Single-Cell Mapping of Brain Myeloid Cell Subsets Reveals Key Transcriptomic Changes Favoring Neuroplasticity after Ischemic Stroke / 神经科学通报·英文版

Interactions between brain-resident and peripheral infiltrated immune cells are thought to contribute to neuroplasticity after cerebral ischemia. However, conventional bulk sequencing makes it challenging to depict this complex immune network. Using single-cell RNA sequencing, we mapped compositional and transcriptional features of peri-infarct immune cells. Microglia were the predominant cell type in the peri-infarct region, displaying a more diverse activation pattern than the typical pro- and anti-inflammatory state, with axon tract-associated microglia (ATMs) being associated with neuronal regeneration. Trajectory inference suggested that infiltrated monocyte-derived macrophages (MDMs) exhibited a gradual fate trajectory transition to activated MDMs. Inter-cellular crosstalk between MDMs and microglia orchestrated anti-inflammatory and repair-promoting microglia phenotypes and promoted post-stroke neurogenesis, with SOX2 and related Akt/CREB signaling as the underlying mechanisms. This description of the brain's immune landscape and its relationship with neurogenesis provides new insight into promoting neural repair by regulating neuroinflammatory responses.

Soybean isoflavones alleviate cerebral ischemia/reperfusion injury in rats by inhibiting ferroptosis and inflammatory cascade reaction / 南方医科大学学报

OBJECTIVE@#To explore the mechanism that mediates the effect of soybean isoflavones (SI) against cerebral ischemia/reperfusion (I/R) injury in light of the regulation of regional cerebral blood flow (rCBF), ferroptosis, inflammatory response and blood-brain barrier (BBB) permeability.@*METHODS@#A total of 120 male SD rats were equally randomized into sham-operated group (Sham group), cerebral I/R injury group and SI pretreatment group (SI group). Focal cerebral I/R injury was induced in the latter two groups using a modified monofilament occlusion technique, and the intraoperative changes of real-time cerebral cortex blood flow were monitored using a laser Doppler flowmeter (LDF). The postoperative changes of cerebral pathological morphology and the ultrastructure of the neurons and the BBB were observed with optical and transmission electron microscopy. The neurological deficits of the rats was assessed, and the severities of cerebral infarction, brain edema and BBB disruption were quantified. The contents of Fe2+, GSH, MDA and MPO in the ischemic penumbra were determined with spectrophotometric tests. Serum levels of TNF-α and IL-1βwere analyzed using ELISA, and the expressions of GPX4, MMP-9 and occludin around the lesion were detected with Western blotting and immunohistochemistry.@*RESULTS@#The rCBF was sharply reduced in the rats in I/R group and SI group after successful insertion of the monofilament. Compared with those in Sham group, the rats in I/R group showed significantly increased neurological deficit scores, cerebral infarction volume, brain water content and Evans blue permeability (P < 0.01), decreased Fe2+ level, increased MDA level, decreased GSH content and GPX4 expression (P < 0.01), increased MPO content and serum levels of TNF-α and IL-1β (P < 0.01), increased MMP-9 expression and lowered occludin expression (P < 0.01). All these changes were significantly ameliorated in rats pretreated with IS prior to I/R injury (P < 0.05 or 0.01).@*CONCLUSION@#SI preconditioning reduces cerebral I/R injury in rats possibly by improving rCBF, inhibiting ferroptosis and inflammatory response and protecting the BBB.

Effect of Xiaoxuming Decoction on activation of astrocytes in acute cerebral ischemia/reperfusion injury / 中国中药杂志

This study investigated the effect of Xiaoxuming Decoction(XXMD) on the activation of astrocytes after cerebral ischemia/reperfusion(I/R) injury. The model of cerebral IR injury was established using the middle cerebral artery occlusion method. Fluorocitrate(FC), an inhibitor of astrocyte activation, was applied to inhibit astrocyte activation. Rats were randomly divided into a sham group, a model group, a XXMD group, a XXMD+FC group, and a XXMD+Vehicle group. Neurobehavioral changes at 24 hours after cerebral IR injury, cerebral infarction, histopathological changes observed through HE staining, submicroscopic structure of astrocytes observed through transmission electron microscopy, fluorescence intensity of glial fibrillary acidic protein(GFAP) and thrombospondin 1(TSP1) measured through immunofluorescence, and expression of GFAP and TSP1 in brain tissue measured through Western blot were evaluated in rats from each group. The experimental results showed that neurobehavioral scores and cerebral infarct area significantly increased in the model group. The XXMD group, the XXMD+FC group, and the XXMD+Vehicle group all alleviated neurobehavioral changes in rats. The pathological changes in the brain were evident in the model group, while the XXMD group, the XXMD+FC group, and the XXMD+Vehicle group exhibited milder cerebral IR injury in rats. The submicroscopic structure of astrocytes in the model group showed significant swelling, whereas the XXMD group, the XXMD+FC group, and XXMD+Vehicle group protected the submicroscopic structure of astrocytes. The fluorescence intensity and protein expression of GFAP and TSP1 increased in the model group compared with those in the sham group. However, the XXMD group, the XXMD+FC group, and XXMD+Vehicle group all down-regulated the expression of GFAP and TSP1. The combination of XXMD and FC showed a more pronounced effect. These results indicate that XXMD can improve cerebral IR injury, possibly by inhibiting astrocyte activation and down-regulating the expression of GFAP and TSP1.

