Rat Middle Cerebral Artery Research Articles

Extra-endothelial TRPV1 channels participate in alcohol and caffeine actions on cerebral artery diameter.

Alcohol (ethyl alcohol; ethanol) and caffeine are the two most widely used psychoactive substances in the world. Caffeine and ethanol have both been reported to constrict cerebral arteries in several species, including humans. We have recently shown that application of 10-μM caffeine mixed with 50 mM ethanol to invitro pressurized cerebral arteries of rats reduced ethanol-induced constriction. This effect was dependent on the presence of nitric oxide (NO•) and could be observed in de-endothelialized arteries supplied with the NO donor sodium nitroprusside (SNP). The molecular target(s) of ethanol-caffeine interaction in cerebral arteries has remained unknown. In the present work, we used rat and mouse middle cerebral arteries (MCA) to identify the extra-endothelial effectors of NO-mediated, caffeine-induced protection against ethanol-evoked arterial constriction. Constriction of intact MCA of rat by either 50 mM ethanol or 10 μM caffeine was ablated in the presence of a selective TRPV1 pharmacological blocker. TRPV1 pharmacological block, but not block of TRPA1, PKG, or BK channels, removed caffeine-induced protection against ethanol-evoked rat MCA constriction, whether evaluated in arteries with intact endothelium or in SNP-supplemented, de-endothelialized arteries. In mouse arteries, caffeine-induced protection against ethanol-induced MCA constriction was significantly amplified, resulting in actual vasodilation, upon pharmacological block of TRPV1, and in TRPV1 knock-out arteries. Despite some species-specific differences, our study unequivocally demonstrates the presence of functional, extra-endothelial TRPV1 that participates in both endothelium-independent MCA constriction by separate exposure to ethanol or caffeine and caffeine-induced protection against ethanol-evoked MCA constriction.

Material Properties of Rat Middle Cerebral Arteries at High Strain Rates.

Traumatic brain injury (TBI), resulting from either impact- or nonimpact blast-related mechanisms, is a devastating cause of death and disability. The cerebral blood vessels, which provide critical support for brain tissue in both health and disease, are commonly injured in TBI. However, little is known about how vessels respond to traumatic loading, particularly at rates relevant to blast. To better understand vessel responses to trauma, the objective of this project was to characterize the high-rate response of passive cerebral arteries. Rat middle cerebral arteries (MCAs) were isolated and subjected to high-rate deformation in the axial direction. Vessels were perfused at physiological pressures and stretched to failure at strain rates ranging from approximately 100 to 1300 s-1. Although both in vivo stiffness and failure stress increased significantly with strain rate, failure stretch did not depend on rate.

Effect of laser Doppler flowmetry and occlusion time on outcome variability and mortality in rat middle cerebral artery occlusion: inconclusive results

BackgroundStroke is among the leading causes of death and disability. Although intense research efforts have provided promising treatment options in animals, most clinical trials in humans have failed and the therapeutic options are few. Several factors have been suggested to explain this translational difficulty, particularly concerning methodology and study design. Consistent infarcts and low mortality might be desirable in some, but not all, studies. Here, we aimed to investigate whether the use of laser Doppler flowmetry (LDF) and the occlusion time (60 vs. 45 min) affected outcome variability and mortality in a rat stroke model. Eighty ovariectomized female Wistar rats were subjected to ischemic stroke using intraluminal filament middle cerebral artery occlusion with or without LDF and with occlusion times of 45 or 60 min. Outcome was evaluated by triphenyl tetrazolium chloride staining of brain slices to measure infarct size and a modified sticky tape test.ResultsNeither LDF nor occlusion times of 45 versus 60 min significantly affected mortality, outcome variability or outcome severity.ConclusionsDue to the unexpectedly high mortality and variability the statistical power was very low and thus the results were inconclusive.

2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside, an analog of salidroside, contributes to neuroprotection in cerebral ischemic injury in vitro and in vivo.

