Early Stage Of Cerebral Ischemia Research Articles

The underlying mechanism of calcium toxicity-induced autophagic cell death and lysosomal degradation in early stage of cerebral ischemia.

Cerebral ischemia is the important cause of worldwide disability and mortality, that is one of the obstruction of blood vessels supplying to the brain. In early stage, glutamate excitotoxicity and high level of intracellular calcium (Ca2+) are the major processes which can promote many downstream signaling involving in neuronal death and brain tissue damaging. Moreover, autophagy, the reusing of damaged cell organelles, is affected in early ischemia. Under ischemic conditions, autophagy plays an important role to maintain energy of the brain and its function. In the other hand, over intracellular Ca2+ accumulation triggers excessive autophagic process and lysosomal degradation leading to autophagic process impairment which finally induce neuronal death. This article reviews the association between intracellular Ca2+ and autophagic process in acute stage of ischemic stroke.

Limb Remote Ischemic Postconditioning Improves Glymphatic Dysfunction After Cerebral Ischemia–Reperfusion Injury

BackgroundDelayed neuronal damage can be caused or aggravated after cerebral ischemia–reperfusion (I/R) injury. Recent studies have shown that glymphatic system dysfunction after cerebral ischemia–reperfusion injury is involved in ischemic brain edema and neuroinflammation, thereby regulating cerebral ischemia–reperfusion injury. The aim of this study is to investigate the changes of glymphatic system after cerebral ischemia–reperfusion injury and whether limb remote ischemic postconditioning (LRIP) can improve the function of glymphatic system to protect the brain. MethodsTo establish a focal brain I/R injury mouse model, this study utilized the middle cerebral artery occlusion/reperfusion (MCAO/R) method. The present study classified eight-week-old C57BL/6 male mice into three groups. The changes in glymphatic function in different periods of ischemia and reperfusion were analyzed through immunofluorescence, transmission electron microscopy (TEM), and Western-Blot (WB) assays. The contents of the evaluation included cerebrospinal fluid flow, swelling degree of brain tissue, aquaporin-4 (AQP4) expression and polarization, and amyloid-β (Aβ) excretion. ResultsIn the early stages of cerebral ischemia, cerebrospinal fluid (CSF) flow is disturbed, accompanied by a decrease in AQP4 polarization. The polarity of AQP4 decreased from 12 h to 72 h of reperfusion, the Aβ deposition. LRIP can increase the expression of β-DG and AQP4 polarization, reduce the deposition of Aβ, improve the function of the glymphatic system, and reduce the expression of AQP4 to play A protective role in brain. ConclusionGlymphatic system impaired after cerebral ischemia–reperfusion injury in mice. LRIP may play a neuroprotective role by improving glymphatic function after I/R.

VEGF, a Key Factor for Blood Brain Barrier Injury After Cerebral Ischemic Stroke.

Blood brain barrier (BBB) injury is an important factor affecting the prognosis of ischemic stroke. Extensive research on BBB injury has revealed that blood vessels and neural networks are interdependent and interrelated during and after the development of the brain. An array of signaling molecules, known as angioneurins, can affect both blood vessels and neural networks simultaneously. Angioneurins not only regulate the angiogenesis and remodeling process of the vascular system, but also act as neurotrophic and neuroprotective factors, or serve as guide molecules for axons. Vascular endothelial growth factor (VEGF) is a type of angioneurin that is expressed in neurons, astrocytes, macrophages, and vascular endothelial cells in ischemic and hypoxic brain tissues after cerebral ischemia. VEGF can increase and induce the destruction of the endothelial barrier in the early stages of cerebral ischemia. Both the upregulation of endogenous VEGF levels and the use of exogenous VEGF are harmful in the acute stage of stroke. However, the harmful effects of VEGF on vascular integrity are transient. Several studies have shown that VEGF regulates angiogenesis, neurogenesis, neurite growth and brain edema after cerebral ischemia. Therefore, it is crucial to understand the dual role of VEGF in ischemic stroke. The following will focus on the damage caused by VEGF to the BBB in the context of cerebral ischemic stroke, as well as therapeutic studies targeting VEGF.

