Time course, distribution and cell types of induction of transforming growth factor betas following middle cerebral artery occlusion in the rat brain.
ABSTRACT: 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:Transforming growth factor-?s (TGF-?s) regulate cellular proliferation, differentiation, and survival. TGF-?s bind to type I (TGF-?RI) and II receptors (TGF-?RII), which are transmembrane kinase receptors, and an accessory type III receptor (TGF-?RIII). TGF-? may utilize another type I receptor, activin-like kinase receptor (Alk1). TGF-? is neuroprotective in the middle cerebral artery occlusion (MCAO) model of stroke. Recently, we reported the expression pattern of TGF-?1-3 after MCAO. To establish how TGF-?s exert their actions following MCAO, the present study describes the induction of TGF-?RI, RII, RIII and Alk1 at 24 h, 72 h and 1 mo after transient 1 h MCAO as well as following 24 h permanent MCAO using in situ hybridization histochemistry. In intact brain, only TGF-?RI had significant expression: neurons in cortical layer IV contained TGF-?RI. At 24 h after the occlusion, no TGF-? receptors showed induction. At 72 h following MCAO, all four types of TGF-? receptors were induced in the infarct area, while TGF-?RI and RII also appeared in the penumbra. Most cells with elevated TGF-?RI mRNA levels were microglia. TGF-?RII co-localized with both microglial and endothelial markers while TGF-?RIII and Alk1 were present predominantly in endothels. All four TGF-? receptors were induced within the lesion 1 mo after the occlusion. In particular, TGF-?RIII was further induced as compared to 72 h after MCAO. At this time point, TGF-?RIII signal was predominantly not associated with blood vessels suggesting its microglial location. These data suggest that TGF-? receptors are induced after MCAO in a timely and spatially regulated fashion. TGF-? receptor expression is preceded by increased TGF-? expression. TGF-?RI and RII are likely to be co-expressed in microglial cells while Alk1, TGF-?RII, and RIII in endothels within the infarct where TGF-?1 may be their ligand. At later time points, TGF-?RIII may also appear in glial cells to potentially affect signal transduction via TGF-?RI and RII.
Project description:<h4>Aim</h4>To explore whether the synthetic cannabinoid receptor agonist WIN55,212-2 could protect oligodendrocyte precursor cells (OPCs) in stroke penumbra, thereby providing neuroprotection following permanent focal cerebral ischemia in rats.<h4>Methods</h4>Adult male SD rats were subjected to permanent middle cerebral artery occlusion (p-MCAO). The animals were administered WIN55,212-2 at 2 h, and sacrificed at 24 h after the ischemic insult. The infarct volumes and brain swelling were assessed. The expression of cannabinoid receptor type 1 (CB1) in the stroke penumbra was examined using Western blot assay. The pathological changes and proliferation of neural glial antigen 2-positive OPCs (NG2(+) cells) in the stroke penumbra were studied using immunohistochemistry staining.<h4>Results</h4>p-MCAO significantly increased the expression of CB1 within the stroke penumbra with the highest level appearing at 2 h following the ischemic insult. Administration of WIN55,212-2 (9 mg/kg, iv) significantly attenuated the brain swelling, and reduced the infarct volume as well as the number of tau-immunoreactive NG2(+) cells (tau-1(+)/NG2(+) cells) in the stroke penumbra. Moreover, WIN55,212-2 significantly promoted the proliferation of NG2(+) cells in the stroke penumbra and in the ipsilateral subventricular zone at 24 h following the ischemic insult. Administration of the selective CB1 antagonist rimonabant (1 mg/kg, iv) partially blocked the effects caused by WIN55,212-2.<h4>Conclusion</h4>Tau-1 is expressed in NG2(+) cells following permanent focal cerebral ischemic injury. Treatment with WIN55,212-2 reduces the number of tau-1(+)/NG2(+) cells and promotes NG2(+) cell proliferation in the stroke penumbra, which are mediated partially via CB1 and may contribute to its neuroprotective effects.