2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxyb-D-pyranoside (code-named SalA-4g), an analog of salidroside, has potent neuroprotective effects. In this study, the pharmacological properties of SalA-4g were evaluated in primary cortical neurons exposed to oxygen and glucose deprivation and in a rat model of transient middle cerebral artery occlusion. The results of pharmacokinetic and brain distribution studies indicated that SalA-4g could pass through the blood-brain barrier with a relatively short elimination time. 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and Annexin V staining collectively showed that SalA-4g inhibited neuronal viability loss and apoptosis in a concentration-dependent manner in an oxygen and glucose deprivation model. Fluorine-18-fluorodeoxyglucose PET/CT imaging indicated that SalA-4g improved metabolic recovery in the ischemic hemisphere in a rat middle cerebral artery occlusion model. Our findings provide further evidence of the potential therapeutic applications of SalA-4g for the treatment of cerebral ischemic injury.

Abstract 40: Biocompatible, Aminocaproic Acid Stabilized Ceria Nanoparticles Rescue the Injured Brain After Subarachnoid Hemorrhage

Background and aims: Despite early aneurysm repair and aggressive management for complications, subarachnoid hemorrhage (SAH) results in at least 25% mortality rate and 50% persistent neurologic deficit. Thus, a novel therapeutic agent that directly targets brain damages after SAH is strongly required. For this purpose, we developed aminocaproic acid stabilized ceria nanoparticles (Amicar-CeNPs), which have incomparable anti-oxidative activity and biocompatibility. Methods: Uniform water-dispersed Amicar-CeNPs were prepared from short sol-gel reaction of cerium (III) ions in aqueous phase with aminocaproic acid. SAH was induced by endovascular perforation of middle cerebral artery of rats. A single dose of Amicar-CeNPs (0.5mg/kg) or saline control were randomly administered intravenously at an hour post-SAH. Neuronal death, macrophage infiltration, SAH grade and brain water content were evaluated at 72 hours. Mortality and neurologic function were assessed for 14 days. Results: Amicar-CeNPs, 3-nm in size and with high Ce 3+ to Ce 4+ ratio, demonstrated very potent anti-oxidative, cytoprotective, and anti-inflammatory activities in vitro. In rodent SAH model, Amicar-CeNPs significantly reduced neuronal death (1.1% vs. 90.1%; P<0.01), macrophage infiltration (74 vs. 188; P<0.01) and brain water content (80.2% vs. 80.7%; P<0.05) 72 hours post-SAH as compared with the saline control. SAH grades were comparable between the two groups (13.1±1.7 vs. 12.3±2.6). Amicar-CeNPs reduced mortality (11% vs. 79%; P<0.01) and improved neurological functions (median 9 vs. 3; modified Garcia score; P<0.01) post-SAH. Conclusions: Amicar-CeNPs, totally synthesized in aqueous phase, demonstrated very promising results against SAH via potent anti-oxidative, neuroprotective and anti-inflammatory activities. Given the obvious limitations of current therapies for SAH, Amicar-CeNPs are strongly required to be tested in SAH patients as an investigational new drug.

Cerebral Artery Vasoconstriction is Endothelin-1 Dependent Requiring Neurogenic and Adrenergic Crosstalk

The regulation of cerebral arterial vasomotor tone involves several mechanisms. The role of sympathetic nerves and the adrenergic neurotransmitter, noradrenaline (NA), has been the subject of debate for decades. Moreover, the specific role of endothelin-1 (ET-1) in cerebral arterial vasoconstriction has not been elucidated to date. In this study, we evaluated the contribution of NA and ET-1 to cerebral artery vasoconstriction. Arterial responses of rat middle cerebral arteries, and human pial cerebral arteries to cumulative concentrations of NA and ET-1, and to Electrical Field Stimulation (EFS), were evaluated. To assess the role of NA and ET-1 when EFS was applied, experiments were performed in the presence of adrenergic, neurogenic, and endothelin-1 receptor modulators. We found that vasoconstriction of cerebral arteries following EFS requires the application of exogenous NA, whereas neither EFS nor NA alone induced vasoconstriction. The observed vasoconstriction was abolished by α-adrenoreceptor antagonist, catecholamine-release inhibitor, blockade of the perivascular neurons, and by the endothelin-2 receptor antagonist (BQ123). Based on our results, cerebral artery vasoconstriction requires simultaneous neurogenic and adrenergic activation and is ET-1 dependent. We hypothesize that NA modulates the release of ET-1. Upon release, ET-1 binds to the ETA-receptor on smooth muscle cells inducing cerebral artery vasoconstriction.

