Inflammasome Regulation at the Level of Cellular and Molecular Function affect by Recombinant Tissue Plasminogen Activator and Exosomes Treatment in Ischemic Stroke Animal Model

In the current research, neuroprotection and performance improvement have been investigated in MCAO animal model treated with exosomes combined with rt-PA. Middle cerebral artery occlusion (MCAO) was induced in 25 adult male Wistar rats. Rats received rt-PA with a dose of 100 µg/kg in a single dose or drived-exosome from bone marrow MSCs. The Garcia scoring system and elevated body test were employed as behavioral tests for the functional recovery assessment. Cresyl violet staining was applied to evaluate the cell death degree in brain tissues. Immunohistochemical analysis was performed for the detection of GFAP and IBA1-positive cells. Results Cresyl violet staining revealed that the population of dark cells was significantly reduced in all treatment groups. A considerable increase (P ≤ 0.05) in catalase enzyme was observed in the combination therapy group compared whit the MCAO group (P ≤ 0.05). The amount of GPX despite the increase in all treatment groups compared to MCAO group was not statistically significant (P ≥ 0.05). Our conclusion was approved by the Nlrp1 and Nlrp3 downregulation during combination therapy in MCAO model by reduction in cell death rate. The Density of GFAP-positive cells Showed a decrease in the exosome with or without rt-PA experimental groups in comparison with the MCAO group (P < 0.05). Our observation indicated that exosomes, in combination with rt-PA, resulted in the noticeable functional recovery, neuronal regeneration, and reduction of neuronal cell death after a 7-day period of the MCAO induction. This novel therapeutic strategy could provide a better treatment option for those patients suffered from stroke.

Comparison of age‐dependent alterations in thioredoxin 2 and thioredoxin reductase 2 expressions in hippocampi between mice and rats

Background Aging is one of major causes triggering neurophysiological changes in many brain substructures, including the hippocampus, which has a major role in learning and memory. Thioredoxin (Trx) is a class of small redox proteins. Among the Trx family, Trx2 plays an important role in the regulation of mitochondrial membrane potential and is controlled by TrxR2. Hitherto, age-dependent alterations in Trx2 and TrxR2 in aged hippocampi have been poorly investigated. Therefore, the aim of this study was to examine changes in Trx2 and TrxR2 in mouse and rat hippocampi by age and to compare their differences between mice and rats. Results Trx2 and TrxR2 levels using Western blots in mice were the highest at young age and gradually reduced with time, showing that no significant differences in the levels were found between the two subfields. In rats, however, their expression levels were the lowest at young age and gradually increased with time. Nevertheless, there were no differences in cellular distribution and morphology in their hippocampi when it was observed by cresyl violet staining. In addition, both Trx2 and TrxR2 immunoreactivities in the CA1-3 fields were mainly shown in pyramidal cells (principal cells), showing that their immunoreactivities were altered like changes in their protein levels. Conclusions Our current findings suggest that Trx2 and TrxR2 expressions in the brain may be different according to brain regions, age and species. Therefore, further studies are needed to examine the reasons of the differences of Trx2 and TrxR2 expressions in the hippocampus between mice and rats.

Abstract 64: Scar-Forming Glial Cells Engulf Synapse and Worsen Stroke Outcome Through Megf10 and Mertk