Project description:<h4>Aims</h4>To precisely characterize the penumbra by MRI based on a modified photothrombotic stroke mouse model.<h4>Methods</h4>The proximal middle cerebral artery was occluded by a convenient laser system in conjunction with an intravenous injection of Rose Bengal in mice. And the suture MCAO model was performed in seven mice as a comparison of the reproducibility. One hour after occlusion, the penumbra was defined in six random photothrombotic stroke mice by mismatch between perfusion-weighted imaging and the apparent diffusion coefficient map on a home-made workstation. After imaging, three random mice of them were chosen to perform the reperfusion surgery. And the other three mice were sacrificed to stain for several potential penumbra markers, such as c-fos and heart shock protein 90. In the remaining mice, the evolution of the lesions was detected on the apparent diffusion coefficient map, diffusion-weighted imaging and T2-weighted imaging at 1, 3, 6, 12 and 24 hours. After evaluating the neurological deficit scores, the brains were sectioned and stained by triphenyltetrazolium chloride and Nissl.<h4>Results</h4>The mice subjected to photothrombosis showed significant behavioral deficits. One hour after occlusion, the low perfusion areas on the perfusion-weighted imaging interlaced with the hypointense areas on the apparent diffusion coefficient map, demonstrating that the penumbra was located both surrounding and inside the lesions. This phenomenon was subsequently confirmed by the c-fos and heart shock protein 90 staining. The final T2-weighted images of the mice subjected to the reperfusion surgery were also consistent with the penumbra images at one hour. At early stages, the lesions were clearly identified on the apparent diffusion coefficient map; the volumes of the lesions on the diffusion-weighted imaging and T2-weighted imaging did not reach a maximum until 12 hours. The coefficient of variation (CV) of the final lesions in the photothrombotic stroke mice was 21.7% (0.08 of 0.37) on T2-weighted imaging and 27.8% (0.10 of 0.35) on triphenyltetrazolium chloride, representing a high reproducibility (n = 7). While the CV of the lesions in the MCAO stroke mice was only 70% (0.24 of 0.34, n = 4).<h4>Conclusions</h4>This study has provided a precise imaging definition of the penumbra based on a reproducible photothrombotic stroke mouse model.
Project description:<h4>Background</h4>The steroid hormone estrogen (17-β-estradiol, E2) provides neuroprotection against cerebral ischemic injury by activating estrogen receptors. The novel estrogen receptor G protein-coupled receptor 30 (GPR30) is highly expressed in the brain and provides acute neuroprotection against stroke. However, the underlying mechanisms remain unclear.<h4>Methods</h4>In this study, ovariectomized female mice were subjected to middle cerebral artery occlusion (MCAO), and E2, G1, and ICI182780 were administered immediately upon reperfusion. The infarction volume, neurological scores, and neuronal injuries were examined. Primary microglial cells were subjected to oxygen-glucose deprivation (OGD), and the drugs were administered immediately upon reintroduction. The pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in penumbra and microglia were assessed by ELISA. The cell viability and lactose dehydrogenase (LDH) release of neurons co-cultured with microglia were analyzed using cell counting kit-8 (CCK8) and LDH release assays. Microglial activation as well as GPR30, Iba1, and Toll-like receptor 4 (TLR4) protein expression and TLR4 mRNA expression were detected. Additionally, NF-κB activity was detected in lipopolysaccharide (LPS)-activated microglia after the activation of GPR30.<h4>Results</h4>GPR30 was highly expressed in microglia and significantly increased after ischemic injury. The activation of GPR30 significantly reduced the infarction volume, improved the neurological deficit, and alleviated neuronal injuries. Moreover, GPR30 activation significantly reduced the release of TNF-α, IL-1β, and IL-6 from ischemic penumbra and microglia subjected to OGD and alleviated neuronal injury as assessed using the CCK8 and LDH assays. Finally, the activation of GPR30 relieved microglial activation, reduced Iba1 and TLR4 protein expression and TLR4 mRNA levels, and inhibited NF-κB activity.<h4>Conclusions</h4>Microglial GPR30 exerts acute neuroprotective effects by inhibiting TLR4-mediated microglial inflammation, which indicates that GPR30 may be a potential target for the treatment of ischemic stroke.
Project description: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:Cellular and humoral inflammations play important roles in ischemic brain injury. The effectiveness of immunomodulatory therapies may critically depend on the chosen experimental model. Our purpose was to compare the post-ischemic neuroinflammation among murine permanent and transient middle cerebral artery occlusion (MCAO) models. Permanent MCAO was induced by transtemporal electrocoagulation and 30 minutes or 90 minutes transient MCAO was induced by intraluminal filament in C57BL/6 mice. Infiltration of leukocyte subpopulations was quantified by immunohistochemistry and fluorescence-activated cell sorting. Cerebral cytokine and adhesion molecule expression was measured by real-time polymerase chain reaction (RT-PCR). Neutrophil infiltration was noted at 24 h after transient MCAO, but did not further increase until 5 days in the permanent MCAO model. Few T cells were observed in both MCAO models at 24 h, but permanent MCAO demonstrated much more infiltrating T cells at 5 days. Pronounced microglial activation was evident at 24 h and 5 days after permanent but not after transient MCAO. The number of invading NK cells and expression of MHCII on CD11b+ cells did not differ among the three groups. Five days after MCAO, the expression of IL-1, TNF-α and IFN-γ and of the adhesion molecules ICAM-1 and VCAM-1 was significantly higher in the permanent than in the transient MCAO groups. Cellular and humoral inflammation differs substantially among commonly used MCAO models. Neuroinflammation is more pronounced after permanent electrocoagulatory MCAO compared with 30 minutes and 90 minutes filament-MCAO.
