Delayed recanalization at 3 days after permanent MCAO attenuates neuronal apoptosis through FGF21/FGFR1/PI3K/Caspase-3 pathway in rats.
ABSTRACT: Reperfusion exceeded time window may induce ischemia/reperfusion injury, increase hemorrhagic transformation, and deteriorate neurological outcomes in ischemic stroke models. However, the increasing clinical evidences supported that reperfusion even within 6-24?h may salvage ischemic tissue and improve neurological outcomes in selected large vessel occlusion patients, without inducing serious ischemia/reperfusion injury and hemorrhagic transformation. The underlying molecular mechanisms are less clear. In present study, we demonstrated that delayed recanalization at 3?days after permanent middle cerebral artery occlusion (MCAO) decreased infarct volumes and improved neurobehavioral deficits in rats, with no increasing animal mortality and intracerebral hemorrhage. Meanwhile, we observed that endogenous neuroprotective agent fibroblast growth factor 21 (FGF21) significantly increased in serum after MCAO, but which did not synchronously increase in penumbra due to permanent MCAO. Recanalization dramatically increased the endogenous FGF21 expression on neurons in penumbra after MCAO. We confirmed that FGF21 activated the FGFR1/PI3K/Caspase-3 signaling pathway, which attenuated neuronal apoptosis in penumbra. Conversely, knockdown of FGFR1 via FGFR1 siRNA abolished the anti-apoptotic effects of FGF21, and in part abrogated beneficial effects of recanalization on neurological outcomes. These findings suggested that delayed recanalization at 3?days after MCAO improved neurological outcomes in rats via increasing endogenous FGF21 expression and activating FGFR1/PI3K/Caspase-3 pathway to attenuate neuronal apoptosis in penumbra. Delayed recanalization at 3?days after ischemic stroke onset may be a promising treatment strategy in selected patients.
Project description:<h4>Background</h4>In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood.<h4>Methods</h4>To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4?h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1<sup>-/-</sup> mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion.<h4>Results</h4>We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ib? in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion.<h4>Conclusions</h4>Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization.
Project description:Perfusion imaging is crucial in imaging of ischemic stroke to determine 'tissue at risk' for infarction. In this study we compared the volumetric quantification of the perfusion deficit in two rat middle-cerebral-artery occlusion (MCAO) models using two gadolinium-based contrast agents (P1152 (Guerbet) and Magnevist (Bayer-Schering, Pittsburgh, PA, USA)) as compared with our well established continuous arterial spin labeling (CASL) perfusion imaging technique. Animals underwent either permanent MCAO or transient MCAO with 80-min reperfusion. Imaging was performed at four different time points after MCAO. A region-of-interest (ROI) analysis of the subregions of the ischemic zone (core, penumbra, transient reversal (TR), and sustained reversal (SR)) using P1152 showed significant reduction in blood flow in the core and TR subregions relative to the penumbral and SR subregions while occluded. After reperfusion, a significant increase in blood flow was recorded at all time points after reperfusion in all regions except TR. From the ROI analysis the threshold for the penumbra was determined to be -62+/-11% and this value was subsequently used for quantification of the volumetric deficit. The ischemic volume as defined by dynamic susceptibility contrast (DSC), was only statistically different from the CASL-derived ischemic volume when using Magnevist at post-reperfusion time points.
Project description:Previous studies have shown that the pial microcirculation remodeling improves neurological outcome after middle cerebral artery occlusion (MCAO), accompanied by higher expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS), modulating in vivo angiogenesis. This study was aimed to assess the effects of bone marrow mesenchymal stem cells (BM-MSCs) infused after MCAO on rat pial microcirculation. Animals were subjected to 2 h MCAO followed by BM-MSCs infusion into internal carotid artery. Pial microcirculation was observed at different reperfusion times by fluorescence microscopy. Geometric characteristics of arteriolar networks, permeability increase, leukocyte adhesion, perfused capillary density, VEGF, and endothelial nitric oxide synthase (e-NOS) expression were evaluated. Green fluorescent protein (GFP)-BM-MSCs were used to evaluate their distribution and cell phenotype development during reperfusion. BM-MSCs stimulated a geometric rearrangement of pial networks with formation of new anastomotic vessels sprouting from preexistent arterioles in the penumbra at 7-14-28 days of reperfusion. At the same time VEGF and eNOS expression increased. GFP-BM-MSCs appear to be involved in endothelial and smooth muscle cell programming in the infarcted area. In conclusion, transient MCAO induced pial vascular remodeling characterized by arteriolar anastomotic arcades (originated from preexistent arterioles in penumbra area) able to overlap the ischemic core supplying blood to the neuronal tissue. BM-MSCs appear to accelerate angiogenic processes facilitating new vessel formation; this mechanism was promoted by an increase in VEGF and eNOS expression.