Chrysin alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis in rats / 中国中药杂志

The purpose of this study is to investigate whether chrysin reduces cerebral ischemia-reperfusion injury(CIRI) by inhi-biting ferroptosis in rats. Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups(200, 100, and 50 mg·kg~(-1)), and a positive drug group(Ginaton, 21.6 mg·kg~(-1)). The CIRI model was induced in rats by transient middle cerebral artery occlusion(tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation. The neurological deficit score was used to detect neurological function. The 2,3,5-triphenyl tetrazolium chloride(TTC) staining was used to detect the cerebral infarction area. Hematoxylin-eosin(HE) staining and Nissl staining were used to observe the morphological structure of brain tissues. Prussian blue staining was used to observe the iron accumulation in the brain. Total iron, lipid pero-xide, and malondialdehyde in serum and brain tissues were detected by biochemical reagents. Real-time quantitative polymerase chain reaction(RT-qPCR), immunohistochemistry, and Western blot were used to detect mRNA and protein expression of solute carrier fa-mily 7 member 11(SLC7A11), transferrin receptor 1(TFR1), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long chain family member 4(ACSL4), and prostaglandin-endoperoxide synthase 2(PTGS2) in brain tissues. Compared with the model group, the groups with drug intervention showed restored neurological function, decreased cerebral infarction rate, and alleviated pathological changes. The low-dose chrysin group was selected as the optimal dosing group. Compared with the model group, the chrysin groups showed reduced content of total iron, lipid peroxide, and malondialdehyde in brain tissues and serum, increased mRNA and protein expression levels of SLC7A11 and GPX4, and decreased mRNA and protein expression levels of TFR1, PTGS2, and ACSL4. Chrysin may regulate iron metabolism via regulating the related targets of ferroptosis and inhibit neuronal ferroptosis induced by CIRI.

Bombyx Batryticatus extract promotes microglia polarization to improve neuron injury and behaviors of cerebral ischemia/reperfusion rats / 中国中药杂志

This study aims to investigate the effect of Bombyx Batryticatus extract(BBE) on behaviors of rats with global cerebral ischemia reperfusion(I/R) and the underlying mechanism. The automatic coagulometer was used to detect the four indices of human plasma coagulation after BBE intervention for quality control of the extract. Sixty 4-week-old male SD rats were randomized into sham operation group(equivalent volume of normal saline, ip), model group(equivalent volume of normal saline, ip), positive drug group(900 IU·kg~(-1) heparin, ip), and low-, medium-, and high-dose BBE groups(0.45, 0.9, and 1.8 mg·g~(-1)·d~(-1) BBE, ip). Except the sham operation group, rats were subjected to bilateral common carotid artery occlusion followed by reperfusion(BCCAO/R) to induce I/R. The administration lasted 7 days for all the groups. The behaviors of rats were examined by beam balance test(BBT). Morphological changes of brain tissue were observed based on hematoxylin-eosin(HE) staining. Immunofluorescence method was used to detect common leukocyte antigen(CD45), leukocyte differentiation antigen(CD11b), and arginase-1(Arg-1) in cerebral cortex(CC). The protein expression of interleukin-1β(IL-1β), interleukin-4(IL-4), interleukin-6(IL-6), and interleukin-10(IL-10) was detected by enzyme-linked immunosorbent assay(ELISA). The non-targeted metabonomics was employed to detect the levels of metabolites in plasma and CC of rats after BBE intervention. The results of quality control showed that the BBE prolonged the activated partial thromboplastin time(APTT), prothrombin time(PT), and thrombin time(TT) of human plasma, which was similar to the anticoagulation effect of BBE obtained previously. The results of behavioral test showed that the BBT score of the model group increased compared with that of the sham operation group. Compared with the model group, BBE reduced the BBT score. As for the histomorphological examination, compared with the sham operation group, the model group showed morphological changes of a lot of nerve cells in CC. The nerve cells with abnormal morphology in CC decreased after the intervention of BBE compared with those in the model group. Compared with the sham operation group, the model group had high average fluorescence intensity of CD45 and CD11b in the CC. The average fluorescence intensity of CD11b decreased and the average fluorescence intensity of Arg-1 increased in CC in the low-dose BBE group compared with those in the model group. The average fluorescence intensity of CD45 and CD11b decreased and the average fluorescence intensity of Arg-1 increased in medium-and high-dose BBE groups compared with those in the model group. The expression of IL-1β and IL-6 was higher and the expression of IL-4 and IL-10 was lower in the model group than in the sham operation group. The expression of IL-1β and IL-6 was lower and the expression of IL-4 and IL-10 was higher in the low-dose, medium-dose, and high-dose BBE groups than in the model group. The results of non-targeted metabonomics showed that 809 metabolites of BBE were identified, and 57 new metabolites in rat plasma and 45 new metabolites in rat CC were found. BBE with anticoagulant effect can improve the behaviors of I/R rats, and the mechanism is that it promotes the polarization of microglia to M2 type, enhances its anti-inflammatory and phagocytic functions, and thus alleviates the damage of nerve cells in CC.

Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats / 中国中药杂志

This study compared the ameliorating effects of L-borneol, natural borneol, and synthetic borneol on the injury of different brain regions in the rat model of acute phase of cerebral ischemia/reperfusion(I/R) for the first time, which provides a reference for guiding the rational application of borneol in the early treatment of ischemic stroke and has important academic and application values. Healthy specific pathogen-free(SPF)-grade SD male rats were randomly assigned into 13 groups: a sham-operation group, a model group, a Tween model group, a positive drug(nimodipine) group, and high-, medium-, and low-dose(0.2, 0.1, and 0.05 g·kg~(-1), respectively) groups of L-borneol, natural borneol, and synthetic borneol according to body weight. After 3 days of pre-administration, the rat model of I/R was established by suture-occluded method and confirmed by laser speckle imaging. The corresponding agents in different groups were then administered for 1 day. The body temperature was monitored regularly before pre-administration, days 1, 2, and 3 of pre-administration, 2 h after model awakening, and 1 d after model establishment. Neurological function was evaluated based on Zea-Longa score and modified neurological severity score(mNSS) 2 h and next day after awakening. The rats were anesthetized 30 min after the last administration, and blood was collected from the abdominal aorta. Enzyme-linked immunoassay assay(ELISA) was employed to determine the serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-4, and transforming growth factor-beta1(TGF-β1). The brain tissues were stained with triphenyltetrazolium chloride(TTC) for the calculation of cerebral infarction rate, and hematoxylin-eosin(HE) staining was used for observing and semi-quantitatively evaluating the pathological damage in different brain regions. Immunohistochemistry was employed to detect the expression of ionized calcium binding adapter molecule 1(IBA1) in microglia. q-PCR was carried out to determine the mRNA levels of iNOS and arginase 1(Arg1), markers of polarization phenotype M1 and M2 in microglia. Compared with the sham-operation group, the model group and the Tween model group showed significantly elevated body temperature, Zea-Longa score, mNSS, and cerebral infarction rate, severely damaged cortex, hippocampus, and striatum, increased serum levels of IL-6 and TNF-α, and decreased serum levels of IL-4 and TGF-β1. The three borneol products had a tendency to reduce the body temperature of rats 1 day after modeling. Synthetic borneol at the doses of 0.2 and 0.05 g·kg~(-1), as well as L-borneol of 0.1 g·kg~(-1), significantly reduced Zea-Longa score and mNSS. The three borneol products at the dose of 0.2 g·kg~(-1) significantly reduced the cerebral infarction rate. L-borneol at the doses of 0.2 and 0.1 g·kg~(-1) and natural borneol at the dose of 0.1 g·kg~(-1) significantly reduced the pathological damage of the cortex. L-borneol and natural borneol at the dose of 0.1 g·kg~(-1) attenuated the pathological damage of hippocampus, and 0.2 g·kg~(-1) L-borneol attenuated the damage of striatum. The 0.2 g·kg~(-1) L-borneol and the three doses of natural borneol and synthetic borneol significantly reduced the serum level of TNF-α, and the 0.1 g·kg~(-1) synthetic borneol reduced the level of IL-6. L-borneol and synthetic borneol at the dose of 0.2 g·kg~(-1) significantly inhibited the activation of cortical microglia, and 0.2 g·kg~(-1) L-borneol up-regulated the expression of Arg1 and down-regulated the expression level of iNOS. In conclusion, the three borneol products may alleviate inflammation to ameliorate the pathological damage of brain regions of rats in the acute phase of I/R by inhibiting the activation of microglia and promoting the polarization of microglia from M1 type to M2 type. The protective effect on brain followed a trend of L-borneol > synthetic borneol > natural borneol. We suggest L-borneol the first choice for the treatment of I/R in the acute phase.

Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury / 中国中药杂志

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.

Compound from Magnolia officinalis Ameliorates White Matter Injury by Promoting Oligodendrocyte Maturation in Chronic Cerebral Ischemia Models / 神经科学通报·英文版

Chronic cerebral hypoperfusion leads to white matter injury (WMI), which subsequently causes neurodegeneration and even cognitive impairment. However, due to the lack of treatment specifically for WMI, novel recognized and effective therapeutic strategies are urgently needed. In this study, we found that honokiol and magnolol, two compounds derived from Magnolia officinalis, significantly facilitated the differentiation of primary oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes, with a more prominent effect of the former compound. Moreover, our results demonstrated that honokiol treatment improved myelin injury, induced mature oligodendrocyte protein expression, attenuated cognitive decline, promoted oligodendrocyte regeneration, and inhibited astrocytic activation in the bilateral carotid artery stenosis model. Mechanistically, honokiol increased the phosphorylation of serine/threonine kinase (Akt) and mammalian target of rapamycin (mTOR) by activating cannabinoid receptor 1 during OPC differentiation. Collectively, our study indicates that honokiol might serve as a potential treatment for WMI in chronic cerebral ischemia.