Project description:Cerebral ischemia is a severe, acute condition, normally caused by cerebrovascular disease, and results in high rates of disability, and death. Phagoptosis is a newly recognized form of cell death caused by phagocytosis of viable cells, and has been reported to contribute to neuronal loss in brain tissue after ischemic stroke. Previous data indicated that exposure of phosphatidylserine to viable neurons could induce microglial phagocytosis of such neurons. Phosphatidylserine can be reversibly exposed to viable cells as a result of a calcium-activated phospholipid scramblase named TMEM16F. TMEM16F-mediated phospholipid scrambling on platelet membranes is critical for hemostasis and thrombosis, which plays an important role in Scott syndrome and has been confirmed by much research. However, few studies have investigated the association between TMEM16F and phagocytosis in ischemic stroke. In this study, a middle-cerebral-artery occlusion/reperfusion (MCAO/R) model was used in adult male Sprague-Dawley rats in vivo, and cultured neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate cerebral ischemia-reperfusion (I/R) injury in vitro. We found that the protein level of TMEM16F was significantly increased at 12 h after I-R injury both in vivo and in vitro, and reversible phosphatidylserine exposure was confirmed in neurons undergoing I/R injury in vitro. Additionally, we constructed a LV-TMEM16F-RNAi transfection system to suppress the expression of TMEM16F during and after cerebral ischemia. As a result, TMEM16F knockdown alleviated motor function injury and decreased the microglial phagocytosis of viable neurons in the penumbra through inhibiting the "eat-me" signal phosphatidylserine. Our data indicate that reducing neuronal phosphatidylserine-exposure via deficiency of TMEM16F blocks phagocytosis of neurons and rescues stressed-but-still-viable neurons in the penumbra, which may contribute to reducing infarct volume and improving functional recovering.
Project description:Acute ischemic stroke (AIS) is a leading cause of disability and mortality worldwide. By high-throughput sequencing of infarct and ischemic penumbra tissue from middle cerebral artery embolization (MCAO) mice, we identified the CircRNA expression was dramatically and selectively regulated in the penumbra tissues.
Project description:BACKGROUND AND PURPOSE:The migratory efficiency of mesenchymal stem cells (MSC) toward cerebral infarct after transplantation is limited. Valproate (VPA) and lithium enhance in vitro migration of MSC by upregulating CXC chemokine receptor 4 and matrix metalloproteinase-9, respectively. Ability of VPA and lithium to promote MSC homing and to improve functional recovery was assessed in a rat model of cerebral ischemia. METHODS:MSC primed with VPA (2.5 mmol/L, 3 hours) and/or lithium chloride (2.5 mmol/L, 24 hours) were transplanted into rats 24 hours after transient middle cerebral artery occlusion (MCAO). Neurological function was assessed via rotarod test, Neurological Severity Score, and body asymmetry test for 2 weeks. Infarct volume was analyzed by MRI. The number of homing MSC and microvessel density in the infarcted regions were measured 15 days after MCAO using immunohistochemistry. RESULTS:Priming with VPA or lithium increased the number of MSC homing to the cerebral infarcted regions, and copriming with VPA and lithium further enhanced this effect. MCAO rats receiving VPA-primed and/or lithium-primed MSC showed improved functional recovery, reduced infarct volume, and enhanced angiogenesis in the infarcted penumbra regions. These beneficial effects of VPA or lithium priming were reversed by AMD3100, a CXC chemokine receptor 4 antagonist, and GM6001, a matrix metalloproteinase inhibitor, respectively. CONCLUSIONS:Priming with VPA and/or lithium promoted the homing and migration ability of MSC, improved functional recovery, reduced brain infarct volume, and enhanced angiogenesis in a rat MCAO model. These effects were likely mediated by VPA-induced CXC chemokine receptor 4 overexpression and lithium-induced matrix metalloproteinase-9 upregulation.
Project description:Direct stimulation of the vagus nerve in the neck via surgically implanted electrodes is protective in animal models of stroke. We sought to determine the safety and efficacy of a non-invasive cervical VNS (nVNS) method using surface electrodes applied to the skin overlying the vagus nerve in the neck in a model of middle cerebral artery occlusion (MCAO).nVNS was initiated variable times after MCAO in rats (n = 33). Control animals received sham stimulation (n = 33). Infarct volume and functional outcome were assessed on day 7. Brains were processed by immunohistochemistry for microglial activation and cytokine levels. The ability of nVNS to activate the nucleus tractus solitarius (NTS) was assessed using c-Fos immunohistochemistry.Infarct volume was 43.15 ± 3.36 percent of the contralateral hemisphere (PCH) in control and 28.75 ± 4.22 PCH in nVNS-treated animals (p < 0.05). The effect of nVNS on infarct size was consistent when stimulation was initiated up to 4 hours after MCAO. There was no difference in heart rate and blood pressure between control and nVNS-treated animals. The number of c-Fos positive cells was 32.4 ± 10.6 and 6.2 ± 6.3 in the ipsilateral NTS (p < 0.05) and 30.4 ± 11.2 and 5.8 ± 4.3 in the contralateral NTS (p < 0.05) in nVNS-treated and control animals, respectively. nVNS reduced the number of Iba-1, CD68, and TNF-? positive cells and increased the number of HMGB1 positive cells.nVNS inhibits ischemia-induced immune activation and reduces the extent of tissue injury and functional deficit in rats without causing cardiac or hemodynamic adverse effects when initiated up to 4 hours after MCAO.