Rat Model Of Experimental Stroke Research Articles

Hemorrhagic transformation in patients with large-artery atherosclerotic stroke is associated with the gut microbiota and lipopolysaccharide.

Abstract JOURNAL/nrgr/04.03/01300535-202407000-00032/figure1/v/2023-11-20T171125Z/r/image-tiff Hemorrhagic transformation is a major complication of large-artery atherosclerotic stroke (a major ischemic stroke subtype) that worsens outcomes and increases mortality. Disruption of the gut microbiota is an important feature of stroke, and some specific bacteria and bacterial metabolites may contribute to hemorrhagic transformation pathogenesis. We aimed to investigate the relationship between the gut microbiota and hemorrhagic transformation in large-artery atherosclerotic stroke. An observational retrospective study was conducted. From May 2020 to September 2021, blood and fecal samples were obtained upon admission from 32 patients with first-ever acute ischemic stroke and not undergoing intravenous thrombolysis or endovascular thrombectomy, as well as 16 healthy controls. Patients with stroke who developed hemorrhagic transformation (n = 15) were compared to those who did not develop hemorrhagic transformation (n = 17) and with healthy controls. The gut microbiota was assessed through 16S ribosomal ribonucleic acid sequencing. We also examined key components of the lipopolysaccharide pathway: lipopolysaccharide, lipopolysaccharide-binding protein, and soluble CD14. We observed that bacterial diversity was decreased in both the hemorrhagic transformation and non-hemorrhagic transformation group compared with the healthy controls. The patients with ischemic stroke who developed hemorrhagic transformation exhibited altered gut microbiota composition, in particular an increase in the relative abundance and diversity of members belonging to the Enterobacteriaceae family. Plasma lipopolysaccharide and lipopolysaccharide-binding protein levels were higher in the hemorrhagic transformation group compared with the non-hemorrhagic transformation group. lipopolysaccharide, lipopolysaccharide-binding protein, and soluble CD14 concentrations were associated with increased abundance of Enterobacteriaceae. Next, the role of the gut microbiota in hemorrhagic transformation was evaluated using an experimental stroke rat model. In this model, transplantation of the gut microbiota from hemorrhagic transformation rats into the recipient rats triggered higher plasma levels of lipopolysaccharide, lipopolysaccharide-binding protein, and soluble CD14. Taken together, our findings demonstrate a noticeable change in the gut microbiota and lipopolysaccharide-related inflammatory response in stroke patients with hemorrhagic transformation. This suggests that maintaining a balanced gut microbiota may be an important factor in preventing hemorrhagic transformation after stroke.

Agrin Involvement in Synaptogenesis Induced by Exercise in a Rat Model of Experimental Stroke

Background Agrin is a proteoglycan that aggregates nicotinic acetylcholine receptors (AChRs) on neuromuscular junctions and takes part in synaptogenesis in the development of the central nervous system. However, its effects on neural repair and synaptogenesis after stroke are still unclear. Objective This study aimed to investigate the effects of agrin on neural repair and synaptogenesis after stroke and the effects of exercise on this process in vivo and in vitro. Methods Exercise with gradually increased intensity was initiated at 1 day after middle cerebral artery occlusion (MCAO) for a maximum of 14 days. Neurological deficit scores and foot fault tests were used to assess the behavioral recovery. Western blotting, immunofluorescence, and electron microscopic images were used to detect the expression of agrin, synaptogenesis-related proteins, and synaptic density in vivo. In vitro, the ischemic neuron model was established via oxygen-glucose deprivation (OGD). The lentivirus overexpressed agrin and CREB inhibitor were used to investigate the mechanism by which agrin promoted synaptogenesis. Results Exercise promoted behavioral recovery and this beneficial role was linked to the upregulated expression of agrin and increased synaptic density. Overexpressed agrin promoted synaptogenesis in OGD neuron, CREB inhibitor downregulated the expression of agrin and hampered synaptogenesis in cultured neurons. Conclusions These results indicated that exercise poststroke improved the recovery of behavioral function after stroke. Synaptogenesis was an important and beneficial factor, and agrin played a critical role in this process and could be a potential therapeutic target for the treatment of stroke and other nervous system diseases.