Objective: Previous studies showed that microglia and astrocytes actively engulf synapse through MEGF10 and MERTK in the developing brain. But whether microglia and astrocytes in ischemic stroke (IS) or hemorrhagic stroke (HS) mice are still phagocytotic and how they affect the outcome of stroke remain unclear. Methods: Microglia or astrocyte-specific MEGF10 or MERTK knockout mice were generated, and subjected to transient middle cerebral artery occlusion (tMCAO), or collagenase-induced intracerebral hemorrhage. Immunostaining, Transmission electron microscope and Western blot were used to evaluate synapse engulfment, and dendritic spines were quantified using Golgi staining. Cresyl violet staining was used to evaluate atrophy volume and lateral ventricle enlargement of stroke mice. Neurobehavioral test including mNSS , rotarod, grid-walking and smart cage were performed to assess neurofunctional recovery. Single-cell RNA sequencing was further conducted to compare gene expression between IS and HS mice. Results: We showed that 14 days after IS, 25% of microglia and 50% of astrocytes were phagocytotic and engulfed synapses in the glial scar. However, in HS, 25% of microglia and 5% of astrocytes were phagocytotic, and only microglia were found to engulf synapses. We further demonstrated that MEGF10 and MERTK were upregulated in both astrocytes and microglia of mice subjected to IS and HS, and knockout of MEGF10 or MERTK in astrocytes or microglia inhibited synapse engulfment, which further increased synaptic density, reduced brain atrophy volume and improved neurobehavioral recovery after IS. However, knockout of MEGF10 or MERTK in microglia but not astrocytes in HS mice resulted in increased synaptic density and neurobehavioral recovery improvement. Single-cell RNA sequencing revealed that phagocytosis-related processes were downregulated in a subtype of astrocytes in HS brain than that in IS brain. Conclusion: Our study revealed a previously unknown role for astrocytes and microglia in engulfing synapse in the glial scar in stroke brain, and showed phagocytic astrocytes and microglia contribute differently to brain function recovery of mice that subjected to IS and HS, opening a new avenue to develop effective strategy for stroke treatment.

Effect of Low Intensity Magnetic Field Stimulation on Calcium-Mediated Cytotoxicity After Mild Spinal Cord Contusion Injury in Rats

Background: Magnetic field (MF) stimulation has the potential to reduce secondary damage and promote functional recovery after neural tissue injury. The study aimed to observe the effect of very low intensity (17.96µT) MF on general body condition, secondary damage, pain status, and locomotion. Methods: We exposed rats to MF (2 h/day × 3 weeks) after 6.25 mm contusion spinal injury. Locomotor behavior was evaluated by BBB score, pain assessment was done by recording threshold for tail flick, expression of voltage-gated calcium channels and extent of secondary damage in the spinal cord was assessed by immunofluorescence and Cresyl violet staining, respectively. Results: A significant ( p ≤ .001) improvement in bladder function as well as BBB score was observed after MF exposure in comparison with sham and SCI over the observation period of 3 weeks. SCI group showed an increase in the threshold for vocalization after discharge, which decreased following MF exposure. Cresyl violet staining showed significantly higher tissue sparing (73%) at the epicenter after MF exposure when compared to SCI group. This was accompanied with a significant decrease in calcium channel expression in MF group as compared to SCI. Conclusion: The results suggest facilitation of sensory-motor recovery after MF exposure, which could be due to attenuation of secondary damage and calcium-mediated excitotoxicity in a mild contusion rat model of SCI.

YES-10, A Combination of Extracts from Clematis mandshurica RUPR. and Erigeron annuus (L.) PERS., Prevents Ischemic Brain Injury in A Gerbil Model of Transient Forebrain Ischemia

Clematis mandshurica RUPR. (CMR) and Erigeron annuus (L.) PERS. (EALP) have pharmacological effects including anti-inflammatory activity and been used in traditional medicines in Asia. However, neuroprotective effects of CMR and/or EALP extracts against brain ischemic insults have never been addressed. Thus, the aim of this study was to examine neuroprotective effects of YES-10, a combination of extracts from CMR and EALP (combination ratio, 1:1), in the hippocampus following ischemia/reperfusion in gerbils. Protection of neurons was investigated by cresyl violet staining, fluoro-jade B histofluorescence staining and immunohistochemistry for neuronal nuclei. In addition, attenuation of gliosis was studied by immunohistochemistry for astrocytic and microglial markers. Treatments with 50 or 100 mg/kg YES-10 failed to protect neurons in the hippocampus after ischemia/reperfusion injury. However, administration of 200 mg/kg YES-10 protected neurons from ischemia/reperfusion injury and attenuated reactive gliosis. These findings strongly suggest that a combination of extracts from CMR and EALP can be used as a prevention approach/drug against brain ischemic damage.