Project description:Stroke is one of the main causes of disease‑related mortality worldwide. Buyang Huanwu Decoction (BHD) has been used to protect against stroke and stroke‑induced disability for several years in China. Studies have shown that BHD can relieve neuronal damage in rats with cerebral ischemia/reperfusion (I/R) injury. However, the mechanism remains unclear. A middle cerebral artery occlusion and reperfusion (MCAO‑R) model was used in the present study. The animals were treated with BHD (5, 10 and 20 g/kg) or rapamycin. Infarct size and modified neurological severity score were calculated on day 5 following MCAO‑R surgery. Cellular changes around the ischemic penumbra were revealed by hematoxylin and eosin and Nissl staining. The protein expression levels of nestin, brain‑derived neurotrophic factor (BDNF), doublecortin on the X chromosome (DCX) and autophagy‑related proteins (beclin 1, LC3‑II and p62) in the peri‑ischemic area of the brain were detected. The results demonstrated that post‑surgical treatment with BHD reduced the brain infarct size and improved neurological deficits in MCAO‑R rats. BHD protected against MCAO‑R‑induced neuronal impairment and promoted neurogenesis, increased the protein expression of nestin, BDNF and DCX and markedly enhanced autophagy by increasing beclin 1 and LC3‑II and decreasing p62. Meanwhile, BHD promoted the expression of sirtuin 1 (SIRT1), an important regulator of autophagy. In conclusion, the present study suggested that post‑surgical treatment with BHD could protect rat brains from I/R injury, potentially through the SIRT1/autophagy pathway.
Project description:Tissue perfusion is a necessary condition for vessel survival that can be compromised under ischemic conditions. Following stroke, delayed effects of early brain reperfusion on the vascular substrate necessary for remodeling, perfusion and maintenance of proper peri-lesional hemodynamics are unknown. Such aspects of ischemic injury progression may be critical for neurological recovery in stroke patients. This study aims to describe the impact of early, non-thrombolytic reperfusion on the vascular brain component and its potential contribution to tissue remodeling and long-term functional recovery beyond the acute phase after stroke in 3-month-old male C57bl/6 mice. Permanent (pMCAO) and transient (60 min, tMCAO) brain ischemia mouse models were used for characterizing the effect of early, non-thrombolytic reperfusion on the brain vasculature. Analysis of different vascular parameters (vessel density, proliferation, degeneration and perfusion) revealed that, while early middle cerebral artery recanalization was not sufficient to prevent sub-acute vascular degeneration within the ischemic brain regions, brain reperfusion promoted a secondary wave of vascular remodeling in the peri-lesional regions, which led to improved perfusion of the ischemic boundaries and late-phase neurological recovery. This study concluded that acute, non-thrombolytic artery recanalization following stroke favors late-phase vascular remodeling and improves peri-lesional perfusion, contributing to secondary functional recovery.
