Rat Transient Middle Cerebral Artery Research Articles

Edaravone Dexborneol protects against blood-brain barrier disruption following cerebral ischemia/reperfusion by upregulating pericyte coverage via vitronectin-integrin and PDGFB/PDGFR-β signaling

BackgroundRecent advancements in brain cytoprotection therapies following cerebral ischemia-reperfusion (I/R) injury have become an emerging interest. Pericytes were vulnerable during the early stages of ischemia. This study aims to explore the protective effects of Edaravone borneol (Eda.B) on pericyte loss, as well as and the underlying mechanisms, given its potential in alleviating I/R injury. Methods: The rat transient middle cerebral artery occlusion (tMCAO) model was established. Rats were randomly divided into Sham group (Sham, n = 24), tMCAO group (tMCAO, n = 24), Edaravone group (Eda, n = 24), Dexborneol group (Dexborneol, n = 24), and Eda.B group (Eda.B, n = 24). Neurological function recovery, infarct volume, and blood-brain barrier (BBB) disruption were assessed using Zea-Longa scoring, TTC staining, and Evans Blue extravasation, respectively. Alterations in Basement membrane (BM) and pericyte coverage were assessed by transmission electron microscopy (TEM). The expression levels of pericyte marker NG2 and PDGFR-β in the ischemic region, as well as BBB transcellular transport-related proteins vitronectin (VTN), α5 and PDGFB were detected by western blotting. Furthermore, a specific inhibitor of PDGFB, MOR8457, was employed (Eda.B + MOR8457, n = 8) to explore the protective effects of Eda.B on pericyte injury via PDGFB/PDGFR-β. ResultsEda.B significantly reduced cerebral infarct volume and promoted neurological function recovery in comparison to the tMCAO, Eda and Dexborneol groups. Additionally, Eda.B significantly ameliorated BBB leakage, mitigated the decrease in pericyte coverage, and reduced vesicle density in endothelial cells and BM thickness following I/R. Mechanically, Eda.B inhibited the downregulation of NG2, PDGFB/PDGFR-β, VTN, while preventing upregulation of α5 protein expression in tMCAO rats. Blocking PDGFB with MOR8457 demonstrated that Eda.B improved pericyte loss and BBB permeability by activating PDGFB/PDGFR-β signaling. ConclusionsWe elucidated that vitronectin-integrin and PDGFB/PDGFR-β signaling contributed to Eda.B's protective effects against pericyte loss and BBB permeability following I/R injury, unraveling new insights into mechanisms of pericyte as a promising therapeutic target.

Cell-Free Supernatant of Lactobacillus rhamnosus and Bifidobacterium breve Ameliorates Ischemic Stroke-Generated Neurological Deficits in Rats.

The beneficial effects of probiotics, postbiotics, and paraprobiotics have already been registered in managing ischemic stroke-generated neuroinflammation and gut dysbiosis. Herein, we examined the impact of cell-free supernatant (CFS) obtained from probiotics (Lactobacillus rhamnosus UBLR-58 and Bifidobacterium breve UBBr-01) in a rat transient middle cerebral artery occlusion (MCAO) model of focal cerebral injury. Pre-MCAO supplementation of probiotics (2 × 109CFU/mL) for 21days or CFS (1mL/rat) for 7days protect the MCAO-induced somatosensory and motor impairments recorded at 24h and 72h after reperfusion in foot-fault, rotarod, adhesive removal, and vibrissae-evoked forelimb placing tests. We also noted the reduced infarct area and neuronal degradation in the right hemisphere of probiotics- and CFS-recipient MCAO-operated animals. Moreover, MCAO-induced altered concentrations of glial-fibrillary acidic protein, NeuN, zonula occludens-1 (ZO-1), TLR4, IL-1β, IL-6, and TNF-α, as well as matrix metalloproteinase-9 (MMP9) were reversed in the treatment groups. Probiotics and CFS treatment ameliorated the elevated levels of IL-6, IL-1β, and MMP9 in the blood plasma of rats. The disrupted microbial phyla, Firmicutes-to-Bacteroides ratio, villi/crypt ratio, and decreased mucin-producing goblet cells, ZO-1, and occludin in the colon of MCAO-operated rats were recovered following probiotics and CFS treatment. NMR characterization of CFS and rat blood plasma revealed the presence of several important bacterial metabolites. These findings suggest that the CFS obtained from Lactobacillus rhamnosus UBLR-58 and Bifidobacterium breve UBBr-01 has the propensity to improve MCAO-generated neurological dysfunctions in rats by dampening neuroinflammation and modulating the gut-brain axis modulators.