Memory Impairment Allied to Temporal Lobe Epilepsy and its Deterioration by Phenytoin: A Highlight on Ameliorative Effects of Levetiracetam in Mouse Model

Background/Objectives Memory impairment (MI) and epilepsy go hand in hand, mainly in conditions of temporal lobe epilepsy (TLE). This disease comorbidity has been reported to worsen upon treatment. Hence this study aims to evaluate the extent of aggravating effect of phenytoin (PHT) at normal and reduced doses on MI associated with TLE and additionally assesses the protective effect of levetiracetam (LEV) on these adverse effects. Methods Swiss albino mice of either sex (n = 36) were used for this study in which seizures were induced by intraperitoneal administration of pilocarpine (300 mg/kg i.p.) followed by evaluation of antiepileptic activity by technique of Racine's scale for convulsive scores. Errors (a factor denoting MI) were assessed using radial arm maze. Finally brain biochemical measures of acetylcholinesterase and glutamate along with cresyl violet staining and estimation of total neuronal number of the hippocampus were performed. Results Exacerbation of MI by PHT was observed, where the extent of MI was found to be lesser in the reduced dose approach (PHTR: 28.50 ± 1.03; p ≤ 0.05). However, this tactic in dose reduction was interfered with the antiepileptic potential of the drug. Attenuation of MI upon combining with LEV, without an interference in the principal treatment, was observed equally in the behavioral and brain aspects of the study (PHTN + LEV: 1.33 ± 0.33 and PHTR + LEV: 1.00 ± 0.17; p ≤ 0.05). Conclusions The promising effects of LEV could thus aid in proposing a new management remedy for TLE to minimize the adverse effect associated with it.

Prenatal Curcumin Administration Reverses Behavioral and Neurochemical Effects and Decreases iNOS and COX-2 Expressions in Ischemic Rat Pups

The objectives were to evaluate alterations in ischemic rat pups, from dams administered with curcumin (25 and 50 mg/kg). Ten-day-old male pups were subjected or not (SO) to brain ischemia and reperfusion, for 1, 7, and 14 days (this last group was submitted to behavioral evaluation). After that, pups were euthanized for determinations of striatal DA and DOPAC in SO, ischemic from untreated (ICUD) or curcumin treated (ICTD) dams, as well as hippocampal immunohistochemistry assays for iNOS and COX-2 and cresyl violet staining. At the 14th postischemia day, the ICUD group showed increased locomotor activity and rearing behavior, which were reversed in ICTD animals. ICUD pups presented decreased striatal DA and DOPAC levels, relatively to SO, mainly at the 1st postischemia day, but also at the 7th and 14th days which were partially reversed in ICTD pups. A greater number of viable neurons were observed in ICTD, as related to the ICUD group. Ischemia increased iNOS and COX-2 expressions, in CA1 and CA3 areas, at the 1st, 7th, and 14th postischemia days, and these effects were minimized in ICTD pups. In conclusion, the prenatal curcumin treatment was shown to be neuroprotective where the drug anti-inflammatory and antioxidant effects probably play a role.

Neuronal nitric oxide synthase expression in resected epileptic dysplastic neocortex

Object Nitric oxide has been associated with epileptogenesis. Previous studies have shown increased expression of N-methyl-d-aspartate (NMDA) subunit NR2B receptors in epileptic dysplastic human neocortex. The expression of neuronal nitric oxide synthase (nNOS), and its relation to this subunit NR2B in epileptic dysplastic tissue has never been addressed. Methods Ten patients with medically intractable epilepsy caused by focal cortical dysplasia (CD), and 2 patients with mesial temporal sclerosis (control group) underwent pre- and/or intraoperative invasive monitoring evaluations. Cortical samples from epileptogenic and nonepileptogenic areas were collected from each patient intraoperatively. Samples were processed for cresyl violet staining, immunocytochemical tests with nNOS, NeuN, and NR2B, and immunofluorescence analyses to evaluate colocalized immunoreactivity between nNOS and NR2B. Results . All samples obtained in the patients with epilepsy revealed CD in various degrees. In the nonepileptic sample group, cresyl violet staining revealed normal cortical architecture in 9 samples, but a mild degree of CD in 3. The density and intensity of nNOS-stained neurons was remarkably increased in the epileptic tissue compared with nonepileptic samples (p < 0.05). Two types of nNOS-stained neurons were identified: Type I, expressing strong nNOS immunoreactivity in larger neurons; and Type II, expressing weak nNOS immunoreactivity in slightly smaller neurons. Different from Type I neurons, Type II nNOS-stained neurons revealed immunoreactivity colocalized with NR2B antibody. Conclusions The overexpression of nNOS in the epileptic samples and the immunoreactivity colocalization between nNOS and NR2B may suggest a possible role of nNOS and NO in the pathophysiological mechanisms related to in situ epileptogenicity.