The Circadian System Is Essential for the Crosstalk of VEGF-Notch-mediated Endothelial Angiogenesis in Ischemic Stroke / 神经科学通报·英文版

Ischemic stroke is a major public health problem worldwide. Although the circadian clock is involved in the process of ischemic stroke, the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear. In the present study, we determined that environmental circadian disruption (ECD) increased the stroke severity and impaired angiogenesis in the rat middle cerebral artery occlusion model, by measuring the infarct volume, neurological tests, and angiogenesis-related protein. We further report that Bmal1 plays an irreplaceable role in angiogenesis. Overexpression of Bmal1 promoted tube-forming, migration, and wound healing, and upregulated the vascular endothelial growth factor (VEGF) and Notch pathway protein levels. This promoting effect was reversed by the Notch pathway inhibitor DAPT, according to the results of angiogenesis capacity and VEGF pathway protein level. In conclusion, our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which Bmal1 regulates angiogenesis through the VEGF-Notch1 pathway.

Total Saponins of Panax notoginseng Activate Akt/mTOR Pathway and Exhibit Neuroprotection in vitro and in vivo against Ischemic Damage / 中国结合医学杂志

OBJECTIVE@#To reveal the neuroprotective effect and the underlying mechanisms of a mixture of the main components of Panax notoginseng saponins (TSPN) on cerebral ischemia-reperfusion injury and oxygen-glucose deprivation/reoxygenation (OGD/R) of cultured cortical neurons.@*METHODS@#The neuroprotective effect of TSPN was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and live/dead cell assays. The morphology of dendrites was detected by immunofluorescence. Middle cerebral artery occlusion (MCAO) was developed in rats as a model of cerebral ischemia-reperfusion. The neuroprotective effect of TSPN was evaluated by neurological scoring, tail suspension test, 2,3,5-triphenyltetrazolium chloride (TTC) and Nissl stainings. Western blot analysis, immunohistochemistry and immunofluorescence were used to measure the changes in the Akt/mammalian target of rapamycin (mTOR) signaling pathway.@*RESULTS@#MTT showed that TSPN (50, 25 and 12.5 µ g/mL) protected cortical neurons after OGD/R treatment (P<0.01 or P<0.05). Flow cytometry and live/dead cell assays indicated that 25 µ g/mL TSPN decreased neuronal apoptosis (P<0.05), and immunofluorescence showed that 25 µ g/mL TSPN restored the dendritic morphology of damaged neurons (P<0.05). Moreover, 12.5 µ g/mL TSPN downregulated the expression of Beclin-1, Cleaved-caspase 3 and LC3B-II/LC3B-I, and upregulated the levels of phosphorylated (p)-Akt and p-mTOR (P<0.01 or P<0.05). In the MCAO model, 50 µ g/mL TSPN improved defective neurological behavior and reduced infarct volume (P<0.05). Moreover, the expression of Beclin-1 and LC3B in cerebral ischemic penumbra was downregulated after 50 µ g/mL TSPN treatment, whereas the p-mTOR level was upregulated (P<0.05 or P<0.01).@*CONCLUSION@#TSPN promoted neuronal survival and protected dendrite integrity after OGD/R and had a potential therapeutic effect by alleviating neurological deficits and reversing neuronal loss. TSPN promoted p-mTOR and inhibited Beclin-1 to alleviate ischemic damage, which may be the mechanism that underlies the neuroprotective activity of TSPN.

Echinacoside Alleviates Cognitive Impairment in Cerebral Ischemia Rats through α 7nAChR-Induced Autophagy / 中国结合医学杂志

OBJECTIVES@#To evaluate the effect of echinacoside (ECH) on cognitive dysfunction in post cerebral stroke model rats.@*METHODS@#The post stroke cognitive impairment rat model was created by occlusion of the transient middle cerebral artery (MCAO). The rats were randomly divided into 3 groups by a random number table: the sham group (sham operation), the MCAO group (received operation for focal cerebral ischemia), and the ECH group (received operation for focal cerebral ischemia and ECH 50 mg/kg per day), with 6 rats in each group. The infarct volume and spatial learning were evaluated by triphenyl tetrazolium chloride staining and Morris water maze. The expression of α7nAChR in the hippocampus was detected by immunohistochemistry. The contents of acetylcholine (ACh), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), activities of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and catalase (CAT) were evaluated by enzyme linked immunosorbent assay. The neural apoptosis and autophagy were determined by TUNEL staining and LC3 staining, respectively.@*RESULTS@#ECH significantly lessened the brain infarct volume and ameliorated neurological deficit in infarct volume and water content (both P<0.01). Compared with MCAO rats, administration of ECH revealed shorter escape latency and long retention time at 7, 14 and 28 days (all P<0.01), increased the α7nAChR protein expression, ACh content, and ChAT activity, and decreased AChE activity in MCAO rats (all P<0.01). ECH significantly decreased MDA content and increased the GSH content, SOD, and CAT activities compared with MCAO rats (all P<0.05). ECH suppressed neuronal apoptosis by reducing TUNEL-positive cells and also enhanced autophagy in MCAO rats (all P<0.01).@*CONCLUSION@#ECH treatment helped improve cognitive impairment by attenuating neurological damage and enhancing autophagy in MCAO rats.

