Cathepsin L acutely alters microvessel integrity within the neurovascular unit during focal cerebral ischemia

During focal cerebral ischemia, the degradation of microvessel basal lamina matrix occurs acutely and is associated with edema formation and microhemorrhage. These events have been attributed to matrix metalloproteinases (MMPs). However, both known protease generation and ligand specificities suggest other participants. Using cerebral tissues from a non-human primate focal ischemia model and primary murine brain endothelial cells, astrocytes, and microglia in culture, the effects of active cathepsin L have been defined. Within 2 hours of ischemia onset cathepsin L, but not cathepsin B, activity appears in the ischemic core, around microvessels, within regions of neuron injury and cathepsin L expression. In in vitro studies, cathepsin L activity is generated during experimental ischemia in microglia, but not astrocytes or endothelial cells. In the acidic ischemic core, cathepsin L release is significantly increased with time. A novel ex vivo assay showed that cathepsin L released from microglia during ischemia degrades microvessel matrix, and interacts with MMP activity. Hence, the loss of microvessel matrix during ischemia is explained by microglial cathepsin L release in the acidic core during injury evolution. The roles of cathepsin L and its interactions with specific MMP activities during ischemia are relevant to strategies to reduce microvessel injury and hemorrhage.     

Acute neurovascular unit protection by simvastatin in transient cerebral ischemia

Abstract

Acute vascular- and neuroprotective effects of simvastatin were evaluated in a rat model of transient, focal cerebral ischemia. Male, Wistar rats (n=12) underwent transient middle cerebral artery (MCA) occlusion for 3 hours followed by 3 hours of reperfusion. After 30 minutes of MCA occlusion, four rats each were subcutaneously injected with either 20 or 40 mg/kg of simvastatin. At the end of 3 hours of reperfusion, tissue injury and blood–brain barrier (BBB) opening were quantified by histology and [¹⁴C]-alpha-aminoisobutyric acid (AIB)-based quantitative autoradiography (QAR), respectively. Compared with untreated rats, those treated with simvastatin (20 mg/kg) had reduced volumes of AIB leakage, tissue pallor and distribution space for AIB (p<0.05). No additional effects were seen with the higher drug dose (40 mg/kg). These data suggest that the acute neuroprotective effects of statins are in part owing to attenuation of stroke-induced changes in BBB permeability.     

Acute neurovascular unit protection by simvastatin in transient cerebral ischemia

Acute vascular- and neuroprotective effects of simvastatin were evaluated in a rat model of transient, focal cerebral ischemia. Male, Wistar rats (n=12) underwent transient middle cerebral artery (MCA) occlusion for 3 hours followed by 3 hours of reperfusion. After 30 minutes of MCA occlusion, four rats each were subcutaneously injected with either 20 or 40 mg/kg of simvastatin. At the end of 3 hours of reperfusion, tissue injury and blood-brain barrier (BBB) opening were quantified by histology and [(14)C]-alpha-aminoisobutyric acid (AIB)-based quantitative autoradiography (QAR), respectively. Compared with untreated rats, those treated with simvastatin (20 mg/kg) had reduced volumes of AIB leakage, tissue pallor and distribution space for AIB (p<0.05). No additional effects were seen with the higher drug dose (40 mg/kg). These data suggest that the acute neuroprotective effects of statins are in part owing to attenuation of stroke-induced changes in BBB permeability.     

脑缺血机制再认识及神经血管单元的保护

脑缺血是导致患者死亡或残疾的三大疾病之一.目前除了急性期4.5 h内进行溶栓治疗被证实对其有效外,尚缺乏其他有效的神经保护治疗措施.为探讨新的治疗方法,人们必须不断深入地理解脑缺血后导致神经元死亡的一系列病理生理机制.因此,本文就脑缺血机制及神经血管单元保护的新进展进行综述,以期加深对其的理解并寻求脑缺血治疗的新思路.     