Project description:Ischemic stroke, accompanied with high mortality and morbidity, may produce heavy economic burden to societies and families. Therefore, it is of great significance to explore effective therapies. Hyperbaric oxygen (HBO) is a noninvasive, nondrug treatment method that has been proved able to save ischemic penumbra by improving hypoxia, microcirculation, and metabolism and applied in various ischemic diseases. Herewith, we fully evaluated the effect of HBO on ischemic stroke and investigated its potential mechanism in the rat ischemia/reperfusion(I/R) model. Sixty Sprague-Dawley male rats were randomly divided into three groups-sham group, MCAO group, and MCAO+HBO group. In the latter two groups, the middle cerebral artery occlusion was performed (MCAO) for 2 hours, and then the occlusion was removed in order to establish the ischemic/reperfusion model. Subsequently, HBO was performed immediately after I/R (2 hours per day for 3 days). 72 hours after MCAO, the brain was dissected for our experiment. Finally, the data from three groups were analyzed by one-way analysis of variance (ANOVA) and followed by a Bonferroni test. In this article, we reported that HBO effectively reduced the infarction and edema and improved neurological functions to a certain extent. As shown by western blot analysis, HBO significantly reduced autophagy by regulating autophagy-related proteins (mTOR, p-mTOR, Atg13, LC3B II and LC3B II) in the hippocampus 72 hours after I/R, which was accompanied by inhibiting the expression of hypoxia inducible factor-1<i>α</i> (HIF-1<i>α</i>) in hippocampus. The results suggest that HBO may improve cerebral I/R injury, possibly via inhibiting HIF-1<i>α</i>, the upstream molecule of autophagy, and therefore, subsequently inhibiting autophagy in the rat model of ischemic stroke.
Project description:<h4>Objectives</h4>During mechanical recanalization of large vessel occlusions (LVO), the use of proximal flow arrest with balloon guide catheters (BGC) was shown to be associated with better angiographic and even clinical outcome. The aim of the study was to analyze the impact of BGC use on microstructural alterations in the salvaged penumbra.<h4>Methods</h4>All patients who underwent mechanical recanalization of LVO of the anterior circulation were reviewed within a prospective stroke registry of a single comprehensive stroke center. Fifty-two patients received an admission CT perfusion together with post-interventional diffusion tensor imaging. Technical details such as BGC usage were correlated with microstructural integrity changes of the salvaged gray matter through the mean diffusivity (MD) index. Moderation analysis was performed to test the interaction of BGC on the correlation between angiographic and clinical outcomes.<h4>Results</h4>For all patients with complete reperfusion, microstructural integrity changes with lowered MD index were found within the salvaged penumbra for cases of non-BGC usage (mean - 0.02) compared to cases with BGC usage (0.01, p = 0.04). The importance of complete reperfusion for good clinical outcome is predominantly based on patients treated with BGC (effect 2.78, p = 0.01 vs. for non-BGC: 0.3, p = 0.71).<h4>Conclusions</h4>The lowered MD index early after mechanical recanalization without BGC usage can be interpreted as microstructural ischemic damage of the salvaged penumbra. It was shown that achieving complete reperfusion in a setting of BGC usage with proximal flow arrest minimizes penumbral damage and improves long-term outcomes.<h4>Key points</h4>• Microstructural ischemic damage can be reduced by using proximal flow arrest during endovascular treatment with balloon guide catheter. • Complete reperfusion in a setting of balloon guide catheter minimizes penumbral damage and improves long-term outcome.
Project description:Transforming growth factor-?s (TGF-?1-3) are cytokines that regulate the proliferation, differentiation, and survival of various cell types. The present study describes the induction of TGF-?1-3 in the rat after focal ischemia at 3 h, 24 h, 72 h and 1 month after transient (1 h) or permanent (24 h) middle cerebral artery occlusion (MCAO) using in situ hybridization histochemistry and quantitative analysis. Double labeling with different markers was used to identify the localization of TGF-? mRNA relative to the penumbra and glial scar, and the types of cells expressing TGF-?s. TGF-?1 expression increased 3 h after MCAO in the penumbra and was further elevated 24 h after MCAO. TGF-?1 was present mostly in microglial cells but also in some astrocytes. By 72 h and 1 month after the occlusion, TGF-?1 mRNA-expressing cells also appeared in microglia within the ischemic core and in the glial scar. In contrast, TGF-?2 mRNA level was increased in neurons but not in astrocytes or microglial cells in layers II, III, and V of the ipsilateral cerebral cortex 24 h after MCAO. TGF-?3 was not induced in cells around the penumbra. Its expression increased in only a few cells in layer II of the cerebral cortex 24 h after MCAO. The levels of TGF-?2 and -?3 decreased at subsequent time points. Permanent MCAO further elevated the levels of all 3 subtypes of TGF-?s suggesting that reperfusion is not a major factor in their induction. TGF-?1 did not co-localize with either Fos or ATF-3, while the co-localization of TGF-?2 with Fos but not with ATF-3 suggests that cortical spreading depolarization, but not damage to neural processes, might be the mechanism of induction for TGF-?2. The results imply that endogenous TGF-?s are induced by different mechanisms following an ischemic attack in the brain suggesting that they are involved in distinct spatially and temporally regulated inflammatory and neuroprotective processes.