Baicalin treats cerebral ischemia reperfusion-induced brain edema in rats by inhibiting TRPV4 and AQP4 of astrocytes / 中国中药杂志

This study aims to explore the pharmacodynamic effect of baicalin on rat brain edema induced by cerebral ischemia reperfusion injury and discuss the mechanism from the perspective of inhibiting astrocyte swelling, which is expected to serve as a refe-rence for the treatment of cerebral ischemia with Chinese medicine. To be specific, middle cerebral artery occlusion(suture method) was used to induce cerebral ischemia in rats. Rats were randomized into normal group, model group, high-dose baicalin(20 mg·kg~(-1)) group, and low-dose baicalin(10 mg·kg~(-1)) group. The neurobehavior, brain index, brain water content, and cerebral infarction area of rats were measured 6 h and 24 h after cerebral ischemia. Brain slices were stained with hematoxylin and eosin(HE) for the observation of pathological morphology of cerebral cortex after baicalin treatment. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of total L-glutathione(GSH) and glutamic acid(Glu) in brain tissue, Western blot to measure the content of glial fibrillary acidic protein(GFAP), aquaporin-4(AQP4), and transient receptor potential vanilloid type 4(TRPV4), and immunohistochemical staining to observe the expression of GFAP. The low-dose baicalin was used for exploring the mechanism. The experimental results showed that the neurobehavioral scores(6 h and 24 h of cerebral ischemia), brain water content, and cerebral infarction area of the model group were increased, and both high-dose and low-dose baicalin can lower the above three indexes. The content of GSH dropped but the content of Glu raised in brain tissue of rats in the model group. Low-dose baicalin can elevate the content of GSH and lower the content of Glu. According to the immunohistochemical staining result, the model group demonstrated the increase in GFAP expression, and swelling and proliferation of astrocytes, and the low-dose baicalin can significantly improve this situation. The results of Western blot showed that the expression of GFAP, TRPV4, and AQP4 in the cerebral cortex of the model group increased, and the low-dose baicalin reduce their expression. The cerebral cortex of rats in the model group was severely damaged, and the low-dose baicalin can significantly alleviate the damage. The above results indicate that baicalin can effectively relieve the brain edema caused by cerebral ischemia reperfusion injury in rats, possibly by suppressing astrocyte swelling and TRPV4 and AQP4.

Effects of Zhongfeng capsule on autophagy related proteins expressions of brain tissue in rats with cerebral ischemia/reperfusion injury / 中国应用生理学杂志

Objective: To investigate the effects of Zhongfeng capsule on the autophagy-related proteins expression in rats with cerebral ischemia/reperfusion injury (CI/ RI), and to explore its neural protection mechanisms of the decoction. Methods: Rat middle cerebral artery ischemia/reperfusion injury model (ischemia for 2 h, reperfusion for 24 h) was prepared by the improved line plug method. Sixty male SD rats were randomly divided into sham operation group, model group, butylphthalide group(0.054 g/kg), Zhongfeng capsule high-dose groups (1.08 g/kg), Zhongfeng capsule middle-dose groups (0.54 g/kg), Zhongfeng capsule low-dose groups (0.27 g/kg), with 10 rats in each group. Rats were treated with Zhongfeng capsule by gavage once a day for 10 days. The rats were sacrificed and the brain tissue was obtained after the experiment in each group. Score neurological deficit was evaluated after 24 h of the last intervention in rat of each group. The pathological changes of brain tissue were observed by HE staining. The serum levels of estradiol (E2) and follicle stimulating hormone (FSH) were determined by ELISA. The expressions of key genes and proteins of PI3K/Akt/Beclin1 signaling pathway in brain tissue were detected by qRT-PCR and Western blot respectively. Results: Compared with the sham operation group, the body weight and protein expressions of p-PI3k and p-Akt in brain tissue of rats were decreased significantly in the model group, while the brain index, neurological deficit score, gene and protein expressions of Beclin1 and LC3 were increased markedly in the model group(P＜0.05 or P＜0.01). In the model group, nerve cells of brain tissue were loosely packed, interstitial edema, triangular in shape, nuclear pyknosis and dark-blue staining were observed. Compared with the model group, the body weight of rats was increased obviously, the neurological deficit score was decreased significantly and the pathological injury of brain tissue was alleviated evidently in high-dose of Zhongfeng capsule group (P＜0.05). The brain index, the gene and protein expressions of Beclin1 and LC3 were decreased apparently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01), while the expressions of p-PI3k and p-Akt in brain tissue were increased evidently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01). Conclusion: Zhongfeng capsule can inhibit autophagy and improve brain neurons lesion of CIRI rats, the mechanism may be related to regulate the expression of Beclin1 and LC3 in PI3K/Akt/Beclin1 signaling pathway.