Hypothermia selectively protects the anterior forebrain mesocircuit during global cerebral ischemia

Hypothermia is an important protective strategy against global cerebral ischemia following cardiac arrest.However,the mechanisms of hypothermia underlying the changes in different regions and connections of the brain have not been fully elucidated.This study aims to identify the metabolic nodes and connection integrity of specific brain regions in rats with global cerebral ischemia that are most affected by hypothermia treatment.18F-fluorodeoxyglucose positron emission tomography was used to quantitatively determine glucose metabolism in different brain regions in a rat model of global cerebral ischemia established at 31–33℃.Diffusion tensor imaging was also used to reconstruct and explore the brain connections involved.The results showed that,compared with the model rats established at 37–37.5℃,the rat models of global cerebral ischemia established at 31–33℃had smaller hypometabolic regions in the thalamus and primary sensory areas and sustained no obvious thalamic injury.Hypothermia selectively preserved the integrity of the anterior forebrain mesocircuit,exhibiting protective effects on the brain during the global cerebral ischemia.The study was approved by the Institutional Animal Care and Use Committee at Capital Medical University(approval No.XW-AD318-97-019)on December 15,2019.     

Protective effect of synaptic inhibition during cerebral ischemia in rats and rabbits.

BACKGROUND: Excitatory neurotransmitters appear to cause cell death during ischemia by inducing depolarization, influx of ions, and metabolic failure in the postsynaptic neuron. If this hypothesis is correct, then postsynaptic membrane hyperpolarization and inhibition of metabolism may be protective. Antagonists of the excitotoxic amino acid glutamate protect neurons in culture and in animal models of stroke but appear to cause unacceptable side effects in humans. We propose an alternative strategy of protection using agonists of the inhibitory neurotransmitter gamma-aminobutyric acid. METHODS: We caused multifocal cerebral ischemia in rats and rabbits by injecting microspheres into the carotid circulation. We administered saline, muscimol, or MK-801 within 5 minutes of stroke onset. We used a bioassay to measure outcome. In rats, we also used learning to assess cortical function, and we performed detailed quantitative brain morphometry 3 months after infarction. RESULTS: Using the bioassay, we found that muscimol exerted a protective effect in rats (p less than 0.01). There was a dose-response effect seen in muscimol-treated rabbits. Rats treated with muscimol or MK-801 exhibited significantly better visual-spatial learning compared with saline-treated subjects (p less than 0.001). Hemisphere volume after ischemia was comparable in all groups. CONCLUSIONS: Agonists of gamma-aminobutyric acid and antagonists of glutamate appear to protect brain during ischemia. Since agonists of gamma-aminobutyric acid are known to have fewer side effects in humans, they may prove more useful in the clinical setting as neuroprotective agents.     

Hyodeoxycholic acid protects the neurovascular unit against oxygen-glucose deprivation and reoxygenation-induced injury in vitro

Calculus bovis is commonly used for the treatment of stroke in traditional Chinese medicine. Hyodeoxycholic acid(HDCA) is a bioactive compound extracted from calculus bovis. When combined with cholic acid, baicalin and jas-minoidin, HDCA prevents hypoxia-reoxygenation-induced brain injury by suppressing endoplasmic reticulum stress-mediated apoptotic signaling. However, the effects of HDCA in ischemic stroke injury have not yet been studied. Neurovascular unit(NVU) dysfunction occurs in ischemic stroke. Therefore, in this study, we investigated the effects of HDCA on the NVU under ischemic conditions in vitro. We co-cultured primary brain microvascular endothelial cells, neurons and astrocytes using a transwell chamber co-culture system. The NVU was pre-treated with 10.16 or 2.54 μg/mL HDCA for 24 hours before exposure to oxygen-glucose deprivation for 1 hour. The cell counting kit-8 assay was used to detect cell activity. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were used to assess apoptosis. Enzyme-linked immunosorbent assay was used to measure the expression levels of inflammatory cytokines, including interleukin-1β, interleukin-6 and tumor necrosis factor-α, and neurotrophic factors, including brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. Oxidative stress-related factors, such as superoxide dismutase, nitric oxide, malondialdehyde and γ-glutamyltransferase, were measured using kits. Pretreatment with HDCA significantly decreased blood-brain barrier permeability and neuronal apoptosis, significantly increased transendothelial electrical resistance and γ-glutamyltransferase activity, attenuated oxidative stress damage and the release of inflammatory cytokines, and increased brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression. Our findings suggest that HDCA maintains NVU morphological integrity and function by modulating inflammation, oxidation stress, apoptosis, and the expression of neurotrophic factors. Therefore, HDCA may have therapeutic potential in the clinical management of ischemic stroke. This study was approved by the Ethics Committee of Experimental Animals of Beijing University of Chinese Medicine(approval No. BUCM-3-2016040201-2003) in April 2016.     

