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Article in Chinese | WPRIM | ID: wpr-928023


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.

Animals , Rats , Aquaporin 4/genetics , Astrocytes , Brain Edema/drug therapy , Brain Ischemia/metabolism , Flavonoids , Infarction, Middle Cerebral Artery/drug therapy , Rats, Sprague-Dawley , Reperfusion , TRPV Cation Channels/therapeutic use
Article in Chinese | WPRIM | ID: wpr-927892


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.

Animals , Male , Rats , Autophagy-Related Proteins/pharmacology , Beclin-1/metabolism , Body Weight , Brain , Brain Ischemia/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats, Sprague-Dawley , Reperfusion Injury/drug therapy
Article in English | WPRIM | ID: wpr-939794


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.

Animals , Rats , Acetylcholinesterase , Autophagy , Brain Ischemia/metabolism , Cerebral Infarction , Cognitive Dysfunction/drug therapy , Glutathione/metabolism , Glycosides , Infarction, Middle Cerebral Artery/drug therapy , Neuroprotective Agents/therapeutic use , Rats, Sprague-Dawley , Reperfusion Injury/drug therapy , Stroke/drug therapy , Superoxide Dismutase/metabolism , alpha7 Nicotinic Acetylcholine Receptor
Article in English | WPRIM | ID: wpr-928942


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.

Animals , Rats , Beclin-1 , Brain Ischemia/metabolism , Glucose , Infarction, Middle Cerebral Artery/drug therapy , Mammals/metabolism , Neuroprotection , Neuroprotective Agents/therapeutic use , Oxygen , Panax notoginseng , Proto-Oncogene Proteins c-akt/metabolism , Reperfusion Injury/metabolism , Saponins/therapeutic use , TOR Serine-Threonine Kinases/metabolism
Int. j. morphol ; 39(3): 754-758, jun. 2021. graf
Article in English | LILACS-Express | LILACS | ID: biblio-1385408


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.

Animals , Rats , Exercise , Brain Ischemia/physiopathology , Brain Ischemia/metabolism , MicroRNAs/metabolism , Brain Ischemia/genetics , Disease Models, Animal , Real-Time Polymerase Chain Reaction
Int. j. morphol ; 38(3): 523-529, June 2020. graf
Article in English | LILACS | ID: biblio-1098282


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.

Animals , Rats , Brain Ischemia/genetics , Brain Ischemia/metabolism , Immunohistochemistry , Brain Ischemia/pathology , Brain Ischemia/therapy , Ketoprofen/pharmacology , Apoptosis/genetics , Neuroprotective Agents/pharmacology , Disease Models, Animal , Caspase 3/genetics , Caspase 8/genetics , Real-Time Polymerase Chain Reaction , Hypothermia, Induced
Int. j. morphol ; 38(3): 616-621, June 2020. graf
Article in English | LILACS | ID: biblio-1098296


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.

Animals , Rats , Brain Ischemia/metabolism , Alcoholism/metabolism , Immunohistochemistry , Brain Ischemia/genetics , Rats, Wistar , MicroRNAs/metabolism , Disease Models, Animal , Alcoholism/genetics , Apoptosis Inducing Factor/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism
Acta cir. bras ; 35(6): e202000601, 2020. tab, graf
Article in English | LILACS | ID: biblio-1130649


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.

Animals , Male , Biomarkers/metabolism , Reperfusion Injury , Brain Ischemia/metabolism , NF-kappa B , Gerbillinae , Interleukin-6 , Tumor Necrosis Factor-alpha , Models, Animal , Dipeptides , Heme Oxygenase-1
Biol. Res ; 52: 32, 2019. graf
Article in English | LILACS | ID: biblio-1038783


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.

Humans , Reperfusion Injury/metabolism , Carcinoma, Hepatocellular/metabolism , RNA, Long Noncoding/metabolism , Liver Neoplasms/metabolism , Hypoxia/metabolism , Oxygen/metabolism , Autophagy/drug effects , Up-Regulation/physiology , Brain Ischemia/metabolism , Apoptosis/physiology , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology
Acta cir. bras ; 33(2): 117-124, Feb. 2018. tab, graf
Article in English | LILACS | ID: biblio-886260


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.