MicroRNA-130a regulates cerebral ischemia-induced blood-brain barrier permeability by targeting Homeobox A5.

Blood-brain barrier (BBB) disruption plays a critical role in brain injury induced by cerebral ischemia, and preserving BBB integrity during ischemia could alleviate cerebral injury. We examined the role of miR-130a in ischemic BBB disruption by using models of rat middle cerebral artery occlusion and cell oxygen-glucose deprivation. We found that ischemia significantly increased microRNA-130a (miR-130a) level and that miR-130a was predominantly from brain microvascular endothelial cells. Antagomir-130a, an antagonist of miR-130a, could attenuate brain edema, lower BBB permeability, reduce infarct volume, and improve neurologic function. MiR-130a overexpression induced by miR-130a mimic increased monolayer permeability, and intercellular inhibition of miR-130a by a miR-130a inhibitor suppressed oxygen-glucose deprivation-induced increase in monolayer permeability. Moreover, dual luciferase reporter system showed that Homeobox A5 was the direct target of miR-130a. MiR-130a, by inhibiting Homeobox A5 expression, could down-regulate occludin, thereby increasing BBB permeability. Our results suggested that miR-130a might be implicated in ischemia-induced BBB dysfunction and serve as a target for the treatment of ischemic stroke.-Wang, Y., Wang, M.-D., Xia, Y.-P., Gao, Y., Zhu, Y.-Y., Chen, S.-C., Mao, L., He, Q.-W., Yue, Z.-Y., Hu, B. MicroRNA-130a regulates cerebral ischemia-induced blood-brain barrier permeability by targeting Homeobox A5.

An experimental evaluation of the therapeutic window of the neuroprotective activity of a low-molecular nerve growth factor mimetic GK-2

To identify the time interval for the preservation of the effect of GK-2 depending on the start of administration after modeling ischemic stroke by the transient occlusion of the middle cerebral artery in rats. The experiments were performed on 33 wild-type male rats and 81 male Wistar rats. Animals were kept in standard conditions. Ischemic stroke was modelled by thread occlusion of the middle cerebral artery. It was found that GK-2 at a daily dose of 1 mg/kg, intraperitoneally, during 7 days statistically significantly reduces brain infarct volume by 20-60% at the first injection from 4 to 24h, with the highest effect 6-8 hours after surgery. Thus, the 'therapeutic window' of GK-2 detected in the experiment is no less than 24 hours, which exceeds the existing neuroprotective agents.

MiR-24 reduces serum lipid levels and inhibits brain tissue cells apoptosis of rats with cerebral infarction.

To study miR-24 effects on cerebral infarction in rats. A rat middle cerebral artery occlusion model (MCAO) was constructed. Intracerebroventricular stereotactic injection of miR-24 agomir/antagomir was performed in the rat MCAO model. According to different experiences, rats were divided into normal, sham, MCAO, miR-24 agomir and miR-24 antagomir groups. Serum TCH, HDL and TG levels were detected. Comparing the normal and sham groups, we observed decreased relative miR-24 expression (P 0.05) and increased cerebral infarction area percentage, apoptotic cells and relative caspase-3 protein expression (P 0.05) in the MCAO, miR-24 agomir and miR-24 antagomir groups. TC, TG and HDL-C levels of the MCAO and miR-24 antagomir groups were higher than those of normal and sham groups (P 0.05). Compared with the MCAO group, increased relative miR-24 expression (P 0.05) and decreased TC, TG and HDL-C levels, cerebral infarction area percentage, number of apoptotic cells and caspase-3 expression (P 0.05) were found in the miR-24 agomir group, contrasting with the observations from the miR-24 antagomir group. miR-24 reduced serum TCH, HDL and TG levels and inhibited brain tissue cell apoptosis in rats with cerebral infarction.

MiR-24 Reduces Serum Lipid Levels and Inhibits Brain Tissue Cells Apoptosis of Rats with Cerebral Infarction.