Combined Analysis of Surface Protein Profile and microRNA Expression Profile of Exosomes Derived from Brain Microvascular Endothelial Cells in Early Cerebral Ischemia.

Endothelial cell damage is an important pathological basis for the deterioration of acute ischemia stroke. Our previous studies have been exploring the mechanism of blood–brain barrier (BBB) endothelial cell injury in the early stage of cerebral ischemia. Exosomes act as an important intercellular player in neurovascular communication. However, the characteristic of exosomes derived from BBB endothelial cells in early ischemic stroke is poorly understood. We exposed cultured brain microvascular endothelial cells (bEnd.3) to 3 h oxygen glucose deprivation (OGD) to mimic early cerebral ischemia in vitro and compared miRome and surface protein contents of exosomes derived from bEnd.3 cells by miRNA sequencing and the proximity barcoding assay (PBA). A total of 346 differentially miRNA (159 upregulated and 187 downregulated) were identified via miRNA-Seq in bEnd.3 cells after exposure to OGD for 3 h. Moreover, Gene Ontology (GO) and KEGG pathway analyses showed that cell proliferation- and angiogenesis-associated miRNAs were significantly affected. The abnormal changes in top eight miRNAs were further verified by a quantitative polymerase chain reaction (qPCR). PBA experiments showed that the numbers of exosomes carrying the following proteins increased significantly under ischemia, including bFGF, CD146, EPHA2, ABCB5, and ITGB2. These proteins were related to angiogenesis, cell proliferation, and cell inflammation. The network analysis combining PBA data with miRNA-Seq data showed that 79 miRNAs were related to 24 membrane proteins and predicted that there were surface proteins associated with a variety of miRNA molecules, such as ITGA9, XIAP, ADAM1, ITGA2, ITGA3, PDPN, and ITGB1. Meanwhile, there were miRNAs related to various surface proteins including miR-410-3p, miR-378b, and miR-1960. Taken together, our data demonstrated for the first time the changes of exosomal miRNAs and surface protein profiles derived from ischemic microvascular endothelial cells, which may provide new therapeutic targets for BBB protection in ischemic stroke.

MicroRNA-124 expression in the brains of rats during early cerebral ischemia and reperfusion injury is associated with cell apoptosis involving STAT3.

Cerebral ischemia and reperfusion injury is a cause of death and disability in adults. MicroRNA-124 possesses protective effects against apoptosis in cerebral ischemia and reperfusion. To provide insights into the diagnosis and treatment of cerebral ischemia and reperfusion injury, the dynamic changes of microRNA-124 expression during the early stage of cerebral ischemia and reperfusion injury in rats was investigated by quantitative polymerase chain reaction. To elucidate the association between the dynamic expression of microRNA-124 and apoptosis, the expression of proteins associated with apoptosis, including caspase-3, apoptosis regulator Bcl-2 (Bcl-2) and apoptosis regulator Bax (Bax) was analyzed by immunohistochemistry and western blot analyses. As signal transducer and activator of transcription 3 (STAT3) is involved in cell apoptosis and associated with Bcl-2, the protein expression of STAT3 and its active form, phosphorylated (p-)STAT3, were analyzed by western blot analysis. The expression of microRNA-124 increased and the maximum value appeared 12 h after reperfusion. Similarly, the expression of Bcl-2 also peaked 12 h after reperfusion, however the expression of caspase-3 and Bax continued to increase after the 12 h time point. These results indicate that the expression of microRNA-124 is closely associated with Bcl-2 and serves a protective role, inhibiting apoptosis. As the upstream regulator of Bcl-2, the expression of p-STAT3 was in accordance with Bcl-2 expression and peaked 12 h after reperfusion. By contrast, STAT3 was downregulated and the minimum level of STAT3 protein was reached 12 h after reperfusion. In summary, during the early stage of cerebral ischemia and reperfusion, the dynamic expression of microRNA-124 exhibited protective effects through the inhibition of apoptosis via anti-apoptotic proteins Bcl-2 and STAT3. Conversely, caspase-3 and Bax maintain apoptosis. The present study provides evidence to aid in the understanding of cerebral ischemia and reperfusion injury and develops methods of diagnosis and therapy of this condition.