Humans , Animals , Male , Rats , Phosphocreatine/pharmacology , Cardiotonic Agents/pharmacology , Reperfusion Injury/metabolism , Brain Ischemia/metabolism , Mitochondrial Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolism , Random Allocation , Brain Ischemia/prevention & control , Rats, Sprague-Dawley , Apoptosis/drug effects , Neuroprotective Agents/pharmacology , Disease Models, Animal , Drug Evaluation, Preclinical , Apoptosis Regulatory Proteins , Caspase 3/metabolism
Braz. j. med. biol. res ; 51(5): e6714, 2018. tab, graf
Article in English | LILACS | ID: biblio-889083


This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 μM), or norbinaltorphimine (norBIN, 1 μM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

Animals , Male , Rats , Cerebral Arteries/drug effects , Brain Ischemia/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Diterpenes, Clerodane/pharmacology , Signal Transduction , Cerebral Arteries/physiopathology , Brain Ischemia/drug therapy , Morpholines/administration & dosage , Chromones/administration & dosage , Rats, Sprague-Dawley , Cyclic GMP/cerebrospinal fluid , Cyclic GMP/metabolism , NG-Nitroarginine Methyl Ester , Diterpenes, Clerodane/antagonists & inhibitors , Disease Models, Animal , Naltrexone/administration & dosage , Naltrexone/analogs & derivatives
Acta cir. bras ; 32(6): 459-466, June 2017. tab, graf
Article in English | LILACS | ID: biblio-886207


Abstract Purpose: To investigate the effects of dexmedetomidine (DEX) on amino acid contents and the cerebral ultrastructure of rats with cerebral ischemia-reperfusion injury (I/R). Methods: Thirty-six, male, Wistar rats were randomly divided into three groups: the sham operation group (group C), the ischemia-reperfusion group (group I/R), and the DEX group (group D). The middle cerebral artery occlusion model was prepared by the modified Longa method. The time of ischemia was 180 min, and 120 min after reperfusion, the amount of glutamate (Glu), and γ-aminobutyric acid (GABA) in the brain were measured, and the ultrastructure-level changes in the cerebral cortex were examined using electron microscopy. Results: Compared to group C, Glu contents in group D, and I/R significantly increased. Compared to group I/R, Glu contents in group D significantly decreased. Compared to group C, GABA contents in group D, and I/R significantly increased, and those in group D significantly increased, as compared to group I/R. The cerebral ultrastructure was normal in group C. Vacuolar degeneration in the plastiosome and nervous processes, was more critical than in group D. Vascular endothelial cells (VEC) were damaged. On the contrary, these changes in group D significantly improved. Conclusion: Dexmedetomidine is capable of decreasing glutamergic content, and increasing GABAergic content, in order to decrease the injury of the cerebral ultrastructure, following cerebral ischemia-reperfusion injury.

Animals , Male , Rats , Reperfusion Injury/metabolism , Cerebral Cortex/chemistry , Brain Ischemia/drug therapy , Neuroprotective Agents/pharmacology , Dexmedetomidine/pharmacology , Glutamine/metabolism , Cerebral Cortex/ultrastructure , Brain Ischemia/metabolism , Rats, Wistar , gamma-Aminobutyric Acid/drug effects , gamma-Aminobutyric Acid/metabolism , Amino Acids/drug effects , Amino Acids/metabolism
Arq. neuropsiquiatr ; 75(1): 30-35, Jan. 2017. graf
Article in English | LILACS | ID: biblio-838854


ABSTRACT Alcohol consumption aggravates injuries caused by ischemia. Many molecular mechanisms are involved in the pathophysiology of cerebral ischemia, including neurotransmitter expression, which is regulated by microRNAs. Objective: To evaluate the microRNA-219 and NMDA expression in brain tissue and blood of animals subjected to cerebral ischemia associated with alcoholism. Methods: Fifty Wistar rats were divided into groups: control, sham, ischemic, alcoholic, and ischemic plus alcoholic. The expression of microRNA-219 and NMDA were analyzed by real-time PCR. Results: When compared to the control group, the microRNA-219 in brain tissue was less expressed in the ischemic, alcoholic, and ischemic plus alcoholic groups. In the blood, this microRNA had lower expression in alcoholic and ischemic plus alcoholic groups. In the brain tissue the NMDA gene expression was greater in the ischemic, alcoholic, and ischemic plus alcoholic groups. Conclusion: A possible modulation of NMDA by microRNA-219 was observed with an inverse correlation between them.

RESUMO Algumas condições podem agravar os danos causados pelo processo isquêmico, tais como o consumo de álcool, e diversos mecanismos moleculares que estão envolvidos na fisiopatologia da isquemia cerebral, incluindo a expressão de neurotransmissores, e estes podem estar regulados por microRNAs. Objetivo: Avaliar a expressão de NMDA e do microRNA-219 no tecido cerebral e no sangue de animais submetidos à isquemia cerebral associada ao alcoolismo. Métodos: 50 ratos Wistar foram divididos em: controle, sham, isquêmico, alcoólico e isquêmico mais alcoólico. A expressão de microRNA-219 e de NMDA foram analisadas por PCR em tempo real. Resultados: Quando comparado com o grupo controle, o microRNA-219 no tecido cerebral foi menos expresso nos grupos isquêmico, alcoólico e associado. No sangue, este microRNA teve menor expressão no grupo alcoólico e no associado. Em relação à expressão do gene do NMDA, em tecido cerebral foi maior nos grupos isquêmico, alcoólico e no associado. Conclusão: Uma possível modulação de NMDA pelo microRNA-219 foi observada, com uma correlação inversa entre eles.