To study miR-24 effects on cerebral infarction in rats. A rat middle cerebral artery occlusion model (MCAO) was constructed. Intracerebroventricular stereotactic injection of miR-24 agomir/antagomir was performed in the rat MCAO model. According to different experiences, rats were divided into normal, sham, MCAO, miR-24 agomir and miR-24 antagomir groups. Serum TCH, HDL and TG levels were detected. Comparingthe normal and sham groups, we observed decreased relative miR-24 expression (p < 0.05) and increased cerebral infarction area percentage, apoptotic cells and relative caspase-3 protein expression (p < 0.05) in theMCAO, miR-24 agomirand miR-24 antagomir groups. TC, TG and HDL-C levels of the MCAO and miR-24 antagomir groups were higher than those of normal and sham groups (p < 0.05).Compared with the MCAO group, increased relative miR-24 expression (p < 0.05) and decreased TC, TG and HDL-C levels,cerebral infarction area percentage, number of apoptotic cells and caspase-3 expression (p < 0.05) were found in themiR-24 agomir group, contrasting with theobservations from the miR-24 antagomir group. miR-24 reduced serum TCH, HDL and TG levels and inhibited brain tissue cell apoptosis in rats with cerebral infarction.

Catgut implantation at acupoints increases the expression of glutamate aspartate transporter and glial glutamate transporter-1 in the brain of rats with spasticity after stroke

Catgut implantation at acupoints has been shown to alleviate spasticity after stroke in rats. However, the underlying mechanisms are poorly understood. In this study, we used the rat middle cerebral artery occlusion model of stroke. Three days after surgery, absorbable surgical catgut sutures were implanted at Dazhui (GV14), Jizhong (GV6), Houhui, Guanyuan (CV4) and Zhongwan (CV12). The Zea Longa score was used to assess neurological function. The Modified Ashworth Scale was used to evaluate muscle tension. The 2,3,5-triphenyl-tetrazolium chloride assay was used to measure infarct volume. Immunohistochemical staining was performed for glutamate aspartate transporter (GLAST) and glial glutamate transporter-1 (GLT-1) expression. Western blot assay was used to analyze the expression of GLAST and GLT-1. Reverse transcription and polymerase chain reaction were carried out to assess the expression of GLAST and GLT-1 mRNAs. After catgut implantation at the acupoints, neurological function was substantially improved, muscle tension was decreased, and infarct volume was reduced in rats with spasticity after stroke. Furthermore, the expression of GLAST and GLT-1 mRNAs was increased on the injured (left) side. Our findings demonstrate that catgut implantation at acupoints alleviates spasticity after stroke, likely by increasing the expression of GLAST and GLT-1.

Proangiogenic functions of an RGD-SLAY-containing osteopontin icosamer peptide in HUVECs and in the postischemic brain

Osteopontin (OPN) is a phosphorylated glycoprotein secreted into body fluids by various cell types. OPN contains arginine-glycine-aspartate (RGD) and serine-leucine-alanine-tyrosine (SLAY) motifs that bind to several integrins and mediate a wide range of cellular processes. In the present study, the proangiogenic effects of a 20-amino-acid OPN peptide (OPNpt20) containing RGD and SLAY motifs were examined in human umbilical vein endothelial cells (HUVECs) and in a rat focal cerebral ischemia model. OPNpt20 exerted robust proangiogenic effects in HUVECs by promoting proliferation, migration and tube formation. These effects were significantly reduced in OPNpt20-RAA (RGD->RAA)-treated cells, but only slightly reduced in OPNpt20-SLAA (SLAY->SLAA)-treated cells. Interestingly, a mutant peptide without both motifs failed to induce these proangiogenic processes, indicating that the RGD motif is crucial and that SLAY also has a role. In OPNpt20-treated HUVEC cultures, AKT and ERK signaling pathways were activated, but activation of these pathways and tube formation were suppressed by anti-αvβ3 antibody, indicating that OPNpt20 stimulates angiogenesis via the αvβ3-integrin/AKT and ERK pathways. The proangiogenic function of OPNpt20 was further confirmed in a rat middle cerebral artery occlusion model. Total vessel length and vessel densities were markedly greater in OPNpt20-treated ischemic brains, accompanied by induction of proangiogenic markers. Together, these results demonstrate that the 20-amino-acid OPN peptide containing RGD and SLAY motifs exerts proangiogenic effects, wherein both motifs have important roles, and these effects appear to contribute to the neuroprotective effects of this peptide in the postischemic brain.

Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mTOR-ULK1 signaling pathway.

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPK-mTOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-mTOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, mTOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin (50 or 100 mg/kg) reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and pS317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-mTOR and pS757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-mTOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.

Involvement of brain-gut axis in treatment of cerebral infarction by β-asaron and paeonol

Cerebral infarction (CI) causes severe brain damage with high incidence. This study aimed to investigate the involvement of brain-gut axis in the treatment of CI by combined administration of β-asaron and paeonol. Rat middle cerebral artery occlusion (MCAO) model was established, the interleukin-1beta (IL-1β) and tumor necrosis factor α (TNF-α) in the rat peripheral blood were determined by ELISA assay, and brain tissue damage was evaluated by TUNNEL assay. The correlation of cholecystokinin (CCK) and nuclear factor-kappaB (NF-κB) signaling components between intestinal mucosa and prefrontal cortex of MCAO rats treated with β-asaron and paeonol were analyzed by quantitative RT-PCR and western blotting. In vitro transwell co-culture was performed to confirm the correlated expression. The expression of CCK and NF-κB signaling components were closely correlated between the intestinal mucosa and prefrontal cortex of MCAO rats treated with β-asaron and paeonol. The combined administration also regulates the IL-1β and TNF-α in the MCAO rat peripheral blood and ameliorate the brain damage in MCAO rats. Elevated expression of related genes was observed in the cortical neurons co-cultured with intestinal mucosal epithelial cells treated by β-asaron and paeonol. The brain-gut axis mediates the therapeutic effect of β-asaron and paeonol for cerebral infarction through CCK and NF-κB signaling.

Myricetin against ischemic cerebral injury in rat middle cerebral artery occlusion model.

The purpose of the present study was to examine the effects of myricetin on reducing cerebral ischemia injury in a rat model. A rat model of permanent middle cerebral artery occlusion (pMCAO) was used in the present study. Rats were randomized into the following five groups: Sham, model, low‑myricetin (1mg/kg), medium‑myricetin (5mg/kg) and high‑myricetin (25mg/kg) groups. Neurological deficit scores were evaluated by an examiner blinded to the experimental groups. Brain infarct size was estimated macroscopically using 2,3,5‑triphenyltetrazolium chloride staining. The levels of inflammatory factors tumor necrosis factor (TNF)‑α, interleukin (IL)‑6 and IL‑1β, and oxidative stress index superoxide dismutase (SOD), malondiadehyde (MDA), and the glutathione/glutathione disulfide (GSH/GSSG) ratio were measured by ELISA. The degree of brain cell apoptosis was determined using a terminal deoxynucleotidyl transferase dUTP nick‑end labeling assay. Protein expression levels of total or phosphorylated p38 mitogen activated protein kinase (MAPK), nuclear factor (NF)‑κB/p65 and protein kinase B (AKT) were determined using a western blotting assay. The neurological deficit score and infarct area induced by pMCAO decreased in a dose‑dependent manner following myricetin treatment. Furthermore, myricetin reduced the expression levels of IL‑1β, IL‑6, TNF‑α, and MDA, and increased GSH/GSSG ratio and SOD activity. A significant decrease in cell apoptosis was observed in response to myricetin. In addition, myricetin significantly increased the level of phosphorylated AKT protein, and decreased the phosphorylation of p38 MAPK and the level of NF‑κB/p65. Overall, the results of the present study suggested that myricetin exhibits a therapeutic effect by reducing ischemic cerebral injury, and the protective effect of myricetin may be associated with the p38 MAPK, NF‑κB/p65 and AKT signaling pathways.

TSLP/TSLPR promote angiogenesis following ischemic stroke via activation of the PI3K/AKT pathway.