Exosomes from human urine-derived stem cells enhanced neurogenesis via miR-26a/HDAC6 axis after ischaemic stroke.

Endogenous neurogenesis holds promise for brain repair and long‐term functional recovery after ischaemic stroke. However, the effects of exosomes from human urine‐derived stem cells (USC‐Exos) in neurogenesis remain unclear. This study aimed to investigate whether USC‐Exos enhanced neurogenesis and promoted functional recovery in brain ischaemia. By using an experimental stroke rat model, we found that intravenous injection of USC‐Exos enhanced neurogenesis and alleviated neurological deficits in post‐ischaemic stroke rats. We used neural stem cells (NSCs) subjected to oxygen‐glucose deprivation/reoxygenation (OGD/R) as an in vitro model of ischaemic stroke. The in vitro results suggested that USC‐Exos promoted both proliferation and neuronal differentiation of NSCs after OGD/R. Notably, a further mechanism study revealed that the pro‐neurogenesis effects of USC‐Exos may be partially attributed to histone deacetylase 6 (HDAC6) inhibition via the transfer of exosomal microRNA‐26a (miR‐26a). Taken together, this study indicates that USC‐Exos can be used as a novel promising strategy for brain ischaemia, which highlights the application of USC‐Exos.

Astragaloside VI Promotes Neural Stem Cell Proliferation and Enhances Neurological Function Recovery in Transient Cerebral Ischemic Injuryvia Activating EGFR/MAPK Signaling Cascades.

Radix Astragali (AR) is a commonly used medicinal herb for post-stroke disability in Traditional Chinese Medicine but its active compounds for promoting neurogenic effects are largely unknown. In the present study, we tested the hypothesis that Astragaloside VI could be a promising active compound from AR for adult neurogenesis and brain repair via targeting epidermal growth factor (EGF)-mediated MAPK signaling pathway in post-stroke treatment. By using cultured neural stem cells (NSCs) and experimental stroke rat model, we investigated the effects of Astragaloside VI on inducing NSCs proliferation and self-renewal in vitro, and enhancing neurogenesis for the recovery of the neurological functions in post-ischemic brains in vivo. For animal experiments, rats were undergone 1.5h middle cerebral artery occlusion (MCAO) plus 7days reperfusion. Astragaloside VI (2μg/kg) was daily administrated by intravenous injection (i.v.) for 7days. Astragaloside VI treatment promoted neurogenesis and astrogenic formation in dentate gyrus zone, subventricular zone, and cortex of the transient ischemic rat brains in vivo. Astragaloside VI treatment enhanced NSCs self-renewal and proliferation in the cultured NSCs in vitro without affecting NSCs differentiation. Western blot analysis showed that Astragaloside VI up-regulated the expression of nestin, p-EGFR and p-MAPK, and increased neurosphere sizes, whose effects were abolished by the co-treatment of EGF receptor inhibitor gefitinib and ERK inhibitor PD98059. Behavior tests revealed that Astragaloside VI promoted the spatial learning and memory and improved the impaired motor function in transient cerebral ischemic rats. Taken together, Astragaloside VI could effectively activate EGFR/MAPK signaling cascades, promote NSCs proliferation and neurogenesis in transient cerebral ischemic brains, and improve the repair of neurological functions in post-ischemic stroke rats. Astragaloside VI could be a new therapeutic drug candidate for post-stroke treatment.