Project description:Brain damage following cerebral ischemia-reperfusion (I/R) is a complicated pathophysiological course, in which inflammation and oxidative stress have been suggested to serve an important role. Toll-like receptor 4 (TLR4) has been suggested to be involved in secondary inflammatory process in cerebral ischemia. Nuclear factor erythroid 2-related factor 2 (Nrf2), an important regulator of the antioxidant host defense, maintains the cellular redox homeostasis. Tissue kallikrein (TK) has been proven to elicit a variety of biological effects in ischemic stroke through its anti-inflammatory and anti-oxidant properties. However, the mechanisms underlying its beneficial effects remain poorly defined. The present study examined the hypothesis that TK attenuates ischemic cerebral injury via the TLR4/nuclear factor-κB (NF-κB) and Nrf2 signaling pathways. Using a transient rat middle cerebral artery occlusion (MCAO) model, the effects of immediate and delayed TK treatment subsequent to reperfusion were investigated. The neurological deficits, infarct size, and the expression of TLR4/NF-κB and Nrf2 pathway in ischemic brain tissues were measured at 24 following MCAO. The results indicated that TK immediate treatment significantly improved neurological deficits and reduced the infarct size, accompanied by the inhibition of TLR4 and NF-κB levels, and the activation of Nrf2 pathway. Furthermore, TK delayed treatment also exerted neuroprotection against I/R injury. However, the neuroprotective effect of TK immediate treatment was better compared with that of TK delayed treatment. In conclusion, the results indicated that TK protected the brain against ischemic injury in rats after MCAO through its anti-oxidative and anti-inflammatory effects. Suppression of TLR4/NF-κB and activation of the Nrf2 pathway contributed to the neuroprotective effects induced by TK in cerebral ischemia. Therefore, TK may provide an effective intervention with a wider therapeutic window for ischemic stroke.
Project description:The neuroprotective function of macrophage migration inhibitory factor (MIF) in ischemic stroke was rarely evaluated. This study aimed to investigate the effects of early treadmill exercise on recovery from ischemic stroke and to determine whether these effects are associated with the expression levels of MIF and brain-derived neurotrophic factor (BDNF) in the ischemic area. A total of 40 male Sprague-Dawley rats were randomly assigned to the ischemia and exercise group [middle cerebral artery occlusion (MCAO)-Ex, n = 10), ischemia and sedentary group (MCAO-St, n = 10), sham-surgery and exercise group (Sham-Ex, n = 10), or sham-surgery and sedentary group (Sham-St, n = 10). The MCAO-Ex and MCAO-St groups were subjected to MCAO for 60 minutes, whereas the Sham-Ex and Sham-St groups were subjected to an identical operation without MCAO. Rats in the MCAO-Ex and Sham-Ex groups then ran on a treadmill for 30 minutes once a day for 5 consecutive days. After reperfusion, the hanging time tested by the wire hang test was longer and the relative fractional anisotropy determined by MRI was higher in the peri-infarct region of the MCAO-Ex group compared with the MCAO-St group. The expression levels of MIF and BDNF in the peri-infarct region were upregulated in the MCAO-Ex group. Increased MIF and BDNF levels were positively correlated with relative fractional anisotropy changes in the peri-infarct region. There was no significant difference in the levels of MIF and BDNF in the peri-infarct region between the Sham-Ex and Sham-St groups. Our study demonstrated that early exercise (initiated 48 hours after the MCAO) could improve motor and neuronal recovery after ischemic stroke. Furthermore, the increased levels of MIF and BDNF in the peri-infarct region (penumbra) may be one of the mechanisms of enhanced neurological function recovery. All experiments were approved by the Institutional Animal Care and Use Committee in Asan Medical Center in South Korea (2016-12-126).