FDCA Attenuates Neuroinflammation and Brain Injury after Cerebral Ischemic Stroke.

Ischemic stroke is a deleterious cerebrovascular disease with few therapeutic options, and its functional recovery is highly associated with the integrity of the blood-brain barrier and neuroinflammation. The Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor fasudil (F) and the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) have been demonstrated to exhibit neuroprotection in a series of neurological disorders. Hence, we synthesized and biologically examined the new salt fasudil dichloroacetate (FDCA) and validated that FDCA was eligible for attenuating ischemic volume and neurological deficits in the rat transient middle cerebral artery occlusion (tMCAO) model. Additionally, FDCA exerted superior effects than fasudil and dichloroacetate alone or in combination in reducing cerebral ischemic injury. Particularly, FDCA could maintain the blood-brain barrier (BBB) integrity by inhibiting matrix metalloproteinase 9 (MMP-9) protein expression and the degradation of zonula occludens (ZO-1) and Occludin protein. Meanwhile, FDCA could mitigate the neuroinflammation induced by microglia. The in vivo and in vitro experiments further demonstrated that FDCA disrupted the phosphorylations of myosin phosphatase target subunit 1 (MYPT1), mitogen-activated protein kinase (MAPK) cascade, including p38 and c-Jun N-terminal kinase (JNK), and pyruvate dehydrogenase (PDH) and limited excessive lactic acid metabolites, resulting in inhibition of BBB disruption and neuroinflammation. In addition, FDCA potently mitigated inflammatory response in human monocytes isolated from ischemic stroke patients, which provides the possibilities of a clinical translation perspective. Overall, these findings provided a therapeutic potential for FDCA as a candidate agent for ischemic stroke and other neurological diseases associated with BBB disruption and neuroinflammation.

Neuroprotective effects of a hemoglobin-based oxygen carrier (stroma-free hemoglobin nanoparticle) on ischemia reperfusion injury

The application of hemoglobin (Hb)-based oxygen carriers (HBOCs) to the treatment of cerebral ischemia has been investigated. A cluster of 1 Hb and 3 human serum albumins (Hb-HSA3) was found to exert neuroprotective effects on ischemia/reperfusion injury. Stroma-free hemoglobin nanoparticles (SFHbNP), a subsequently developed HBOC consisting of a spherical polymerized stroma-free Hb core with a HSA shell, contains the natural antioxidant enzyme catalase and, thus, is expected to exert additive effects. We herein investigated whether SFHbNP exerted enhanced neuroprotective effects in a rat transient middle cerebral artery occlusion (tMCAO) model. Rats were subjected to 2-hour tMCAO and divided into the following 3 groups with the intravenous administration of the respective reagents: (1) phosphate-buffered saline (PBS), as a vehicle (2) Hb-HSA3, and (3) SFHbNP. After 24-hour reperfusion, infarct and edema volumes decreased in the order of the PBS, Hb-HSA3, and SFHbNP groups, with a significant difference (p < 0.05) between the PBS and SFHbNP groups. Similar reductions were observed in oxidative stress, leukocyte recruitment, and blood–brain barrier disruption in the order of the PBS, Hb-HSA3, and SFHbNP groups. In the early phase of reperfusion within 6 h, microvascular HBOC perfusion and cerebral blood flow were maintained at high levels during the reperfusion period in the Hb-HSA3 and SFHbNP groups. However, a difference was observed in tissue oxygen partial pressure levels, which significantly decreased after 6-hour reperfusion in the Hb-HSA3 group, but remained high in the SFHbNP group. A superior oxygen transport ability appears to be related to the enhanced neuroprotective effects of SFHbNP.

Astragaloside IV alleviates stroke-triggered early brain injury by modulating neuroinflammation and ferroptosis via the Nrf2/HO-1 signaling pathway.