GlyT1 Inhibitor NFPS Exerts Neuroprotection via GlyR Alpha1 Subunit in the Rat Model of Transient Focal Cerebral Ischaemia and Reperfusion

Background/Aims: Glycine is a strychnine-sensitive inhibitory neurotransmitter in the central nervous system (CNS), especially in the spinal cord, brainstem, and retina. The objective of the present study was to investigate the potential neuroprotective effects of GlyT1 inhibitor N [3-(4'-fluorophenyl)-3-(4'-phenylphenoxy) propyl] sarcosine (NFPS) in the rat model of experimental stroke. Methods: In vivo ischaemia was induced by transient middle cerebral artery occlusion (tMCAO). The methods of Western Blotting, Nissl Staining and Morris water maze methods were applied to analyze the anti-ischaemia mechanism. Results: The results showed that high dose of NFPS (H-NFPS) significantly reduced infarct volume, neuronal injury and the expression of cleaved caspase-3, enhanced Bcl-2/Bax, and improved spatial learning deficits which were administered three hours after transient middle cerebral artery occlusion (tMCAO) induction in rats, while, low dose of NFPS (L-NFPS) exacerbated the injury of ischaemia. These findings suggested that low and high dose of NFPS produced opposite effects. Importantly, it was demonstrated that H-NFPS-dependent neuronal protection was inverted by salicylate (Sal), a specific GlyR ɑ1 antagonist. Such effects could probably be attributed to the enhanced glycine level in both synaptic and extrasynaptic clefts and the subsequently altered extrasynaptic GlyRs and their subtypes. Conclusions: These data imply that GlyT1 inhibitor NFPS may be a novel target for clinical treatment of transient focal cerebral ischaemia and reperfusion which are associated with altered GlyR alpha 1 subunits.

Propofol Protects Against Focal Cerebral Ischemia via Inhibition of Microglia-Mediated Proinflammatory Cytokines in a Rat Model of Experimental Stroke

Ischemic stroke induces microglial activation and release of proinflammatory cytokines, contributing to the expansion of brain injury and poor clinical outcome. Propofol has been shown to ameliorate neuronal injury in a number of experimental studies, but the precise mechanisms involved in its neuroprotective effects remain unclear. We tested the hypothesis that propofol confers neuroprotection against focal ischemia by inhibiting microglia-mediated inflammatory response in a rat model of ischemic stroke. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. Propofol (50 mg/kg/h) or vehicle was infused intravenously at the onset of reperfusion for 30 minutes. In vehicle-treated rats, MCAO resulted in significant cerebral infarction, higher neurological deficit scores and decreased time on the rotarod compared with sham-operated rats. Propofol treatment reduced infarct volume and improved the neurological functions. In addition, molecular studies demonstrated that mRNA expression of microglial marker Cd68 and Emr1 was significantly increased, and mRNA and protein expressions of proinflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6 were augmented in the peri-infarct cortical regions of vehicle-treated rats 24 h after MCAO. Immunohistochemical study revealed that number of total microglia and proportion of activated microglia in the peri-infarct cortical regions were markedly elevated. All of these findings were ameliorated in propofol-treated rats. Furthermore, vehicle-treated rats had higher plasma levels of interleukin-6 and C-reactive protein 24 h after MCAO, which were decreased after treatment with propofol. These results suggest that propofol protects against focal cerebral ischemia via inhibition of microglia-mediated proinflammatory cytokines. Propofol may be a promising therapeutic agent for the treatment of ischemic stroke and other neurodegenerative diseases associated with microglial activation.

Early exercise improves cerebral blood flow through increased angiogenesis in experimental stroke rat model

BackgroundEarly exercise after stroke promoted angiogenesis and increased microvessles density. However, whether these newly formatted vessels indeed give rise to functional vascular and improve the cerebral blood flow (CBF) in impaired brain region is still unclear. The present study aimed to determine the effect of early exercise on angiogenesis and CBF in ischemic region.MethodsAdult male Sprague Dawley rats were subjected to 90 min middle cerebral artery occlusion(MCAO)and randomly divided into early exercise and non-exercised control group 24 h later. Two weeks later, CBF in ischemic region was determined by laser speckle flowmetry(LSF). Meantime, micro vessels density, the expression of tie-2, total Akt and phosphorylated Akt (p-Akt), and infarct volume were detected with immunohistochemistry, 2,3,5 triphenyltetrazolium chloride (TTC) staining and western blotting respectively. The function was evaluated by seven point’s method.ResultsOur results showed that CBF, vessel density and expression of Tie-2, p-Akt in ischemic region were higher in early exercise group compared with those in non-exercise group. Consistent with these results, rats in early exercise group had a significantly reduced infarct volume and better functional outcomes than those in non-exercise group.ConclusionsOur results indicated that early exercise after MCAO improved the CBF in ischemic region, reduced infarct volume and promoted the functional outcomes, the underlying mechanism was correlated with angiogenesis in the ischemic cortex.