The AMPAR antagonist perampanel protects the neurovascular unit against traumatic injury via regulating Sirt3

IntroductionPerampanel is a highly selective and noncompetitive α‐amino‐3 ‐hydroxy‐5‐methyl‐4‐isoxazole propionate receptor (AMPAR) antagonist, which has been used as an orally administered antiepileptic drug in more than 55 countries. Recently, perampanel was shown to exert neuroprotective effects in hemorrhagic and ischemic stroke models via regulating blood–brain barrier (BBB) function.AimHere, the protective effects of perampanel were investigated in an in vitro neurovascular unit (NVU) system established using a triple cell co‐culture model (neurons, astrocytes, and brain microvascular endothelial cells) and in an in vivo traumatic brain injury (TBI) model.ResultsNeurons in the NVU system exhibit a more mature morphological phenotype compared with neurons cultured alone, and the co‐culture system mimicked an impermeable barrier in vitro. Perampanel protects the NVU system against traumatic and excitotoxic injury, as evidenced by reduced lactate dehydrogenase (LDH) release and apoptotic rate. Treatment with perampanel attenuated lipid peroxidation and expression of inflammatory cytokines. In addition, perampanel increased Sirt3 protein expression, enhanced the activities of mitochondrial enzyme IDH2 and SOD2, and preserved BBB function in vitro. Knockdown of Sirt3 using specific siRNA (Si‐Sirt3) partially reserved the effects of perampanel on neuronal injury and BBB function. Treatment with perampanel in vivo attenuated brain edema, preserved neurological function, inhibited apoptosis and microglia activation after TBI. Furthermore, perampanel increased the expression of Sirt3 and preserved BBB function after TBI. The effect of perampanel on BBB function and brain edema was abolished by knockdown of Sirt3 in vivo.ConclusionOur results indicate that the noncompetitive AMPAR antagonist perampanel protects the NVU system and reduces brain damage after TBI via activating the Sirt3 cascades.     

Intercellular adhesion molecule-5 protects PAJU cells during in vitro ischemia following cerebral infarction

BACKGROUND: Intercellular adhesion molecule-5 (ICAM-5) relieves the damage of beta-amyloid protein to PAJU cells. However, little is known about how ICAM-5 works as a neurotrophic factor, or whether ICAM-5 lessens neuronal damage under ischemic conditions following cerebral infarction. OBJECTIVE: To investigate the effects of ICAM-5 on PAJU cells growth in serum-free medium under ischemic conditions following cerebral infarction. DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Central Laboratory, Second Xiangya Hospital, Central South University, China, in June 2009. MATERIALS: Human ICAM-5 gene transfected into PAJU-TLN cells was supplied by the Life Science College, Helsinki University, Finland. Empty vector transfected PAJU-NEO cells were established by the Gene Center, Second Xiangya Hospital, Central South University, China. METHODS: PAJU-TLN cells transfected with human ICAM-5 or empty vector were incubated in serum-free medium. MAIN OUTCOME MEASURES: Phase contrast microscopy was used to observe changes in PAJU cell morphology. 3-(4,5-dimethylthiazolzyl)-2,5-diphenyltetrazolium bromide was used to determine cell viability. Hoechst 33258 was used to stain cell nuclei. Flow cytometry was utilized to measure the apoptosis rate of both PAJU-TLN and PAJU-NEO cells.RESULTS: Both PAJU-TLN and PAJU-NEO cells were injured by cultivating in serum-free medium, but the survival rate of PAJU-TLN cells was significantly higher.CONCLUSION: ICAM-5 protects PAJU-TLN cells from serum deprivation-induced apoptosis, induces the outgrowth of PAJU cells, and diminishes their morphologic impairment.     