Stroke is an acute cerebrovascular disease. Astragaloside IV (AS-IV) is an active ingredient extracted from Astragalus membranaceus with an established therapeutic effect on central nervous system diseases. This study examined the neuroprotective properties and possible mechanisms of AS-IV in stroke-triggered early brain injury (EBI) in a rat transient middle cerebral artery occlusion (MCAO) model. The neurological scores and brain water content were analyzed. 2,3,5-triphenyl tetrazolium chloride (TTC) staining was utilized to determine the infarct volume, neuroinflammatory cytokine levels, and ferroptosis-related genes and proteins, and neuronal damage and molecular mechanisms were evaluated by terminal deoxynucleotidyl transferase dutp nick-end labeling (TUNEL) staining, western blotting, and real-time polymerase chain reaction. AS-IV administration decreased the infarct volume, brain edema, neurological deficits, and inflammatory cytokines TNF-α, interleukin-1β (IL-1β), IL-6, and NF-κB, increased the levels of SLC7A11 and glutathione peroxidase 4 (GPX4), decreased lipid reactive oxygen species (ROS) levels, and prevented neuronal ferroptosis. Meanwhile, AS-IV triggered the Nrf2/HO-1 signaling pathway and alleviated ferroptosis due to the induction of stroke. Hence, the findings of this research illustrate that AS-IV administration can improve delayed ischemic neurological deficits and decrease neuronal death by modulating nuroinflammation and ferroptosis via the Nrf2/HO-1 signaling pathway.

Bone Marrow-derived Mesenchymal Stem Cells Promote Microglia/Macrophage M2 Polarization and Enhance Neurogenesis in the Acute and Chronic Stages after Ischemic Stroke

• BMSCs promote microglia/macrophage M2 polarization in acute and chronic stages after stroke. • BMSCs enhance neurogenesis after cerebral ischemia/reperfusion. • The enhancement of neurogenesis by BMSCs could be associated with microglia M2 polarization. : Ischemic stroke has been regarded as a major cause of disability and death around the world due to limited effective therapies. Accumulating evidence have shown that although microglia are polarized to an anti-inflammatory M2 phenotype in the early stage of ischemia, they transform progressively into a proinflammatory M1 phenotype. Bone marrow-derived mesenchymal stem cells (BMSCs) may be used to treat ischemic injury through regulating the poststroke inflammatory response. However, the mechanism by which BMSCs can treat ischemic stroke remains unclarified. : This study aimed to investigate whether BMSCs shift M1-to-M2 phenotype transformation of microglia/macrophages and enhance neurogenesis in a rat transient middle cerebral artery occlusion (tMCAO) model. : Ninety-minute tMCAO was applied to the rats, followed by reperfusion. BMSCs were transplanted into the rats via intravenous injection at 24 h after tMCAO. After being randomly divided into the sham group, the MCAO group, and the BMSCs group, the rats’ behavior was assessed at 1, 3, 7, and 14 days following tMCAO. qRT-PCR, double-immunofluorescence staining, and Western blot were performed at 3 and 14 days after tMCAO to determine M1/M2 polarization of microglia/macrophages. Neurogenesis was examined by double-immunofluorescence staining at 14 days after tMCAO. Expression of brain-derived neurotrophic factor (BDNF) was measured on the protein level by immunofluorescence staining at 3 and 14 days after tMCAO. : We found that BMSCs treatment promoted the recovery of neurological function after tMCAO, inhibited the expression of TNF-α, iNOS and CD16/32, which are markers of M1 microglia/macrophage, and enhanced the expression of IL-10, TGF-β and CD206 that are markers of M2 microglia/macrophage. Moreover, BMSCs treatment promoted neurogenesis and M2-derived BDNF expression after tMCAO. : It is indicated by the results that BMSCs modulate neuroinflammation and enhance neurogenesis, which could be due to transforming microglia/macrophages from the M1 polarization state towards M2 in a rat tMCAO model.