Tissue-type plasminogen activator protects the postsynaptic density in the ischemic brain

Cerebral ischemia causes the presynaptic release of tissue-type plasminogen activator (tPA). The postsynaptic density (PSD) is a postsynaptic structure that provides a matrix where signaling transduction of excitatory synapses takes place. The postsynaptic density protein-95 (PSD-95) is the most abundant scaffolding protein in the postsynaptic density (PSD), where it modulates the postsynaptic response to the presynaptic release of glutamate by regulating the anchoring of glutamate receptors to the PSD. We found that tPA induces the local translation of PSD-95 mRNA and the subsequent recruitment of PSD-95 protein to the PSD, via plasminogen-independent activation of TrkB receptors. Our data show that PSD-95 is removed from the PSD during the early stages of cerebral ischemia, and that this effect is abrogated by either the release of neuronal tPA, or intravenous administration of recombinant tPA (rtPA). We report that the effect of tPA on PSD-95 is associated with inhibition of the phosphorylation and recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to the PSD, known to amplify the effect of the excitotoxic injury, and that this is followed by TrkB-mediated protection of dendritic spines from the harmful effects of the hypoxic insult. These data reveal that tPA is a synaptic protector in the ischemic brain.

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.

Hypothermia exerts early neuroprotective effects involving protein conjugation of SUMO‑2/3 in a rat model of middle cerebral artery occlusion.

How hypothermia serves an early protective role against cerebral ischemia remains to be determined. The small ubiquitin-related modifier protein (SUMO) functions as a post-translational modification system and SUMO-2/3 subtypes are often activated in early stress. The present study investigated changes in the protein level of SUMO using western blotting and immunocytochemistry when neurons were exposed to oxygen-glucose deprivation (OGD) in vitro, as well as in a rat model of middle cerebral artery occlusion (MCAO) in vivo. The results demonstrated that a large number of proteins were conjugated to SUMO-2/3 in OGD-injured neurons (within 10 min, peaking at 12 h), and was markedly enhanced under conditions of hypothermia (33°C). Concordantly, lactate dehydrogenase (LDH) release and the apoptosis rate, as determined by LDH and TUNEL assays, respectively, were lower in hypothermia-treated neurons. Similar results were obtained in a rat model of MCAO. Neurological deficit scores were lower in the hypothermia group than in the sham group in the early stage of cerebral ischemia (P<0.05). However, no significant differences in neurological deficit scores were detected between the hypothermia group and the sham group in the late stage of ischemia (21 days; P>0.05). This study implicates a role for SUMO-2/3 in early hypothermia-induced neuroprotection against stroke. The development of small molecule therapeutics based on SUMO-2/3 may benefit patients with cerebral ischemia.

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 Role of GluN2A in Cerebral Ischemia: Promoting Neuron Death and Survival in the Early Stage and Thereafter

Over-activation of NMDA receptors is a crucial step required for brain damage following a stroke. Although clinical trials for NMDA receptor blockers have failed, the role of GluN2A subunit in cerebral ischemia has been extensively evaluated in recent years. However, the effect of GluN2A on neuron damage induced by cerebral ischemia remains a matter of controversy. The underlying reason may be that GluN2A mediates both pro-death and pro-survival effects. These two effects result from two mutually excluding pathways, Ca2+ overload-dependent pro-death signaling and C-terminal-dependent pro-survival signaling, respectively. During the early stage of cerebral ischemia, over-activation of GluN2A plays an important role in Ca2+ overload. Under this condition, pro-death signaling might overcome pro-survival signaling. When GluN2A activity is restored almost to the normal level over time, pro-survival signaling of GluN2A will be dominant. The hypothesis that GluN2A promotes neuron death and survival in the early stage of cerebral ischemia and thereafter will be introduced in detail in this review.

Hypoxic Preconditioning Increases Blood-Brain Barrier Disruption in the Early Stages of Cerebral Ischemia.

