Genetic Deletion of mGlu3 Metabotropic Glutamate Receptors Amplifies Ischemic Brain Damage and Associated Neuroinflammation in Mice

Backgroud: Type-3 metabotropic glutamate (mGlu3) receptors are found in both neurons and glial cells and regulate synaptic transmission, astrocyte function, and microglial reactivity. Here we show that the genetic deletion of mGlu3 receptors amplifies ischemic brain damage and associated neuroinflammation in adult mice. An increased infarct size was observed in mGlu3−/− mice of both CD1 and C57Black strains 24 h following a permanent occlusion of the middle cerebral artery (MCA) as compared to their respective wild-type (mGlu3+/+ mice) counterparts. Increases in the expression of selected pro-inflammatory genes including those encoding interleukin-1β, type-2 cycloxygenase, tumor necrosis factor-α, CD86, and interleukin-6 were more prominent in the peri-infarct region of mGlu3−/− mice. In contrast, the expression of two genes associated with the anti-inflammatory phenotype of microglia (those encoding the mannose-1-phosphate receptor and the α-subunit of interleukin-4 receptor) and the gene encoding the neuroprotective factor, glial cell line-derived neurotrophic factor, was enhanced in the peri-infarct region of wild-type mice, but not mGlu3−/− mice, following MCA occlusion. In C57Black mice, the genetic deletion of mGlu3 receptors worsened the defect in the paw placement test as assessed in the contralateral forepaw at short times (4 h) following MCA occlusion. These findings suggest that mGlu3 receptors are protective against ischemic brain damage and support the way to the use of selective mGlu3 receptor agonists or positive allosteric modulators in experimental animal models of ischemic stroke.

Vascular protection and regenerative effects of intranasal DL-3-N-butylphthalide treatment after ischaemic stroke in mice

ObjectiveTo investigate the effects of DL-3-N-butylphthalide (NBP) via intranasal delivery after ischaemic stroke in mice.MethodsC57BL/6 mice were divided into three groups: sham, stroke with vehicle and stroke with NBP treatment. Ischaemic stroke was induced by permanent ligation of right middle cerebral artery with 7 min common carotid artery occlusion. NBP (100 mg/kg) or vehicle was intranasally administered at 1 hour after stroke and repeated once a day until sacrifice. Bromodeoxyuridine (BrdU) (50 mg/kg/day) was given from the third day until sacrifice. Sensorimotor function was tested during 1–21 days after stroke. Local cerebral blood flow in the ischaemic and peri-infarct regions was measured using laser Doppler flowmetry before, during and 3 days after ischaemia. Expressions of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase as well as regenerative marker BrdU in the peri-infarct region were analysed by western blotting and immunohistochemical methods.ResultsCompared with the vehicle group, NBP treatment significantly increased the VEGF expression in the poststroke brain. Stroke mice that received NBP showed significantly less vascular damage after stroke and more new neurons and blood vessels in the peri-infarct region at 21 days after stroke. In the adhesive removal test, the sensorimotor function of stroke mice treated with NBP performed significantly better at 1, 3 and 7 days after stroke compared with vehicle controls.ConclusionDaily intranasal NBP treatment provides protective and neurogenic/angiogenic effects in the poststroke brain, accompanied with functional improvements after a focal ischaemic stroke in mice.

Aryl hydrocarbon receptor antagonism before reperfusion attenuates cerebral ischaemia/reperfusion injury in rats

Aryl hydrocarbon receptor (AhR) antagonism can mitigate cellular damage associated with cerebral ischaemia and reperfusion (I/R) injury. This study investigated the neuroprotective effects of AhR antagonist administration before reperfusion in a rat stroke model and influence of the timing of AhR antagonist administration on its neuroprotective effects. Magnetic resonance imaging (MRI) was performed at baseline, immediately after, and 3, 8, and 24 h after ischaemia in the sham, control (I/R injury), TMF10 (trimethoxyflavone [TMF] administered 10 min post-ischaemia), and TMF50 (TMF administered 50 min post-ischaemia) groups. The TMF treatment groups had significantly fewer infarcts than the control group. At 24 h, the relative apparent diffusion coefficient values of the ischaemic core and peri-infarct region were significantly higher and relative T2 values were significantly lower in the TMF10 groups than in the control group. The TMF treatment groups showed significantly fewer terminal deoxynucleotidyl transferase dUTP nick-end labelling positive (+) cells (%) in the peri-infarct region than the control group. This study demonstrated that TMF treatment 10 or 50 min after ischaemia alleviated brain damage. Furthermore, the timing of AhR antagonist administration affected the inhibition of cellular or vasogenic oedema formation caused by a transient ischaemic stroke.

Administration of hATSCs results in recovery of cerebral infarction animal model

To examine pathway of stem cell transplanted to the brain, stem cells were infected with flourescence. hATSCs in the infarct region were mostly located at the border between intact brain tissue and the area of the infarction and in other sections within the infarct cavity. Examination of section with flourescence indicated that there was significant gliosis or infiltration of leukocytes around the implantation site of the stem cell. Implanted stem cell integrated and migrated to multiple areas of the brain including the contrallateral cortex. The cells persisted in the sites to which they migrated at 30 days after implantation. The heaviest concentrations of cells were transplanted into rats at 24hr after MCAO, more cells were migrated into injured area of brain cortex. Stem cell in the infarct region were found at the border between intact brain tissue and the area of infarction and within the infarct cavity.

Epicortical Brevetoxin Treatment Promotes Neural Repair and Functional Recovery after Ischemic Stroke

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory–inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na+]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca2+) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

Abstract TP114: Histone Deacetylase Inhibitors Panobinostat and Entinostat for Motor Recovery After Ischemic Stroke in Mice

In this study, we investigated the potential of two histone deacetylase (HDAC) inhibitors, panobinostat and entinostat, to enhance recovery of motor function after ischemic stroke in CD-1 male mice. Panobinostat, which is a pan-HDAC inhibitor, is an FDA-approved drug for certain cancers, whereas entinostat is a class-I HDAC inhibitor and is widely expected to get approval for clinical use in near future. Stroke was induced by photothrombosis and the drugs were administered intraperitoneally every other day (panobinostat at 10 mg/kg or 30 mg/kg; entinostat at 5 mg/kg or 15 mg/kg) starting from day 5 to day 15 after stroke. The control group received only vehicle following the same regimen. In addition, all drug or vehicle-treated mice exercised for 2 hours (voluntary wheel running) starting from day 9 to day 41 after stroke. Additional control groups included sham-operated animals, and mice which had stroke but were not drug/vehicle-treated or allowed to run. Motor function of the mice was evaluated by blinded investigators using gridwalk and cylinder tests before and after stroke. Acetylation of histone 3 in the peri-infarct region of the brain samples was measured by immunoblotting. Our results indicate that lower dose of both panobinostat and entinostat marginally improved motor function in mice by day 42 after stroke, although it did not reach statistical significance. Notably, this improvement trend was lost with the higher dose of both drugs which also showed some toxicity. No statistical difference was observed in running distance of mice among experimental groups. Likewise, we did not observe statistically significant difference in stroke volumes among the experimental groups. Immunoblotting experiments indicated that both panobinostat and entinostat dose-dependently increased the level of acetylated histone in the cortical, peri-infarct region of drug-treated animals compared to the non-treated groups. In summary, our results indicate that both panobinostat and entinostat do not facilitate improvement of motor function after stroke at the tested doses. However, it is likely that lower doses of these drugs may enhance recovery of motor function after stroke.