GLP-1R Agonist Exendin-4 Protects Against Hemorrhagic Transformation Induced by rtPA After Ischemic Stroke via the Wnt/β-Catenin Signaling Pathway

Tissue plasminogen activator (tPA) is recommended by the FDA to dissolve intravascular clots after acute ischemic stroke (AIS). However, it may contribute to hemorrhagic transformation (HT). The Wnt/β-catenin signaling pathway plays an important role in regulating the blood–brain barrier (BBB) formation in the central nervous system. We explored whether glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (EX-4) reduces the risk of HT after rtPA treatment via the Wnt/β-catenin pathway by using a rat transient middle cerebral artery occlusion (MCAO) model in vivo and an oxygen–glucose deprivation plus reoxygenation (OGD/R) model in vitro. Our results showed that EX-4 attenuated neurological deficits, brain edema, infarct volume, BBB disruption, and rtPA-induced HT in ischemic stroke. EX-4 suppressed the production of ROS and the activation of MMP-9 to protect the integrity of the BBB by activating the Wnt/β-catenin signaling pathway. PRI-724, a selective inhibitor of β-catenin, was able to reverse the therapeutic effect of EX-4 in vivo and in vitro. Therefore, our results indicate that the GLP-1R agonist may be a potential therapeutic agent to decrease the risk of rtPA-induced HT after ischemic stroke via the Wnt/β-catenin signaling pathway.

Fingolimod protects against cerebral ischemia reperfusion injury in rats by reducing inflammatory cytokines and inhibiting the activation of p38 MAPK and NF-κB signaling pathways

Fingolimod (FTY720) is a sphingosine 1-phosphate (S1P) receptor agonist. Here, to understand biological activity of FTY720 pretreatment and post-treatment on cerebral ischemia reperfusion injury (CIRI), rat transient middle cerebral artery occlusion/reperfusion (tMCAO/R) model was generated. Neurological deficit scoring was assessed after tMCAO/R. Four groups were established including sham-operated control group, operated group, and two FTY720-treated groups. Neuron damage was observed by Nissl staining. Gene expression was measured using qPCR and western blot analysis. Tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) levels were evaluated by enzyme-linked immunosorbent assay (ELISA). We uncovered that neurological score in two FTY720-treated groups was significantly lower than that in the operated group. FTY720 pretreatment or posttreatment groups had a significantly increased number of Nissl bodies in cerebral cortex as compared with the operated group, indicating that FTY720 administration reduced neuronal damage. Besides, FTY720 posttreatment improved memory impairment induced by tMCAO/R. In addition, IL-1β, IL-6, and TNF-α levels in the cerebral cortex and hippocampus of two FTY720-treated groups were significantly decreased in comparison to the operated group, showing that FTY720 could reduce the release of inflammatory cytokines in brain tissue. Furthermore, phosphorylation of p38MAPK and NF-κB pathway-related molecules in ischemic brain tissues of FTY720 group were markedly down-regulated compared to the operated group. Together, FTY720 pretreatment or posttreatment improved the neurological deficit of middle cerebral ischemia/reperfusion rat model and reduced neuronal damage by decreasing the levels of inflammatory cytokines and attenuating the phosphorylation levels of p38MAPK and NF-κB pathway-associated molecules. FTY720 exhibits neuroprotective effects against ischemic reperfusion injury in rats.

Brain Protein Expression Profile Confirms the Protective Effect of the ACTH(4-7)PGP Peptide (Semax) in a Rat Model of Cerebral Ischemia-Reperfusion.

The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

Neuroprotective effects of combination therapy of regional cold perfusion and hemoglobin-based oxygen carrier administration on rat transient cerebral ischemia

Regional cold perfusion and hemoglobin-based oxygen carrier administration both exert neuroprotective effects against cerebral ischemia reperfusion injury. We herein investigated whether the combination of these two therapies leads to stronger neuroprotective effects. Combination therapy was performed with the regional perfusion of cold HemoAct, a core-shell structured hemoglobin-albumin cluster, in a rat transient middle cerebral artery occlusion model. The effects of combination therapy, the intra-arterial administration of 10 °C HemoAct (10H) initiated at the onset of reperfusion, were compared with those of monotherapies, the intra-arterial administration of 10 °C saline (10S) and 37 °C HemoAct (37H), and an untreated control under the condition of 2-hour ischemia/24-hour reperfusion. The durability of therapeutic effects and the therapeutic time window of combination therapy were assessed based on comparisons with the 10H and control groups. Significantly better neurological findings and smaller infarct volumes were observed in the three treated (10S, 37H, and 10H) groups than in the control group. Among the 3 treated groups, only the 10H group showed significant improvements over the control group in the other items examined, including cerebral blood flow reduction, brain edema, and protein extravasation. The significant therapeutic effects of combination therapy on neurological disabilities and infarct volumes were confirmed at least until 7 days after reperfusion. Furthermore, combination therapy ameliorated neurological disabilities and hemorrhagic transformation in rats subjected to 4- and 5-hour ischemia/24-hour reperfusion. Since therapeutic effects may be expected until at least 5 h of complete ischemia and reperfusion, this combination therapy is a promising neuroprotective strategy against severe ischemic stroke.