Even though hypoxic preconditioning has been reported to produce neuroprotection, its effect on blood-brain barrier (BBB) disruption in the early stages of cerebral ischemia within the therapeutic window is not clear. Since hypoxic preconditioning increases expression of vascular endothelial growth factor (VEGF) that modulates vascular permeability, the effects of hypoxic preconditioning and VEGF on BBB permeability were investigated after one hour of focal cerebral ischemia. Rats were exposed to 8% of oxygen for two hours or room air and then 24 hours later, permanent middle cerebral artery (MCA) occlusion was performed. In some of the hypoxic preconditioned rats, a VEGF-A antibody was applied to the ischemic cortex one hour before MCA occlusion. One hour after MCA occlusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid was determined to measure the degree of BBB disruption. MCA occlusion increased the Ki when compared with the contralateral cortex (14.1 ± 4.0 vs 4.2 ± 1.9 μL/g/min, p < 0.0001). Hypoxic preconditioning further increased the Ki in the ischemic cortex when compared with the control rats (25.1 ± 8.7 μL/g/min, p < 0.01). Application of VEGF antibody to the ischemic cortex of the hypoxic preconditioned animals reduced the Ki to the level of the control rats (13.6 ± 5.1 μL/g/min, p < 0.01). Our data demonstrated that hypoxic preconditioning increased BBB disruption through a VEGF related pathway and suggest the possibility of aggravation of brain edema by hypoxic preconditioning in the early stages of cerebral ischemia.

Potassium Channels in Ischemic Stroke

On average, a stroke occurs every 40s in the United States and 87% of these cases are ischemic strokes. In the acute phase, cerebral edema is a major cause of neuronal death, and then activated microglia/macrophages significantly contribute to the secondary inflammatory damage later in ischemic stroke. Neither the physiological or pathological roles of potassium channels in ischemic stroke have been systematically explored. Our studies reveal that potassium channels are important in cerebral edema formation and microglia/macrophage activation. KCa3.1, a calcium‐activated potassium channel, physiologically regulates ion and fluid secretion in the lung and gastrointestinal tract. It is also expressed on vascular endothelium where it participates in blood pressure regulation. Our recent study of the expression and physiological role of KCa3.1 in blood–brain barrier (BBB) endothelium found that BBB endothelial cells exhibit KCa3.1 protein and activity facilitating Na+ transportation from the blood into the brain transcellularly through the co‐operation of multiple cotransporters, exchangers, pumps, and channels in normal physiological conditions and the early stages of cerebral ischemia, when the BBB is still intact. Pharmacological blockade with KCa3.1 blocker, TRAM‐34, significantly reduced Na+ uptake, and cytotoxic edema measured by MRI in the ischemic rat brain with permanent middle cerebral artery occlusion (MCAO) in the first 3 hours of ischemic stroke. (Chen et el. 2015 Stroke 46:237.) Together with the voltage‐gated potassium channel Kv1.3, KCa3.1 also regulates membrane potential and Ca2+ signaling in microglia and monocyte‐derived macrophages. We recently evaluated acute isolated microglia/macrophage from mice subjected to MCAO with eight days of reperfusion. Microglia from the infarcted area exhibited higher densities of K+ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non‐infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/macrophages. Pharmacological inhibition or genetic deletion of KCa3.1 resulted in significantly smaller infarct areas on day‐8 after MCAO and improved neurological deficits. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. (Chen et al. 2015 JCBFM; epub) In summary, K+ channels can be a potential pharmacological target for ischemic stroke to slow down cerebral edema formation and reduce the secondary inflammatory damage from activated microglia/macrophage.Support or Funding InformationSupported by AHA &amp; NIHGM

Blood-brain barrier KCa3.1 channels: evidence for a role in brain Na uptake and edema in ischemic stroke.