脑缺血后神经血管保护机制和损伤机制研究进展

脑梗死是致死性疾病之一,发病率高,严重危害人类健康。尽管各方面的研究不断深入,但是目前仍然缺乏有效的神经保护药物,脑梗死的治疗依然是一个非常棘手的问题。本文从内源性保护机制、谷氨酸受体的双向作用、非谷氨酸依赖的钙失衡损伤机制进行综述,进一步阐释脑缺血后神经血管的保护机制和损伤机制,以求突破神经保护研究的传统框架,寻找神经血管保护治疗的新理念和新思路。     

Apelin-13 protects neurovascular unit against ischemic injuries through the effects of vascular endothelial growth factor

Apelin-13 has protective effects on many neurological diseases, including cerebral ischemia. Here, we aimed to test Apelin-13's effects on ischemic neurovascular unit (NVU) injuries and investigate whether the effects were dependent on vascular endothelial growth factor (VEGF). We detected the expression of VEGF and its receptors (VEGFRs) induced by Apelin-13 injection at 1 d, 3 d, 7 d and 14 d after middle cerebral artery occlusion (MCAO). Meanwhile, we examined the effects of Apelin-13 on NVU in both in vivo and in vitro experiments as well as whether the effects were VEGF dependent by using VEGF antibody. We also assessed the related signal transduction pathways via multiple inhibitors. We demonstrated Apelin-13 highly increased VEGF and VEGFR-2 expression, not VEGFR-1. Importantly, Apelin-13 led to neurological functions improvement by associating with promotion of angiogenesis as well as reduction of neuronal death and astrocyte activation, which was markedly blocked by VEGF antibody. In cell cultures, Apelin-13 protected neurons, astrocytes and endothelial cells against oxygen-glucose deprivation (OGD) injuries. Moreover, the effect of Apelin-13 to up-regulate VEGF was suppressed by extracellular signal-regulated kinase (ERK) inhibitor U0126 and phosphatidylinositol 3′-kinase (PI3K) inhibitor LY294002. Our data suggest protective effects of Apelin-13 on ischemic NVU injuries are highly associated with the increase of VEGF binding to VEGFR-2, possibly acting through activation of ERK and PI3K/Akt pathways.     

Respiratory chain inhibition induces tolerance to focal cerebral ischemia.

The authors show that the inhibitor of the succinate dehydrogenase, 3-nitroproprionic acid (3-NPA), which in high doses and with chronic administration is a neurotoxin, can induce profound tolerance to focal cerebral ischemia in the rat when administered in a single dose (20 mg/kg) 3 days before ischemia. Infarcts were approximately 70% and 35% smaller in the 3-NPA preconditioned groups of permanent and transient focal cerebral ischemia, respectively. This regimen of 3-NPA preconditioning neither induced necrosis, apoptosis, or any other histologically detectable damage to the brain, nor did it affect behavior of the animals. 3-NPA led to an immediate (1-hour) and long-lasting (3-day) decrease in succinate dehydrogenase activity (30% reduction) throughout the brain, whereas only a short metabolic impairment occurred (ATP decrease of 35% within 30 minutes, recovery within 2 hours). The authors found that 3-NPA induces a burst of reactive oxygen species and the free radical scavenger dimethylthiourea, when administered shortly before the 3-NPA stimulus, completely blocked preconditioning. Inhibition of protein synthesis with cycloheximide given at the time of 3-NPA administration completely inhibited preconditioning. The authors were unsuccessful in showing upregulation of mRNA for the manganese superoxide dismutase, and did not detect increased activities of the copper-zinc and manganese superoxide dismutases, prototypical oxygen free radicals scavenging enzymes, after 3-NPA preconditioning. The authors conclude that it is possible to pharmacologically precondition the brain against focal cerebral ischemia, a strategy that may in principal have clinical relevance. The data show the relevance of protein synthesis for tolerance, and suggests that oxygen free radicals may be critical signals in preconditioning.     