The inhibitory effect of S‐nitrosoglutathione on blood–brain barrier disruption and peroxynitrite formation in a rat model of experimental stroke

The hallmark of stroke injury is endothelial dysfunction leading to blood-brain barrier (BBB) leakage and edema. Among the causative factors of BBB disruption are accelerating peroxynitrite formation and the resultant decreased bioavailability of nitric oxide (NO). S-nitrosoglutathione (GSNO), an S-nitrosylating agent, was found not only to reduce the levels of peroxynitrite but also to protect the integrity of BBB in a rat model of cerebral ischemia and reperfusion (IR). A treatment with GSNO (3 μmol/kg) after IR reduced 3-nitrotyrosine levels in and around vessels and maintained NO levels in brain. This mechanism protected endothelial function by reducing BBB leakage, increasing the expression of Zonula occludens-1 (ZO-1), decreasing edema, and reducing the expression of matrix metalloproteinase-9 and E-selectin in the neurovascular unit. An administration of the peroxynitrite-forming agent 3-morpholino sydnonimine (3 μmol/kg) at reperfusion increased BBB leakage and decreased the expression of ZO-1, supporting the involvement of peroxynitrite in BBB disruption and edema. Mechanistically, the endothelium-protecting action of GSNO was invoked by reducing the activity of nuclear factor kappa B and increasing the expression of S-nitrosylated proteins. Taken together, the results support the ability of GSNO to improve endothelial function by reducing nitroxidative stress in stroke.

Abstract 2741: S-Nitrosoglutathione Increases Benefit of Motor Exercise on Functional Recovery and Stimulates Neurorepair Mechanisms Following Experimental Stroke in Rats

Background: Stroke is the leading cause of long-term physical disability and life-long neurologic deficits. The disability stems from acute neurovascular injury and injury-induced compromised neuroplasticity. This neuroplasticity can be restored by stimulating neurotrophic factors via two possible modalities: rehabilitation activity and neurorepair therapy. Improvement of neurologic function has been achieved following brain trauma by the neurovascular protective agent S-nitrosoglutathione (GSNO). Therefore, we investigated whether GSNO stimulates the expression neurotrophic factors and enhances the benefits of motor exercise, leading to functional recovery in a rat model of experimental stroke. Methods: Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) for 60 min followed by reperfusion in adult male rats. Injured animals were either treated with vehicle (IR group), GSNO (0.25 mg/kg, GSNO group) or underwent rotarod exercise (EX group). In the third treatment group, GSNO was combined with rotarod exercise (GSNO+EX group). The groups were compared in terms of brain infarction, neurological score, motor function, cell death, tissue structure, and the expression of the neurotrophic factors BDNF, TrKB, and myelin. Results: All three treated groups (GSNO, EX and GSNO+EX) showed reduced infarction, improved motor and neurological functions, and decreased apoptotic neuronal cell death compared to the IR group. However, the combination group GSNO+EX showed a trend toward greater recovery than the GSNO or EX group alone. All the three treated groups also showed enhanced expression of neurotrophic factors and improved tissue histology. A delayed intervention (24 h after IR) by GSNO also aided the functional recovery. Furthermore, the protective effect of GSNO and exercise was blunted in another set of animals by an inhibition of AKT activity using the PI3 kinase inhibitor LY 294002 compound. Conclusion: GSNO, like exercise, aids recovery of functions in a long-term treatment by stimulating the expression of neurotrophic factors, reducing infarctions, and decreasing cell death. A combination of exercise and GSNO shows a greater degree of improvement in neurobehavioral function. Clinical relevance of the therapy is supported by an improved functional recovery even when GSNO was administered 24 h following IR. Mechanistically, the improvements by GSNO and exercise are dependent, at least in part, on AKT activity.