The current study aimed to investigate the effects of the thymic stromal lymphopoietin (TSLP)/TSLP receptor (TSLPR) on angiogenesis following ischemic stroke invivo and invitro. Furthermore, whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway mediates the effects of TSLP/TSLPR on angiogenesis was explored. A rat middle cerebral artery occlusion (MCAO) model was established, and it was demonstrated that the expression levels of TSLP and TSLPR were significantly increased in the infarct area between 12 and 72h after MCAO, as determined by ELISA and western blot analyses. TSLP injection was revealed to upregulate vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang‑2) expression levels in the infarct area following MCAO, as determined by western blot analysis. An invitro MCAO model was constructed by exposing human umbilical vein endothelial cells (HUVECs) to oxygen‑glucose deprivation (OGD). It was revealed that the expression levels of TSLP and TSLPR were significantly increased in HUVECs subjected to OGD. TSLP treatment was revealed to induce invitro angiogenesis by promoting cell proliferation and migration, and increasing tube length of OGD‑treated HUVECs, as determined by MTT, Transwell‑migration and tube formation assays, respectively. Furthermore, it was demonstrated that the PI3K/AKT pathway was activated by TSLP treatment. However, it was revealed that PI3K inhibitor, LY294002, could attenuate the effects of TSLP on invitro angiogenesis of OGD‑treated HUVECs. In conclusion, to the best of our knowledge, this study demonstrated for the first time that TSLP/TSLPR promote angiogenesis following ischemic stroke in vivo and invitro. Furthermore, it was demonstrated that the effects of TSLP/TSLPR on angiogenesis were, at least partially, mediated via activation of the PI3K/AKT pathway. TSLP/TSLPR may serve as a potential therapeutic target for ischemic stroke treatment.

Near-infrared diffuse reflectance signals for monitoring spreading depolarizations and progression of the lesion in a male rat focal cerebral ischemia model.

In ischemic stroke research, a better understanding of the pathophysiology and development of neuroprotection methods are crucial, for which in vivo imaging to monitor spreading depolarizations (SDs) and evolution of tissue damage is desired. Since these events are accompanied by cellular morphological changes, light-scattering signals, which are sensitive to cellular and subcellular morphology, can be used for monitoring them. In this study, we performed transcranial imaging of near-infrared (NIR) diffuse reflectance at ∼800 nm, which sensitively reflects light-scattering change, and examined how NIR reflectance is correlated with simultaneously measured cerebral blood flow (CBF) for a rat middle cerebral artery occlusion (MCAO) model. After MCAO, wavelike NIR reflectance changes indicating occurrence of SDs were generated and propagated around the ischemic core for ∼90 min, during which time NIR reflectance increased not only within the ischemic core but also in the peripheral region. The area with increased reflectance expanded with increase in the number of SD occurrences, the correlation coefficient being 0.7686 (n = 5). The area with increased reflectance had become infarcted at 24 hr after MCAO. The infarct region was found to be associated with hypoperfusion or no-flow response to SD, but hyperemia or hypoperfusion followed by hyperemia response to SD was also observed, and the regional heterogeneity seemed to be connected with the rat cerebrovasculature and hence existence/absence of collateral flow. The results suggest that NIR reflectance signals depicted early evolution of tissue damage, which was not seen by CBF changes, and enabled lesion progression monitoring in the present stroke model.

Neuroprotective and Anti-inflammatory Effects of a Dodecamer Peptide Harboring Ninjurin 1 Cell Adhesion Motif in the Postischemic Brain.

It has been reported that the innate immune response plays important roles in brain ischemia and that the infiltration of blood-derived immune cells is a key initiator of this response. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is a cell adhesion molecule responsible for cell-to-cell interactions between immune cells and endothelial cells. In the present study, we investigated the proinflammatory and neuroprotective effects of Ninj1 and a dodecamer peptide harboring Ninj1 N-terminal adhesion motif (N-NAM, Pro26~Asn37) in a rat middle cerebral artery occlusion (MCAO) model of stroke. Ninj1 was predominantly induced in neutrophils and endothelial cells in the ischemic hemispheres around 12h to 1day post-MCAO, which coincided with a massive neutrophil influx. We demonstrated that intranasal administration of Ninj1 small interfering RNA (siRNA) or N-NAM significantly blocked neutrophil infiltration in postischemic brains. In addition, intranasal administration of Ninj1 siRNA or N-NAM reduced the mean infarct volume to 46.5±9.2 or 30.6 ± 11.7% of that of the PBS-treated MCAO controls, respectively, which was accompanied by significant amelioration of neurological and motor deficits. We showed that N-NAM or Ninj1 siRNA effectively blocked the adhesion and transendothelial migration of TNF-α-stimulated human myelocytic leukemia cells to human umbilical vein endothelial cells and similarly suppressed adhesion and migration of monocytes. Activations of phosphoinositide 3-kinase and Ras-related C3 botulinum toxin substrate 1 are involved in these Ninj1-mediated processes and can be inhibited by N-NAM or Ninj1 siRNA. These results indicate that Ninj1 plays an important role in neutrophil infiltration in the postischemic brain and N-NAM confers robust neuroprotective and anti-inflammatory effects by inhibiting Ninj1-mediated infiltration of neutrophils.

