediated brain injury may be relevant to human stroke. Conclusion: These findings indicate that astrocytic IL-15 contributes to post-ischemic brain damage.

Inhibition of Brain Ischemia-Caused Notch Activation in Microglia May Contribute to Isoflurane Postconditioning-Induced Neuroprotection in Male Rats

Prolonged exposure to volatile anesthetics alone may be detrimental to the brain. However, volatile anesthetics, such as isoflurane, can provide neuroprotection against various damaging insults. Application of isoflurane after focal brain ischemia reduces ischemic brain injury. We determined whether this isoflurane postconditioning-induced neuroprotection requires inhibition of brain ischemia-induced Notch signaling activation. Here, we showed that TUNEL-positive staining cell density and active caspase 3 expression were increased in the ischemic penumbral brain tissues of male rats after a 90-min middle cerebral arterial occlusion (MCAO). This increase was inhibited by isoflurane postconditioning and a Notch inhibitor. Isoflurane postconditioning and the Notch inhibitor also inhibited brain ischemia-induced Notch activation and proinflammatory cytokine production. Most cells expressing active Notch also were positive for CD11b, a microglial and white blood cell marker. Isoflurane postconditioning and the Notch inhibitor inhibited 1 ng/ml lipopolysaccharide- and oxygen-glucose deprivation-induced Notch activation and proinflammatory cytokine production from microglial cultures. The inhibition of cytokine production by isoflurane postconditioning, but not by a high concentration of the Notch inhibitor, disappeared in the presence of 10 ng/ml lipopolysaccharide. Our results suggest that Notch activation in microglia contributes to the cell apoptosis in the ischemic brain tissues. Inhibiting this Notch activation may participate in isoflurane postconditioning-induced neuroprotection against transient focal brain ischemia in male rats.

T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice

T lymphocytes are key contributors to the acute phase of cerebral ischemia reperfusion injury, but the relevant T cell-derived mediators of tissue injury remain unknown. Using a mouse model of transient focal brain ischemia, we report that IL-21 is highly up-regulated in the injured mouse brain after cerebral ischemia. IL-21-deficient mice have smaller infarcts, improved neurological function, and reduced lymphocyte accumulation in the brain within 24 h of reperfusion. Intracellular cytokine staining and adoptive transfer experiments revealed that brain-infiltrating CD4(+) T cells are the predominant IL-21 source. Mice treated with decoy IL-21 receptor Fc fusion protein are protected from reperfusion injury. In postmortem human brain tissue, IL-21 localized to perivascular CD4(+) T cells in the area surrounding acute stroke lesions, suggesting that IL-21-mediated brain injury may be relevant to human stroke.

Sulindac for stroke treatment: neuroprotective mechanism and therapy.

Sulindac, a widely used nonsteroidal anti-inflammatory drug (NSAID) is a prodrug that is reduced by methionine sulfoxide reductase to its active form as an inhibitor of cyclooxygenase 1 and 2. The drug has been shown to elicit tissue protection by processes that may include at least three functions: antioxidant, preconditioning and anti-inflammatory. Sulindac demonstrates neuroprotection that involves inhibition of mitochondrial calcium overload or a decrease in protein oxidation. We have demonstrated the induction by sulindac treatment of pro-survival proteins Hsp27, Akt and Bcl-2 in the ischemic penumbra and core of the central nervous system (CNS) infarct in a rat model of ischemic stroke. Our findings point to sulindac acting on the endoplasmic reticulum (ER) to decrease ATF-6 and on the mitochondrion to increase Bcl-2 as well as decrease pro-apoptotic components BAK and PUMA. The resulting decrease in ER stress and reduction in apoptosis underlies the protective effect of sulindac in reducing infarct size following transient focal brain ischemia. The potent neuroprotective effect of sulindac in the stroke model is obtained with low-dose administration of the drug pointing to the potential of sulindac as a valuable neuroprotective agent against oxidative stress in cerebral ischemia.

In vivo monitoring of arterially transplanted bone marrow mononuclear cells in a rat transient focal brain ischemia model using magnetic resonance imaging

Objectives: Intra-arterial transplantation of bone marrow mononuclear cells (BMMCs) effectively improves neuronal function and limits the infarct size. We monitored the fate of BMMCs labeled with super paramagnetic iron oxide (SPIO) until 7 days after the transplantation using high-field magnetic resonance imaging (MRI).Methods: Male Sprague–Dawley rats were subjected to 90-minute focal ischemia using the intraluminal suture technique followed by transplantation of 1×107 BMMCs or vehicle only via the ipsilateral carotid artery immediately after reperfusion. Autologous BMMCs were labeled with SPIO by electroporation prior to ischemia. MRI studies were performed at 1 hour, 24 hours, 3 days, and 7 days after reperfusion on each rat. The total infarct volume and the volume of negative dots were measured on T2-weighted images and T2*-weighted images, respectively.Results: One hour after BMMC transplantation, we confirmed wide spread distribution of BMMCs in the ischemic hemisphere as a negative dot. The volume of negative dot normalized by hemispheric volume decreased rapidly and was seldom seen at the seventh day after transplantation. The infarct volume was significantly smaller in the transplanted group than the vehicle group at 24 hours and 7 days after reperfusion.Discussion: The present study established in vivo monitoring of intra-arterial transplanted SPIO-labeled BMMCs immediately after reperfusion using MRI of a rat transient focal ischemia model. The accumulation of BMMCs in ischemic lesion at the acute stage of ischemia can be part of the conditions to limit the infarct size.

Isoflurane postconditioning reduces ischemia-induced nuclear factor-κB activation and interleukin 1β production to provide neuroprotection in rats and mice

Application of isoflurane, a volatile anesthetic, after brain ischemia can reduce ischemic brain injury in rodents (isoflurane postconditioning). This study is designed to determine whether isoflurane postconditioning improves long-term neurological outcome after focal brain ischemia and whether this protection is mediated by attenuating neuroinflammation. Adult male Sprague–Dawley rats were subjected to a 90-min middle cerebral arterial occlusion (MCAO). Isoflurane postconditioning was performed by exposing rats to 2% isoflurane for 60min immediately after the MCAO. Isoflurane postconditioning reduced brain infarct volumes, apoptotic cells in the ischemic penumbral brain tissues and neurological deficits of rats at 4weeks after the MCAO. Isoflurane postconditioning reduced brain ischemia/reperfusion-induced nuclear transcription factor (NF)-κB (NF-κB) activation as well as interleukin 1β (IL-1β) and interleukin-6 production in the ischemic penumbral brain tissues at 24h after the MCAO. IL-1β deficient mice had smaller brain infarct volumes and better neurological functions than wild-type mice at 24h after a 90-min focal brain ischemia. Isoflurane posttreatment failed to induce neuroprotection in the IL-1β deficient mice. Our results suggest that isoflurane postconditioning improved long-term neurological outcome after transient focal brain ischemia. This protection may be mediated by inhibiting NF-κB activation and the production of the proinflammatory cytokine IL-1β.