MicroRNA-27b expression in rats submitted to physical exercise and experimental cerebral ischemia / Expresión de MicroRNA-27b en ratas sometidas a ejercicio físico e isquemia cerebral experimental

SUMMARY: Cerebral ischemia has not only a high mortality rate, which is the second leading cause of death worldwide, but is also responsible for severe disabilities in working age individuals, generating enormous public expending for treatment and rehabilitation of the affected individuals. The role of microRNAs in the pathophysiology of cerebral ischemia has been highlighted in current investigations. In addition, recent studies have also highlighted physical exercise as a possible protective factor both in the prevention and in the effects of cerebral ischemia, placing it as an important study resource. Thus, we investigated the role of physical exercise in experimental cerebral ischemia associated with the expression of microRNA-27b. 16 animals were used, divided into four experimental groups: Control, Physical Exercise, Cerebral Ischemia and Cerebral Ischemia associated with Physical Exercise. The real-time PCR methodology was used to analyze the expression of microRNA-27b. Although there were no statistically significant differences in the expression of microRNA-27b between the groups studied, the increased expression of microRNA-27b in the Physical Exercise group indicates its neuroprotective role in the pathophysiology of cerebral ischemia.

RESUMEN: La isquemia cerebral no solo tiene una alta tasa de mortalidad y es la segunda causa principal de muerte en todo el mundo, sino también es la causa de enfermedades invalidantes en personas en edad laboral, lo que genera un gasto público enorme para el tratamiento y la rehabilitación de las personas afectadas. El papel de los microARN en la fisiopatología de la isquemia cerebral se ha destacado en las investigaciones actuales. Además, estudios recientes también han destacado el ejercicio físico como un posible factor protector tanto en la prevención como en los efectos de la isquemia cerebral, situándolo como un importante recurso de estudio. Por lo tanto, investigamos el papel del ejercicio físico en la isquemia cerebral experimental asociada con la expresión del microARN-27b. Se utilizaron 16 animales, divididos en cuatro grupos experimentales: Control, Ejercicio Físico, Isquemia Cerebral e Isquemia Cerebral asociada al Ejercicio Físico. Se utili- zó la metodología de PCR en tiempo real para analizar la expresión de microARN-27b. Aunque no se observaron diferencias estadísticamente significativas en la expresión de microARN-27b entre los grupos estudiados, la mayor expresión de microARN-27b en el grupo de Ejercicio Físico indica su papel neuroprotector en la fisiopatología de la isquemia cerebral.

Morphometric analysis and pattern of protein and gene expression of apoptosis in focal cerebral ischemia in rats and the neuroprotetive action of hypothermia and ketoprofen / Análisis morfométrico y modelo de expresión de proteína y genes de apoptosis en isquemia cerebral focal en ratas y la acción neuroprotectora de hipotermia y ketoprofeno

This study aimed to investigate the morphometric and the pattern of protein and gene expression related to the extrinsic apoptotic pathway in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. For this analysis, 120 rats were randomly divided into 3 groups (20 animals each): control - no surgery (20 animals); sham - simulation of surgery (20 animals); ischemic - focal ischemia for 1 hour, without reperfusion (80 animals) and divided into four subgroups with 20 animals each: ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the apoptosis genes (Fas, c-Flip, caspase-8 and caspase-3) and the apoptosis protein caspase-3 were evaluated by quantitative real-time PCR and immunohistochemistry, respectively. Hypo expression of genes of extrinsic pathway of apoptosis was observed: Fas receptor, c-Flip and caspase-8 in the ischemics areas. Increases in the gene and protein caspase-3 in the ischemic areas were also observed, and these increases were reduced by hypothermia and ketoprofen, also noted in the morphometric study. The caspases-3 increase suggests that this gene plays an important role in apoptosis, probably culminating in cell death and that the neuroprotective effect of hypothermia and ketoprofen is involved.