RTN1-C mediates cerebral ischemia/reperfusion injury via ER stress and mitochondria-associated apoptosis pathways

The reticulon family has been found to induce apoptosis, inhibit axon regeneration and regulate protein trafficking. However, little is known about the mechanisms of how reticulon proteins are involved in neuronal death-promoting processes during ischemia. Here, we report that the expression of Reticulon Protein 1-C (RTN1-C) was associated with the progression of cerebral ischemia/reperfusion (I/R) injury. Using a combination of rat middle cerebral artery occlusion (MCAO) stroke and oxygen-glucose deprivation followed by reoxygenation (OGD/R) models, we determined that the expression of RTN1-C was significantly increased during cerebral ischemic/reperfusion. RTN1-C overexpression induced apoptosis and increased the cell vulnerability to ischemic injury, whereas RTN1-C knockdown reversed ischemia-induced apoptosis and attenuated the vulnerability of OGD/R-treated neural cells. Mechanistically, we demonstrated that RTN1-C mediated OGD/R-induced apoptosis through ER stress and mitochondria-associated pathways. RTN1-C interacted with Bcl-xL and increased its localization in the ER, thus reducing the anti-apoptotic activity of Bcl-xL. Most importantly, knockdown of Rtn1-c expression in vivo attenuated apoptosis in MCAO rats and reduced the extent of I/R-induced brain injury, as assessed by infarct volume and neurological score. Collectively, these data support for the first time that RTN1-C may represent a novel candidate for therapies against cerebral ischemia/reperfusion injury.

(−)-Phenserine inhibits neuronal apoptosis following ischemia/reperfusion injury

Stroke commonly leads to adult disability and death worldwide. Its major symptoms are spastic hemiplegia and discordant motion, consequent to neuronal cell death induced by brain vessel occlusion. Acetylcholinesterase (AChE) is upregulated and allied with inflammation and apoptosis after stroke. Recent studies suggest that AChE inhibition ameliorates ischemia-reperfusion injury and has neuroprotective properties. (−)-Phenserine, a reversible AChE inhibitor, has a broad range of actions independent of its AChE properties, including neuroprotective ones. However, its protective effects and detailed mechanism of action in the rat middle cerebral artery occlusion model (MCAO) remain to be elucidated. This study investigated the therapeutic effects of (−)-phenserine for stroke in the rat focal cerebral ischemia model and oxygen-glucose deprivation/reperfusion (OGD/RP) damage model in SH-SY5Y neuronal cultures. (−)-Phenserine mitigated OGD/PR-induced SH-SY5Y cell death, providing an inverted U-shaped dose-response relationship between concentration and survival. In MCAO challenged rats, (−)-phenserine reduced infarction volume, cell death and improved body asymmetry, a behavioral measure of stoke impact. In both cellular and animal studies, (−)-phenserine elevated brain-derived neurotrophic factor (BDNF) and B-cell lymphoma 2 (Bcl-2) levels, and decreased activated-caspase 3, amyloid precursor protein (APP) and glial fibrillary acidic protein (GFAP) expression, potentially mediated through the ERK-1/2 signaling pathway. These actions mitigated neuronal apoptosis in the stroke penumbra, and decreased matrix metallopeptidase-9 (MMP-9) expression. In synopsis, (−)-phenserine significantly reduced neuronal damage induced by ischemia/reperfusion injury in a rat model of MCAO and cellular model of OGD/RP, demonstrating that its anti-apoptotic/neuroprotective/neurotrophic cholinergic and non-cholinergic properties warrant further evaluation in conditions of brain injury.