Brain Repair by Hematopoietic Growth Factors in a Rat Model of Stroke

Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are essential growth factors in hematopoiesis. We determined whether receptors for SCF and G-CSF exist in the brain and whether exogenous SCF and G-CSF are beneficial to brain repair after brain ischemia. A well-established rat model of experimental stroke was used in this study. SCF, G-CSF, SCF+G-CSF, or saline was subcutaneously administered 3 hours to 7 days after brain ischemia. Bromodeoxyuridine was administered simultaneously. Sensorimotor function was evaluated with a limb placement test and foot fault test over time. We observed that receptors for SCF and G-CSF were expressed in both neurogenic regions and neurons. SCF-treated rats showed the best functional restoration at 1 week that was maintained 4, 7, and 10 weeks after the final injection. G-CSF-induced functional recovery was limited and unstable. Interestingly, stable but delayed functional improvement was seen in SCF+G-CSF-treated rats. Infarction size was significantly reduced in all growth factor-treated rats. In addition, SCF and SCF+G-CSF enhanced neural progenitor cell proliferation in the subventricular zone bilaterally, whereas G-CSF and SCF+G-CSF treatment increased bromodeoxyuridine -positive cells in periinfarct areas. SCF and G-CSF are neuroprotective and beneficial to functional restoration when administered during the acute phase after brain ischemia, indicating hematopoietic growth factors play a role in brain repair.

Inhibition of NF-κB activation by 5-lipoxygenase inhibitors protects brain against injury in a rat model of focal cerebral ischemia

BackgroundStroke is one of the leading causes of death worldwide and a major cause of morbidity and mortality in the United States of America. Brain ischemia-reperfusion (IR) triggers a complex series of biochemical events including inflammation. Leukotrienes derived from 5-lipoxygenase (5-LOX) cause inflammation and are thus involved in the pathobiology of stroke injury.MethodsTo test the neuroprotective efficacy of 5-LOX inhibition in a rat model of focal cerebral IR, ischemic animals were either pre- or post-treated with a potent selective 5-LOX inhibitor, (N- [3-[3-(-fluorophenoxy) phenyl]-1-methyl-2-propenyl]-N-hydroxyurea (BW-B 70C). They were evaluated at 24 h after reperfusion for brain infarction, neurological deficit score, and the expression of 5-LOX. Furthermore, the mechanism and the anti-inflammatory potential of BW-B 70C in the regulation of nuclear factor kappa B (NF-κB) and inflammatory inducible nitric oxide synthase (iNOS) were investigated both in vivo and in vitro.Results and discussionBoth pre- and post-treatment with BW-B 70C reduced infarctions and improved neurological deficit scores. Immunohistochemical study of brain sections showed IR-mediated increased expression of 5-LOX in the neurons and microglia. BW-B 70C down-regulated 5-LOX and inhibited iNOS expression by preventing NF-κB activation. Two other structurally different 5-LOX inhibitors were also administered post IR: caffeic acid and 2, 3, 5-trimethyl-6- [12-hydroxy-5, 10-dodecadiynyl]-1, 4-benzoquinone (AA-861). As with BW-B 70C, they provided remarkable neuroprotection. Furthermore, in vitro, BW-B 70C inhibited lipopolysaccharide (LPS) mediated nitric oxide production, iNOS induction and NF-κB activation in the BV2 microglial cell line. Treating rat primary microglia with BW-B70C confirmed blockage of LPS-mediated translocation of the p65 subunit of NF-κB from cytosol to nucleus.ConclusionThe study demonstrates the neuroprotective potential of 5-LOX inhibition through down-regulation of NF-κB in a rat model of experimental stroke.