Neuroprotective effect of salvianolate on cerebral ischaemia-reperfusion injury in rats by inhibiting the Caspase-3 signal pathway

Salvianolate has been widely used for the treatment of cerebrovascular diseases. However, the detailed molecular mechanism of how it alleviates cerebral ischaemia-reperfusion injury is not well understood. In the present study, we investigated the neuroprotective effects of salvianolate in acute cerebral infarction using the PC12 cell oxygen-glucose deprivation (OGD) model in vitro and the rat transient middle cerebral artery occlusion (MCAO) model in vivo. The results showed that the salvianolate significantly reduced the level of reactive oxygen species and inhibited the Caspase-3 signalling pathway in vitro; at the same time, in vivo experiments showed that salvianolate obviously reduced the infarct area (12.9%) and repaired cognitive function compared with the model group (28.28%). In conclusion, our data demonstrated that the salvianolate effectively alleviated cerebral ischaemia-reperfusion injury via suppressing the Caspase-3 signalling pathway.

Cerebrolysin Ameliorates Focal Cerebral Ischemia Injury Through Neuroinflammatory Inhibition via CREB/PGC-1α Pathway.

Neuroinflammation is one of the important factors aggravating brain injury after ischemic stroke. We aimed to investigate the effects of cerebrolysin (CBL) on neuroinflammation in vivo and in vitro and the underlying mechanisms. The gene expressions of pro-inflammatory factors and anti-inflammatory factors were analyzed by real time PCR in rat transient middle cerebral artery occlusion (tMCAO) model, lipopolysaccharides-induced neuroinflammatory mice model and LPS-treated mouse primary microglia cells. The neuroprotective effects of CBL were evaluated by infarct size, Longa test and Rotarod test for long-term functional recovery in rats subjected to ischemia. The role of CREB/PGC-1α pathway in anti-neuroinflammatory effect of CBL was also determined by real time PCR and Western blotting. In the tMCAO model, administration of CBL at 3 h post-ischemia reduced infarct volume, promoted long-term functional recovery, decreased the gene expression of pro-inflammatory factors and increased the gene expression of anti-inflammatory factors. Correspondingly, in LPS-induced neuroinflammatory mice model, CBL treatment attenuated sickness behavior, decreased the gene expression of pro-inflammatory factors, and increased the gene expression of anti-inflammatory factors. In in vitro and in vivo experiments, CBL increased the protein expression levels of PGC-1α and phosphorylated CREB to play anti-inflammatory effect. Additionally, the application of the specific CREB inhibitor, 666-15 compound could effectively reverse the anti-inflammatory effect of CBL in primary mouse microglia cells and anti-ischemic brain injury of CBL in rats subjected to tMCAO. In conclusion, CBL ameliorated cerebral ischemia injury through reducing neuroinflammation partly via the activation of CREB/PGC-1α pathway and may play a therapeutic role as anti-neuroinflammatory agents in the brain disorders associated with neuroinflammation.

Orally Administered Crocin Protects Against Cerebral Ischemia/Reperfusion Injury Through the Metabolic Transformation of Crocetin by Gut Microbiota.

Our pilot study suggested that orally administered crocin was hardly absorbed into circulatory system, but it was effective against cerebral ischemic/reperfusion (I/R) injury. The pharmacologically active component and targeting site of crocin remain elusive. In this study, the cerebral-protective effect of crocin was evaluated on a rat transient middle cerebral artery occlusion (MCAO) model. Our data showed that oral administration of crocin had better effectiveness in cerebral protection than an intravenous injection. Neither crocin nor its metabolite crocetin were determined in the brain of cerebral I/R rats, indicating a target site of periphery. Abundant crocetin was detected in plasma after oral administration instead of intravenous injection of crocin. Meanwhile, orally administered crocetin showed similar cerebral protection to that of crocin, but this exciting effect was not clearly observed by intravenous administration of crocetin, indicating the importance of crocetin in gut. Moreover, orally administered crocin showed less cerebral-protective effect in pseudo germ-free (pGF) MCAO rats. In vitro and in vivo experiments confirmed that crocin could be deglycosylated to crocetin in gut content of normal rats, rather than that of pGF rats, indicating that gut microbiota facilitated the transformation of crocin into crocetin, which played a key role in the activation of the pharmacological effect. Metabolomic study revealed that microbial-host co-metabolic molecules were significantly perturbed after oral administration of crocin, indicating a regulation on intestinal ecosystem. It was further suggested that gut microbiota may be the potential target of the cerebral-protective effect of crocin.