KCa3.1, a calcium-activated potassium channel, regulates ion and fluid secretion in the lung and gastrointestinal tract. It is also expressed on vascular endothelium where it participates in blood pressure regulation. However, the expression and physiological role of KCa3.1 in blood-brain barrier (BBB) endothelium has not been investigated. BBB endothelial cells transport Na(+) and Cl(-) from the blood into the brain transcellularly through the co-operation of multiple cotransporters, exchangers, pumps, and channels. In the early stages of cerebral ischemia, when the BBB is intact, edema formation occurs by processes involving increased BBB transcellular Na(+) transport. This study evaluated whether KCa3.1 is expressed on and participates in BBB ion transport. The expression of KCa3.1 on cultured cerebral microvascular endothelial cells, isolated microvessels, and brain sections was evaluated by Western blot and immunohistochemistry. Activity of KCa3.1 on cerebral microvascular endothelial cells was examined by K(+) flux assays and patch-clamp. Magnetic resonance spectroscopy and MRI were used to measure brain Na(+) uptake and edema formation in rats with focal ischemic stroke after TRAM-34 treatment. KCa3.1 current and channel protein were identified on bovine cerebral microvascular endothelial cells and freshly isolated rat microvessels. In situ KCa3.1 expression on BBB endothelium was confirmed in rat and human brain sections. TRAM-34 treatment significantly reduced Na(+) uptake, and cytotoxic edema in the ischemic brain. BBB endothelial cells exhibit KCa3.1 protein and activity and pharmacological blockade of KCa3.1 seems to provide an effective therapeutic approach for reducing cerebral edema formation in the first 3 hours of ischemic stroke.

Early ischemic blood brain barrier damage: a potential indicator for hemorrhagic transformation following tissue plasminogen activator (tPA) thrombolysis?

Tissue plasminogen activator (tPA) thrombolysis, remains to be the only United States Food and Drug Administration (FDA) approved treatment for acute ischemia stroke. However, the use of tPA has been profoundly constrained due to its narrow therapeutic time window and the increased risk of potentially deadly hemorrhagic complications. TPA-associated hemorrhagic transformation (HT) often occurs as a result of catastrophic failure of the blood brain barrier (BBB), wherein the affected cerebral capillaries can no longer hold blood constituents. Due to its direct contribution to edema and HT, reperfusion-associated BBB damage has been extensively studied, while BBB damage that occurs within the thrombolytic time window is largely neglected. Of note, ischemia-induced BBB damage in the early stroke stages is increasingly appreciated to negatively affect the safety and efficacy profiles of thrombolytic therapy for ischemic stroke. In this review, we discussed the recent findings of spatio-temporal evolution of BBB injury in the early stages of cerebral ischemia and its association with intracerebral hemorrhage following tPA thrombolysis. The increased understanding of early ischemic BBB damage and its close link to tPA-associated HT is of particular importance for developing new preventive and therapeutic strategies to reduce the hemorrhagic complications in stroke thrombolysis.

The Effects of Fludarabine on Rat Cerebral Ischemia

Cerebral ischemic injury involves a variety of cellular and molecular events. Signal transducers and activators of transcription-1 (STAT-1) activation is associated with neuronal cell death and contributes to ischemic injury. The effects of fludarabine, a specific inhibitor of STAT1 protein, on cerebral ischemic/reperfusion (I/R) injury were studied in a rat model. Rats subjected to I/R injury were either treated with intra-cerebroventricular injection of fludarabine (5,000 μM, 10 μl) or saline 20 min before middle cerebral artery occlusion (MCAO). MR examinations including T2-weighted imaging (T2WI), diffusion-weighted imaging (DWI), and perfusion-weighted imaging (PWI) were performed after I/R period. Then rat brains were sectioned for triphenyltetrazolium chloride (TTC) stains, analyzed by Western blot and TUNEL staining of apoptosis. It was found that fludarabine treatment decreased the infarct volume of the cerebrum and the number of apoptotic neural cells in the ischemic brain. Compared to saline-treated group, the apparent diffusion coefficient (ADC) and cerebral blood volume (CBV) in the ischemic region were greater, and the mean transit time (MTT) was shortened in the fludarabine-treated group. Moreover, fludarabine inhibited the expression level of phosphorylated STAT1 (P-STAT1) in neural cells after I/R injury, whereas the expression of phosphorylated STAT3 (P-STAT3) was increased. Therefore, we concluded that fludarabine administrated in early stage of cerebral ischemia had neuroprotective effects, and the underlying mechanism could be mediated through inhibiting STAT1 phosphorylation and activating the cross regulation between STAT1 and STAT3 in neural cells.