Este estudio tuvo como objetivo investigar la morfometría y el patrón de expresión de proteínas y genes relacionados con la vía apoptótica extrínseca en la isquemia cerebral focal experimental y el agujero de neuroprotección con hipotermia y ketoprofeno. Se dividieron aleatoriamente 120 ratas en 3 grupos (20 animales cada uno): control - sin cirugía (20 animales); simulación - simulación de cirugía (20 animales); isquemia isquemia focal durante 1 hora, sin reperfusión (80 animales) y dividida en cuatro subgrupos con 20 animales cada uno: isquemia + hipotermia intraisquémica; isquemia + ketoprofeno intravenoso previo, e isquemia + hipotermia y ketoprofeno. El volumen del infarto se midió utilizando un análisis morfométrico de áreas de infarto definidas por cloruro de trifenil tetrazolio y los patrones de expresión de los genes de apoptosis (Fas, c-Flip, caspase-8 y caspase-3) y la proteína de apoptosis caspase-3 fueron evaluados por PCR cuantitativa en tiempo real e inmunohistoquímica, respectivamente. Se observó hipoexpresión de genes de la vía extrínseca de la apoptosis: receptor Fas, c-Flip y caspasa-8 en las áreas isquémicas. También se observaron aumentos en el gen y la proteína caspasa-3 en las áreas isquémicas y estos aumentos se redujeron por hipotermia y ketoprofeno, también observado por estudio morfométrico. El aumento de caspasas-3 sugiere que este gen tiene un papel importante en la apoptosis, y probable causa de muerte celular, involucrando el efecto neuroprotector de la hipotermia y el ketoprofeno.

AIF, PARP-1 and MicroRNA-9 expression in cerebral ischemia associated to alcoholism experimental model / Expresión de AIF, PARP-1 y MicroRNA-9 de isquemia cerebral asociado a un modelo experimental de alcoholismo

The chronic consumption of alcohol causes a worsening of the events that follow the cerebral ischemia. These events are regulated through the expression of several genes and microRNAs. The aimof this work was To analyze and describe the expression profile of PARP and AIF and miRNA-9 proteins in rats submitted to focal cerebral ischemia, associated or not with chronic alcoholism model. Methods: Twenty adult Wistar rats, subdivided into: control; ischemic; alcoholic and ischemic / alcoholized for immunohistochemical analysis and miRNA-9 gene expression. Results: There was a reduction in the protein expression of PARP-1 and a positive marking for AIF in the ischemic / alcoholized group. The miRNA-9 did not obtain significant expression. The association of ischemia with chronic alcohol use promoted a tendency to low expression of miRNA-9, low expression of PARP-1 and high expression of AIF, indicating an interference in the protective effect of miRNA-9 be observed in the other groups.

El consumo crónico de alcohol provoca un empeoramiento de los eventos que siguen a la isquemia cerebral. Estos eventos están regulados a través de la expresión de varios genes y microRNA. El objetivo de este trabajo fue analizar y describir el perfil de expresión de las proteínas PARP y AIF y microRNA-9 en ratas sometidas a isquemia cerebral focal, asociadas o no, con el modelo de alcoholismo crónico. Veinte ratas Wistar adultas se dividieron en: grupo control, isquémico alcohólico, e isquémico / alcoholizado para análisis inmunohistoquímico y expresión de genes microRNA-9. Resultados: Hubo una reducción en la expresión de proteínas de PARP-1 y un marcado positivo para AIF en el grupo isquémico / alcoholizado. No se observó una expresión significativa en el microRNA-9. La asociación de la isquemia con el consumo crónico de alcohol promovió una tendencia a la baja expresión de microRNA-9, baja expresión de PARP1 y alta expresión de AIF, lo que indica una interferencia en el efecto protector de microRNA-9 en los otros grupos.

Preconditioning with L-Ala-Gln reduces the expression of inflammatory markers (TNF-α, NF-κB, IL-6 and HO-1) in an injury animal model of cerebrovascular ischemia in Meriones unguiculatus (gerbils)

Abstract Purpose To evaluate the neuroprotective effect of L-alanyl-glutamine in a gerbil model of brain ischemia-reperfusion injury based on immunohistochemical quantification of pro-inflammatory and cell activation biomarkers (TNF-α, NF-κB, IL-6 and HO-1). Methods Male gerbils weighing 100-180 g were pretreated with either 0.75 g/kg L-Ala-Gln (n=18) or 2.0 mL saline (n=18) administered i.v. 30 minutes before the bilateral ligation of the common carotid artery during 15 min and then the ligation was removed. Under anesthesia with urethane, brain tissue was harvested at 0 min (T0), 30 min (T30) and 60 min (T60) after reperfusion. The tissue was embedded in 10% formalin overnight and 4-μm sections were prepared for immunostaining with monoclonal antibodies. Immunostained cells were counted by optical microscopy. The statistical analysis used mean values based on 4 sections. Results The pretreatment with L-Ala-Gln animal group 1 demonstrated significantly lower levels of TNF-α, NF-κB and IL-6. On the other hand, the levels of HO-1 were significantly higher, suggesting a protective role in model of brain ischemia-reperfusion injury. Conclusion These findings suggest a protective effect of L-Ala-Gln by decreasing levels of TNF-alpha, IL-6 and NF-κB and Increasing levels of HO-1.