From the Literature

From the Literature

Intra-arterial Drug Delivery to the Ischemic Brain in Rat Middle Cerebral Artery Occlusion Model.

Rat transient middle cerebral artery occlusion (tMCAO) model is one of the most commonly used animal models in ischemic stroke studies. In the model, increasing safety and efficacy of therapeutic agent administration, such as stem cells and drugs directly to the ischemic brain using the internal carotid artery (ICA) is essential, because using the common carotid artery (CCA) for injection can close CCA completely and cause many complications after tMCAO surgery. Also, the pterygopalatine artery (PPA) is an arterial branch of the ICA that supplies blood circulation of the external part of the brain and removing the blood circulation of the PPA is required for more complete induction of ischemia to the brain. Herein, we present the insertion of intra-arterial catheter in the ICA via the external carotid artery (ECA) after the PPA in rats subjected to tMCAO surgery.

AMPA Receptor Antagonist Perampanel Ameliorates Post-Stroke Functional and Cognitive Impairments

Perampanel (PER), a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist, clinically used for seizure control, has been reported to exert neuroprotective effects in experimental models of neurodegenerative diseases. However, few studies have investigated the therapeutic effects of PER in brain injury including stroke. Our aim was to investigate the neuroprotective potential of PER using a rat transient middle cerebral artery occlusion (MCAO) model. Sprague–Dawley rats underwent 90-min MCAO followed by intraperitoneal PER administration at a dose of 1.5 mg/kg. Infarct volumes, neurological deficits, and immunological analyses were performed at 7 days after MCAO. PER significantly reduced infarct volumes (p < 0.05) and improved motor function (p < 0.05) compared with vehicle. Immunological analysis showed that PER significantly inhibited microglial activation, pro-inflammatory cytokine expression, and oxidative stress compared with vehicle. Moreover, PER suppressed neurodegeneration in the cortical ischemic boundary zone, via downregulation of Bcl-2-associated x and upregulation of Bcl-extra-large with Akt activation. In addition, post-stroke secondary neuronal damage and cognitive impairments, using the Y-maze test, were assessed 30 days after MCAO. PER significantly improved spatial working memory, which was accompanied by hippocampal CA1 neuronal loss and cortical thinning, compared with vehicle. These results indicate that PER attenuates infarct volumes and motor function deficits possibly through its anti-inflammatory, antioxidant, and anti-apoptotic activities, mediated via activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathways in the acute ischemic phase, and further ameliorates post-stroke cognitive impairments via the suppression of secondary neuronal damage in the chronic ischemic phase.

Abstract TMP33: TXA302 Promotes Permanent Functional Recovery With Delivery Initiated Up to 4 Weeks After Stroke in Multiple Rodent Models