Cerebrovascular protection by various nitric oxide donors in rats after experimental stroke

The efficacy of nitric oxide (NO) treatment in ischemic stroke, though well recognized, is yet to be tested in clinic. NO donors used to treat ischemic injury are structurally diverse compounds. We have shown that treatment of S-nitrosoglutathione (GSNO) protects the brain against injury and inflammation in rats after experimental stroke [M. Khan, B. Sekhon, S. Giri, M. Jatana, A. G. Gilg, K. Ayasolla, C. Elango, A. K. Singh, I. Singh, S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke, J. Cereb. Blood Flow Metab. 25 (2005) 177-192.]. In this study, we tested structurally different NO donors including GSNO, S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP), methylamine hexamethylene methylamine NONOate (MAHMA), propylamine propylamine NONOate (PAPA), 3-morpholinosydnonimine (SIN-1) and compared their neuroprotective efficacy and antioxidant property in rats after ischemia/reperfusion (I/R). GSNO, in addition to neuroprotection, decreased nitrotyrosine formation and lipid peroxidation in blood and increased the ratio of reduced versus oxidized glutathione (GSH/GSSG) in brain as compared to untreated animals. GSNO also prevented the I/R-induced increase in mRNA expression of ICAM-1 and E-Selectin. SNAP and SNP extended limited neuroprotection, reduced nitrotyrosine formation in blood and blocked increase in mRNA expression of ICAM-1 and E-Selectin in brain tissue. PAPA, MAHMA, and SIN-1 neither protected the brain nor reduced oxidative stress. We conclude that neuroprotective action of NO donors in experimental stroke depends on their ability to reduce oxidative stress both in brain and blood.

Neuroprotective action of halogenated derivatives of L-phenylalanine.

The aromatic amino acid L-Phenylalanine (L-Phe) significantly and reversibly depresses excitatory glutamatergic synaptic transmission (GST) via a unique set of presynaptic and postsynaptic mechanisms. Therefore, we hypothesized that endogenous derivatives of L-Phe, which display potent antiglutamatergic activity, may safely and efficaciously protect the brain during conditions characterized by overactivation of glutamate receptors. We tested this hypothesis in vitro with a combination of patch-clamp and lactate dehydrogenase (LDH) analyses in rat cultured neurons exposed to simulated ischemia, and in vivo using a rat model of experimental stroke caused by transient middle cerebral artery occlusion (MCAO). 3,5-diiodo-L-tyrosine (DIT) and 3,5-dibromo-L-tyrosine (DBrT), endogenous halogenated derivatives of L-Phe, attenuated GST by similar mechanisms as L-Phe, but with greater potency. For example, the IC50s for DIT and DBrT to depress the frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor-mediated mEPSCs were 104.6+/-14.1 micromol/L and 127.5+/-13.3 micromol/L, respectively. Depression of GST by DIT and DBrT persisted during energy deprivation. Furthermore, DBrT significantly reduced LDH release in neuronal cultures exposed to oxygen glucose deprivation. In rats subjected to transient MCAO, DBrT decreased the brain infarct volume and neurological deficit score to 52.7+/-14.1% and 57.1+/-12.0% of control values, respectively. DBrT neither altered atrioventricular nodal and intraventricular conduction in isolated heart, nor heart rate and blood pressure in vivo. DBrT, an endogenous halogenated derivative of L-Phe, shows promise as a representative of a novel class of neuroprotective agents by exerting significant neuroprotection in both in vitro and in vivo models of brain ischemia.

Bile acids: from genomics to disease and therapyEdited by G. Paumgartner, D. Keppler, U. Leuschner, and A. Stiehl. 320 pp. $181.00. Kluwer Academic Publishers: the Netherlands, 2003. ISBN 0-79238-781-3. Web address for ordering: www.wkap.nl

Bile acids: from genomics to disease and therapyEdited by G. Paumgartner, D. Keppler, U. Leuschner, and A. Stiehl. 320 pp. $181.00. Kluwer Academic Publishers: the Netherlands, 2003. ISBN 0-79238-781-3. Web address for ordering: www.wkap.nl