Preconditioning exercise reduces brain damage of ischemic stroke in rats via PI3K-AKT pathway by bioinformatic analysis.

Ischemic stroke is one of the most vital causes of high neurological morbidity and mortality in the world. Preconditioning exercise is considered as the primary prevention of stroke to resistance to subsequent injury. We tried to research the underlying biological mechanisms of this exercise. Forty-two SD rats were randomly divided into three groups: middle cerebral artery occlusion (MCAO) group, exercise group with MCAO (EX + MCAO) group, and sham group, with 14 rats in each group. The EX + MCAO group underwent exercise preconditioning for 3 weeks before occlusion, and the other two groups were fed and exercised normally. After 3 weeks, MCAO model was made by thread plug method in the EX + MCAO group and MCAO group. After successful modeling, the Longa scale was used to evaluate the neurological impairment of rats at day 0, day 1, and day 2. The rats in each group were killed on the third day after modeling. TTC staining measured the infarct volume of each group. The morphology and apoptosis of cortical cells were observed by HE and Tunel staining. Three rats in each group underwent high-throughput sequencing. Bioinformatic analysis was used to find the deferentially expressed genes (DEGs) and predict the transcription factor binding sites (TFBS) of the next-generation sequencing results. Gene enrichment (GSEA) was used to analyze potential functional genes and their corresponding signaling pathways. The Longa scale showed EX + MCAO group had the neurological function better than the modeling group (P < 0.001). TTC staining showed that the infarct size of EX + MCAO group was less than MCAO group (P < 0.05). HE and Tunel staining showed that the cells in the EX + MCAO group and the sham group had normal morphology and fewer apoptotic cells than MCAO group. A new gene named 7994 was discovered and TFBS of this gene was predicted, which could interact with key genes such as Foxd3, Foxa2, NR4A2, SP1, CEBPA, and SOX10. GSEA showed that EX + MCAO group could promote and regulate angiogenesis and apoptosis through PI3K-AKT pathway. Preconditioning exercise could improve nerve function and reduce infarct size in rats. The underlying mechanism is to regulate the PI3K-AKT pathway through several key genes, promote cerebral angiogenesis, and reduce apoptosis.

Pre-ischaemic Treatment with Enriched Environment Alleviates Acute Neuronal Injury by Inhibiting Endoplasmic Reticulum Stress-dependent Autophagy and Apoptosis.

Enriched environment (EE) is effective in preventing cerebral ischemia-reperfusion (I/R) injury. However, little is known about the mechanism underlying the neuroprotection of EE preprocessing. Endoplasmic reticulum (ER) stress has been demonstrated to be extensively involved in I/R injury. We aimed to investigate the potential regulatory mechanism of ER stress in the neuroprotection of pre-ischemic EE. Rats were subjected to middle cerebral artery occlusion (MCAO) or sham surgery after 4 weeks of exposure in standard or enriched environments. We found that EE pretreatment alleviates acute neuronal injury after MCAO, as shown by reduced infarct volume and neurological deficit score. The expression of ER stress-related proteins, markers of autophagy, and apoptosis were detected to investigate the underlying mechanism. Our results showed that pre-ischemic EE inhibited the ER stress, as evidenced by the inactivation of activating transcription factor 6 (ATF6), protein kinase RNA (PKR)-like ER kinase (PERK), and inositol-requiring enzyme 1 (IRE1) pathways. Moreover, the rats reared in EE were detected with lower autophagic activity and apoptosis levels. The decrease in activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and phospho-c-Jun N-terminal kinases (p-JNK) expression suggested EE pretreatment might inhibit autophagy and apoptosis via modulating ER stress-mediated PERK-ATF4-CHOP and IRE1-JNK signal pathways, which provides a new idea for the prevention of the deleterious cerebral and functional consequences of ischemic stroke.

Effects of enriched environment on microglia and functional white matter recovery in rats with post stroke cognitive impairment.

BACKGROUND: White matter damage is an important contributor to cognitive impairment after stroke. This study was designed to explore the beneficial effects of enriched environment (EE) on white matter recovery and cognitive dysfunction after stroke, and further explore the potential mechanism of EE on white matter recovery from the perspective of microglia and microglia-mediated neuroinflammation. METHODS: Male SD rats underwent middle cerebral artery occlusion (MCAO) or sham surgery. During the MCAO operation, a laser Doppler blood flow meter was used to monitor the blood flow to ensure the success of the model. At 72 h after the operation, 3 rats were selected for TTC staining to identify the infarct size. One week after surgery, the rats were randomly assigned into four different groups-MCAO+standard environment (SE), MCAO+enriched environment (EE), Sham+SE and Sham+EE for 4 weeks. At four weeks after MCAO surgery, neurological function deficiency condition and cognitive function were assessed using Longa score and Morris Water Maze prior to euthanasia. The loss or regeneration of myelin was stained with LFB, the expression of myelin regeneration-related protein and microglia protein was quantified by western blot and immunofluorescence, and the level of inflammatory factors was measured by ELISA. RESULTS: EE treatment remarkably decreased the neurological deficit score, ameliorated the cognitive functional deficit in MCAO rats. Furthermore, EE alleviated white matter lesions and demyelination, increased myelin basic protein expression and decreased the number of activated microglia in the hippocampus of MCAO rats. In addition, ELISA analysis indicated that EE decreased the level of IL-1ß, IL-6, which further suggests that EE may reduce the level of pro-inflammatory factors by affecting the expression of microglia marker, IBA1, provide a benefit physiological environment for myelin recovery, and improve post stroke cognitive impairment. CONCLUSIONS: Our results suggest that exposure to EE substantially reduced the damage to brain tissue caused by activation of microglia activation, decreased the level of pro-inflammatory cytokins, which may induced by microglia, protected and promote white matter recovery to improve cognitive function after stroke. Our findings also indicate exposure to EE is beneficial for patients with white matter impairment characterised by white matter disease-related inflammation.

Neuroprotective effects of bone marrow stromal cells on rat organotypic hippocampal slice culture model of cerebral ischemia.

Organotypic hippocampal slice cultures prepared from newborn rats were maintained in vitro for 9 days. Cultures were then exposed to 30 min of combined oxygen-glucose deprivation (OGD). After OGD, the area covered by neurites was decreased. The dead cells of hippocampal slices in the ischemia group were 40.4% at day 3 and 41.6% at day 7 after OGD. The ultrastructure of the CA1 region of the slices was seriously damaged. While hippocampal slices were cultured in combination with bone marrow stromal cells (MSCs), the average area covered by neurites was comparatively increased. The dead cells were only 25.2% at day 3 and 27.1% at day 7 after coculture. The damage of the ultrastructure of the CA1 region in the coculture group was reduced significantly. Thus, in an in vitro model of simulated ischemia, MSCs can promote the outgrowth of neurites from hippocampal slices and alleviate cell damage. The neuroprotective effect might be mediated through diffusible neurotrophic factors secreted from MSCs.