Introduction: TXA302 is a proprietary analogue of angiotensin 1-7 (Ang1-7). Previous studies have shown that intracerebroventricular administration of Ang1-7 as a pre-treatment to experimental occlusive rodent stroke resulted in improved neurological function, decreased infarct volume, enhanced cerebral blood flow and decreased inflammation. Here, systemic administration of TXA302 was evaluated with treatment delayed up to 4 weeks to assess the potential of TXA302 to promote recovery in chronic stroke. Methods and Results: Four independent studies were conducted in the rat transient (2 hour) and permanent middle cerebral artery occlusion (MCAO) models. Daily subcutaneous delivery of TXA302 or vehicle was started either 24 hours or 4 weeks after MCAO and continued for 4, 6 or 8 weeks. Treatment was started at 4 weeks to assess the ability of TXA302 to promote recovery independent of neuroprotection and to model the chronic phase of the injury. Two of the studies employed a dose range of TXA302 from 1 to 500 μg/kg and two studies had a washout period of 4 weeks after the completion of dosing to assess the permanence of the recovery effect. Sensorimotor recovery was measured by limb placing, stepping, and body swing tests along with composite neurological scoring. Cerebral blood flow (CBF) was evaluated by doppler ultrasonography. All studies were conducted in a blinded fashion and recovery of treated animals was compared to vehicle by repeated measures ANOVA with corrections for multiple comparisons. TXA302 treatment resulted in significant, robust and permanent dose-responsive improvements in neurological function and CBF compared to vehicle treated animals. Conclusions: The ability to delay treatment for 4 weeks suggests the changes are not attributable to neuroprotection. Furthermore, this wide therapeutic window enhances translational potential as clinical trials can be conducted without confounding factors of treating, stabilizing and assessing baseline functional measures in acute stroke patients. Ongoing studies are exploring the potential of TXA302 to enhance endogenous remodeling through angiogenesis and neural plasticity. Non-clinical studies and manufacturing development are underway to enable an IND for stroke recovery.

Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury.

Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxidative effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco's modified Eagle's medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen species in astrocytes subjected to ischemia-reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosin (3-NT) in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin improved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.

Inhibition of tPA-induced hemorrhagic transformation involves adenosine A2b receptor activation after cerebral ischemia

Tissue plasminogen activator (tPA) is administered after ischemic stroke to dissolve intravascular clots, but its use can lead to hemorrhagic transformation (HT). Therapeutic strategies to reduce hemorrhagic complications of tPA might be of benefit for stroke patients. Adenosine A2b receptor (A2bR) plays pivotal roles in regulating vascular protection in peripheral organs. This study explored whether A2bR agonist BAY 60-6583 reduces hemorrhage risk after tPA usage. Using a rat transient middle cerebral artery occlusion model, we showed that mRNA and protein expression of A2bR increased to a greater extent after ischemia-reperfusion than did expression of the other three adenosine receptors (A1, A2a, and A3). tPA administration reduced A2bR expression in ischemic brain microvessels. Post-treatment with BAY 60-6583 (1mg/kg) at the start of reperfusion reduced lesion volume in the absence or presence of tPA (10mg/kg) and attenuated brain swelling, blood-brain barrier disruption, and tPA-exacerbated HT at 24h. Additionally, BAY 60-6583 mitigated sensorimotor deficits in the presence of tPA. BAY 60-6583 inhibited tPA-enhanced matrix metalloprotease-9 activation, probably through elevation of tissue inhibitor of matrix metalloproteinases-1 expression, and thereby reduced degradation of tight junction proteins. These effects would likely protect cerebrovascular integrity. A2bR agonists as an adjuvant to tPA could be a promising strategy for decreasing the risk of HT during treatment for ischemic stroke.

Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke

Interleukin (IL)-10 is a contributing factor to neuroprotection of mesenchymal stem cell (MSC) transplantation after ischemic stroke. Our aim was to increase therapeutic effects by combining MSCs and ex vivo IL-10 gene transfer with an adeno-associated virus (AAV) vector using a rat transient middle cerebral artery occlusion (MCAO) model. Sprague-Dawley rats underwent 90 min MCAO followed by intravenous administration of MSCs alone or IL-10 gene-transferred MSCs (MSC/IL-10) at 0 or 3 hr after ischemia reperfusion. Infarct lesions, neurological deficits, and immunological analyses were performed within 7 days after MCAO. 0-hr transplantation of MSCs alone and MSC/IL-10 significantly reduced infarct volumes and improved motor function. Conversely, 3-hr transplantation of MSC/IL-10, but not MSCs alone, significantly reduced infarct volumes (p < 0.01) and improved motor function (p < 0.01) compared with vehicle groups at 72 hr and 7 days after MCAO. Immunological analysis showed that MSC/IL-10 transplantation significantly inhibits microglial activation and pro-inflammatory cytokine expression compared with MSCs alone. Moreover, overexpressing IL-10 suppressed neuronal degeneration and improved survival of engrafted MSCs in the ischemic hemisphere. These results suggest that overexpressing IL-10 enhances the neuroprotective effects of MSC transplantation by anti-inflammatory modulation and thereby supports neuronal survival during the acute ischemic phase.