Neuroprotection of taurine through inhibition of 12/15 lipoxygenase pathway in cerebral ischemia of rats

Background: Cerebral ischemia exhibits a multiplicity of pathophysiological mechanisms. Taurine (Tau), an endogenous substance, possesses a number of cytoprotective properties. The aim of the present study was to examine the neuroprotective effect of Tau, through affecting 12/15 lipoxygenase (12/15-LOX) signal pathway in an acute permanent middle cerebral artery occlusion (MCAO) model of rats.

Methods: Sprague-Dawley rats were randomly divided into 3 groups (n = 10), namely the sham-operated group, MCAO group and Tau group. Tau was intraperitoneally administrated immediately after cerebral ischemia. At 24 h after MCAO, neurological function score, brain water content and infarct volume were assessed. The expression of 12/15-lipoxygenase (12/15-LOX), p38 mitogen-activated protein kinase (p38 MAPK), and cytosolic phospholipase A2 (cPLA2) was measured by Western blot. Enzyme-linked immunosorbent assay was used to evaluate the inflammatory factors TNF-α, IL-1β and IL-6 in serum.

Results: Compared with MCAO group, taurine significantly improved neurological function and significantly reduced brain water content (p < 0.05) and infarct volume (p < 0.05). Consistent with these indices, the overexpressions of 12/15-LOX, p38 MAPK, cPLA2, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly decreased in Tau group (p < 0.05).

Conclusion: Taurine protected the brain from damage caused by MCAO; this effect may be through down-regulation of 12/15-LOX, p38 MAPK, and cPLA2.     

Up-regulation of A-type potassium currents protects neurons against cerebral ischemia

Excitotoxicity is the major cause of many neurologic disorders including stroke. Potassium currents modulate neuronal excitability and therefore influence the pathological process. A-type potassium current (I_A) is one of the major voltage-dependent potassium currents, yet its roles in excitotoxic cell death are not well understood. We report that, following ischemic insults, the I_A increases significantly in large aspiny (LA) neurons but not medium spiny (MS) neurons in the striatum, which correlates with the higher resistance of LA neurons to ischemia. Activation of protein kinase Cα increases I_A in LA neurons after ischemia. Cultured neurons from transgenic mice lacking both Kv1.4 and Kv4.2 subunits exhibit an increased vulnerability to ischemic insults. Increase of I_A by recombinant expression of Kv1.4 or Kv4.2 is sufficient in improving the survival of MS neurons against ischemic insults both in vitro and in vivo. These results, taken together, provide compelling evidence for a protective role of I_A against ischemia.     

The function and integrity of the neurovascular unit rests upon the integration of the vascular and inflammatory cell systems

The neurovascular unit is composed of a microvascular endothelium, neuron, and glial cell elements that are in physical proximity to the endothelium. The vascular system provides oxygen, glucose, and hormones for brain cells and guides the cells to appropriately respond to the local environment. Conversely, the brain cells, especially glial cells, can regulate the function of blood vessels in response to local requirements. The disruption of the neurovascular coordination was observed in a variety of inflammation-related diseases in brain, such as infectious diseases, stroke, vascular dementia, and multiple sclerosis. Inflammatory responses resulting from infections or injury of the brain activate the endothelium and glial cells to various degrees depending on the type, titer, or strength and duration of exposure to the agents or insults. The activation of endothelial and microglial cells may be modulated by the action of cytokines or other substances secreted from these cells. In an effort to understand the pathogenesis and find rational treatments against inflammatory disorders in brain, studies have been separately carried out using either endothelial cells or microglia. Increasing evidence, however, indicates that a crosstalk between these two cell types is important for the brain inflammation. Here, we review recent advances that provide insights into the coordinated interaction between the vascular and microglial systems, including the role of the specialized endothelium in regulating the immune response that occurs within CNS, the influence of microglial cells on the properties of endothelial cells, and the effects of endothelium on the state of microglial activation.