Relationship between the induction of inflammatory processes and infectious diseases in patients with ischemic stroke.

Pro-inflammatory cytokines participate in the induction of ischemic stroke. So far, their participation in the cerebral ischemia was proven for the tumor necrosis factor TNF-α, interleukin-1 (IL-1), and interleukin-6 (IL-6). The release of the pro-inflammatory cytokines into the extracellular space causes the enlargement of the brain damage region, and consequently increases the neurological deficit and negatively affects the survival rate prognoses. That is confirmed by the increased concentration of pro-inflammatory cytokines in blood and the cerebrospinal fluid of patients with brain stroke, as well as by the research on the induced/experimental cerebral ischemia in animals. The pro-inflammatory cytokines participate in the migration of the reactive T lymphocytes to the regions of brain ischemia where they enhance the nerve tissue damage by down-regulation of microcirculation, induce the pro-thrombotic processes and release other neurotoxic cytokines. Also, in the early stage of cerebral ischemia, cytokines activate the axis hypothalamus-pituitary gland-adrenal cortex and increase the cortisol concentration in blood, what results in the decreased resistance to infectious diseases. Administration of the inhibitor of the interleukin-1 receptor (IL-1Ra) inhibits the inflammatory processes in the region of brain ischemia, and subsequently improves the prognosis for the size of the neurological deficit and the survival rate, as well as resistance to infectious diseases.

Green tea polyphenols alleviate early BBB damage during experimental focal cerebral ischemia through regulating tight junctions and PKCalpha signaling

BackgroundIt has been supposed that green tea polyphenols (GTPs) have neuroprotective effects on brain damage after brain ischemia in animal experiments. Little is known regarding GTPs’ protective effects against the blood-brain barrier (BBB) disruption after ischemic stroke. We investigated the effects of GTPs on the expression of claudin-5, occludin, and ZO-1, and the corresponding cellular mechanisms involved in the early stage of cerebral ischemia.MethodsMale Wistar rats were subjected to a middle cerebral artery occlusion (MCAO) for 0, 30, 60, and 120 min. GTPs (400 mg/kg/day) or vehicle was administered by intragastric gavage twice a day for 30 days prior to MCAO. At different time points, the expression of claudin-5, occludin, ZO-1, and PKCα signaling pathway in microvessel fragments of cerebral ischemic tissue were evaluated.ResultsGTPs reduced BBB permeability at 60 min and 120 min after ischemia as compared with the vehicle group. Transmission electron microscopy also revealed that GTPs could reverse the opening of tight junction (TJ) barrier at 60 min and 120 min after MACO. The decreased mRNA and protein expression levels of claudin-5, occludin, and ZO-1 in microvessel fragments of cerebral ischemic tissue were significantly prevented by treatment with GTPs at the same time points after ischemia in rats. Furthermore, GTPs could attenuate the increase in the expression levels of PKCα mRNA and protein caused by cerebral ischemia.ConclusionsThese results demonstrate that GTPs may act as a potential neuroprotective agent against BBB damage at the early stage of focal cerebral ischemia through the regulation of TJ and PKCα signaling.

Relationship of gelatinases-tight junction proteins and blood-brain barrier permeability in the early stage of cerebral ischemia and reperfusion.

Gelatinases matrix metalloproteinase-2 and matrix metalloproteinase-9 have been shown to mediate claudin-5 and occludin degradation, and play an important regulatory role in blood-brain barrier permeability. This study established a rat model of 1.5-hour middle cerebral artery occlusion with reperfusion. Protein expression levels of claudin-5 and occludin gradually decreased in the early stage of reperfusion, which corresponded to the increase of the gelatinolytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. In addition, rats that received treatment with matrix metalloproteinase inhibitor N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpenthanoyl]-L-tryptophan methylamide (GM6001) showed a significant reduction in Evans blue leakage and an inhibition of claudin-5 and occludin protein degradation in striatal tissue. These data indicate that matrix metalloproteinase-2 and matrix metalloproteinase-9-mediated claudin-5 and occludin degradation is an important reason for blood-brain barrier leakage in the early stage of reperfusion. The leakage of the blood-brain barrier was present due to gelatinases-mediated degradation of claudin-5 and occludin proteins. We hypothesized that the timely closure of the structural component of the blood-brain barrier (tight junction proteins) is of importance.