Effect of ginsenosides on serous metabonomic profiles in cerebral ischemia-reperfusion rats based on ~1H-NMR / 中国中药杂志

Serum metabonomic profiles of the model of focal cerebral ischemia reperfusion is established with the suture-occluded method by Longa to study the effect of ginsenosides. In this study, 48 rats were randomly divided into six groups: sham-operated group, pathological model group, positive drug group(6 mg·kg~(-1)·d~(-1)) and high, medium, low-dose ginsenosides groups(200, 100, 50 mg·kg~(-1)·d~(-1)). They are given intragastric administration respectively with same amount of 0.5% CMC-Na,nimodipine and ginsenoside for 5 days. At 2 h after the final administration, the model was established with the suture-occluded method, and free radical-scavenging activity changes of ginsenoside were observed by maillard reaction, and Longa was possible used as a renoprotective agent-occluded method. At the end of 24 h after the reperfusion, the hemolymph of rats in each group was collected, and the ~1H-NMR spectrum was collected after being treated by certain methods, and analyzed by principal component analysis(PCA). Compared with sham-operated group, pathological model group showed significant increases in the levels of lactate, glutamate, taurine, choline, glucose and methionine, but decreases in the levels of 3-hydroxybutyrate and phosphocreatine/creatine in serum. After treatment with ginsenosides, lipid, 3-hydroxybutyrate and phosphocreatine/creatine were increased in the serum of ginsenosides group rats, but with decreases in lactate and glutamate. The results showed that ginsenosides could regulate metabolic disorders in rats with focal cerebral ischemia reperfusion, and promote a recovery in the process of metabolism. It's helpful to promote the metabolic changes in rats with focal cerebral ischemia reperfusion via ~1H-NMR, and lay a foundation to develop ginsenosides as a new drug to treat ischemic cerebral paralysis.

The long non-coding RNA H19 induces hypoxia/reoxygenation injury by up-regulating autophagy in the hepatoma carcinoma cells

BACKGROUND: Long non-coding RNA H19 (H19) plays an important role by regulating protein expression in different tissues and organs of the body. However, whether H19 induces hypoxia/reoxygenation (h/R) injury via increase of autophagy in the hepatoma carcinoma cells is unknown. RESULTS: H19 was expressed in the hepatoma carcinoma cells (Hep G2 and HCCLM3 cells) and its expression was most in 8 h/24R. The knockdown of H19 and 3-MA (an autophagy inhibitor) protected against h/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The knockdown of H19 and 3-MA also decreased the autophagic vesicles (AVs) and the expression of Beclin-1 and the ration of LC3-II/LC3-I, and increased cell viability, the expression of Bcl-2 and P62 and the phosphorylation of PI3K, Akt and mTOR. In addition, chloroquine (CQ, an inhibitor of autophagy flux) markedly decreased formation of autophagy flux (the ration of LC3-II/LC3-I). The results of the knockdown of H19 group were similar to those of the 3-MA (or CQ) group. Rapamycin (a mTOR inhibitor, an autophagy activator) further down-regulated h/R-induced decrease of the phosphorylated PI3K, Akt and mTOR. The knockdown of H19 cancelled the effect of rapamycin. The overexpression of H19 further expanded h/R-induced increase of the ration of LC3-II/LC3-I and decrease of the phosphorylated PI3K, Akt and mTOR. CONCLUSIONS: Our results suggest that the long non-coding RNA H19 induces h/R injury by up-regulation of autophagy via activation of PI3K-Akt-mTOR pathway in the hepatoma carcinoma cells.

Efficacy of phosphocreatine pre-administration on XIAP and Smac in ischemic penumbra of rats with focal cerebral ischemia reperfusion injury

Abstract Purpose: To observe the efficacy of phosphocreatine pre-administration (PCr-PA) on X-linked inhibitor of apoptosis protein (XIAP), the second mitochondia-derived activator of caspase (Smac) and apoptosis in the ischemic penumbra of rats with focal cerebral ischemia-reperfusion injury (CIRI). Methods: A total of 60 healthy male Sprague Dawley (SD) rats were randomly divided into three groups (n=20): group A (the sham operation group), group B <intraperitoneally injected with 20 mg/kg (10 mg/ml) of saline before preparing the ischemia-reperfusion (IR) model>, and group C <intraperitoneally injected with 20 mg/kg (10 mg/ml) of PCr immediately before preparing the IR model>. After 24 h for reperfusion, the neurological function was evaluated and the tissue was sampled to detect expression of XIAP, Smac and caspase-3 positive cells in the ischemic penumbra so as to observe the apoptosis. Results: Compared with group B, neurological deficit scores, numbers of apoptotic cells, expression of Smac,caspase-9 and the numbers of Caspase-3 positive cells were decreased while expression of XIAP were increased in the ischemic penumbra of group C. Conclusions: Phosphocreatine pre-administration may elicit neuroprotective effects in the brain by increasing expression of X-linked inhibitor of apoptosis protein, reducing expression of second mitochondia-derived activator of caspase, and inhibiting the apoptosis in the ischemic penumbra.