The Intrinsic PEDF is Regulated by PPARγ in Permanent Focal Cerebral Ischemia of Rat

Inflammatory damage plays a pivotal role in cerebral ischemia and may represent a target for treatment. Pigment epithelium-derived factor (PEDF) is proven to possess neuroprotective property. But there is little known about the intrinsic PEDF after cerebral ischemia. This study evaluated the time course expression of the intrinsic PEDF and its underlying regulation mechanisms after cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion. Telmisartan (PPARγ agonist) and GW9662 (PPARγ antagonist) were systemically administered to explore the effect on PPARγ, PEDF, NF-κB and MMP-9 expression at 24 h after cerebral ischemia by western blot and qRT-PCR. The neurological deficits, brain water content and infarct volume were measured. Compared with normal group, the expressions of PEDF and PPARγ decreased, and the expression of NF-κB and MMP-9 increased at early stage after ischemia (P < 0.05). Compared with the vehicle group, the decrease of PEDF and PPARγ was significantly up-regulated and the increase of NF-κB and MMP-9 was down-regulated by telmisartan at 24 h (P < 0.05). The neurological deficits, brain water content and infarct volume were dramatically alleviated by telmisartan (P < 0.05). Telmisartan's effects were reversed by GW9662 co-administration (P < 0.05). The expression of intrinsic PEDF was down-regulated at the early stage of cerebral ischemia. The protective effects of intrinsic PEDF by activating PPARγ pathway may be one of the strategic targets for cerebral ischemic therapies.

Matrix metalloproteinase-2 or -9 deletions protect against hemorrhagic transformation during early stage of cerebral ischemia and reperfusion

MMP-9 deficiency protected against photochemical thrombosis-induced brain hemorrhagic transformation (HT), but it did not protect against tissue plasminogen activator-induced brain hemorrhage. The roles of MMP-2 and/or MMP-9 knockout (KO) in mechanical reperfusion induced HT after ischemia have not been investigated. Here we assessed the effects of MMP-2 KO, MMP-9 KO and MMP-2/9 double KO (dKO) in protecting against mechanical reperfusion induced HT and other brain injuries after the early stages of cerebral ischemia in mice of the same genetic background. Middle cerebral artery occlusion (MCAO) was performed in mice. Reperfusion was started at 1 or 1.5h after onset of MCAO. All mice were sacrificed 8h after MCAO. We found that both pro- and active MMP-2 and MMP-9 levels were significantly elevated in the early ischemic brain. After the early stages of ischemia and reperfusion, the hemorrhagic incidence was reduced in the cortex of MMP-2 KO mice (p<0.05 vs. WT). The hemorrhagic volume was significantly decreased in the cortexes of MMP-2 and/or -9 knockout mice (MMP-9 KO vs. WT: p<0.01, MMP-2 KO and dKO vs. WT: p<0.001). In the basal ganglia, MMP-2 KO and MMP-2/9 dKO mice displayed a remarkable decrease in hemorrhagic volume (p<0.01 or 0.05 vs. WT), but MMP-9 KOs did not protect against hemorrhage. MMP-2 and/or -9 knockout mice displayed significantly decreased infarction volume in both the cortex and striatum, in addition to improved neurological function (p<0.001 vs. WT). The results suggested that MMP-2 deficiency and MMP-2 and MMP-9 double deficiency were more protective than MMP-9 deficiency against HT after the early stages of ischemia and reperfusion. These studies increase our understanding of MMP-2 and MMP-9 in HT development and will help to selectively target MMPs to protect the post-ischemic brain from injury and HT.