Animals , Male , Rats , Brain Ischemia/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , MicroRNAs/metabolism , Alcoholism/complications , Immunohistochemistry , Brain Ischemia/etiology , Rats, Wistar , Disease Models, Animal
Acta cir. bras ; 31(9): 629-637, Sept. 2016. graf
Article in English | LILACS | ID: lil-795996


ABSTRACT PURPOSE: To evaluated histopathological changes, morphometric and expression of proteins CASPASE-3, BCL-2 and XIAP related to apoptosis in the cerebellum after induction of temporary focal cerebral ischemia followed by reperfusion, with or without a model of chronic alcoholism. METHODS: Fifty Wistar rats were used and divided into: control group (C), sham group (S), ischemic group (I), alcoholic group (A), and ischemic and alcoholic group (IA). The cerebellum samples collected were stained for histopathological and morphometric analysis and immunohistochemistry study. RESULTS: Histopathological changes were observed a greater degree in animals in groups A and IA. The morphometric study showed no difference in the amount of cells in the granular layer of the cerebellum between the groups. The expression of CASPASE-3 was higher than BCL-2 and XIAP in the groups A and IA. CONCLUSION: We observed correlation between histopathological changes and the occurrence of apoptosis in cerebellar cortex.

Animals , Male , Cerebellum/pathology , Brain Ischemia/pathology , Apoptosis , Ethanol/pharmacology , Alcoholism/pathology , Apoptosis Regulatory Proteins/metabolism , Immunohistochemistry , Reperfusion Injury/pathology , Cerebellum/drug effects , Cerebellum/metabolism , Brain Ischemia/metabolism , Rats, Wistar , Statistics, Nonparametric , Proto-Oncogene Proteins c-bcl-2/metabolism , Disease Models, Animal , Alcoholism/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolism , Caspase 3/metabolism
Braz. j. med. biol. res ; 48(9): 798-804, Sept. 2015. ilus
Article in English | LILACS | ID: lil-756403


Stroke is the third most common cause of death worldwide, and most stroke survivors present some functional impairment. We assessed the striatal oxidative balance and motor alterations resulting from stroke in a rat model to investigate the neuroprotective role of physical exercise. Forty male Wistar rats were assigned to 4 groups: a) control, b) ischemia, c) physical exercise, and d) physical exercise and ischemia. Physical exercise was conducted using a treadmill for 8 weeks. Ischemia-reperfusion surgery involved transient bilateral occlusion of the common carotid arteries for 30 min. Neuromotor performance (open-field and rotarod performance tests) and pain sensitivity were evaluated beginning at 24 h after the surgery. Rats were euthanized and the corpora striata was removed for assay of reactive oxygen species, lipoperoxidation activity, and antioxidant markers. Ischemia-reperfusion caused changes in motor activity. The ischemia-induced alterations observed in the open-field test were fully reversed, and those observed in the rotarod test were partially reversed, by physical exercise. Pain sensitivity was similar among all groups. Levels of reactive oxygen species and lipoperoxidation increased after ischemia; physical exercise decreased reactive oxygen species levels. None of the treatments altered the levels of antioxidant markers. In summary, ischemia-reperfusion resulted in motor impairment and altered striatal oxidative balance in this animal model, but those changes were moderated by physical exercise.

Animals , Male , Rats , Brain Ischemia/complications , Corpus Striatum/metabolism , Motor Disorders/prevention & control , Oxidative Stress/physiology , Physical Conditioning, Animal/physiology , Reperfusion Injury/complications , Brain Ischemia/metabolism , Catalase/metabolism , Disease Models, Animal , Glutathione/metabolism , Lipid Peroxidation , Motor Disorders/etiology , Oxidation-Reduction , Pain/physiopathology , Rats, Wistar , Reactive Oxygen Species/analysis , Superoxide Dismutase/metabolism
Indian J Exp Biol ; 2014 Jun; 52(6): 597-605
Article in English | IMSEAR | ID: sea-153738


Limb remote ischemic postconditioning (LRIP) can reduce ischemia-reperfusion injury (IRI), but its mechanisms are still unclear. We hypothesize that LRIP reduces IRI by reversing eNOS uncoupling. Focal ischemia was induced in Sprague-Dawley rats by middle cerebral artery occlusion for 2 h followed by a 24 h reperfusion. Before this surgery, folic acid (FA) was administered to the drug treatment group by gavage for 11 days. After a 24 h reperfusion, behavioural testing, vascular function, NO concentration and superoxide dismutase activity in the serum were determined. In addition, the infarct size of the brain was also detected. The mRNA of eNOS, nNOS, GTP cyclohydrolase I (GTPCH), P22phox and xanthine oxidase (XO) in the ischemic region were detected by RT-PCR, and nitrotyrosine (Tyr-NO2) was detected using Western blot analysis. The results showed that LRIP, FA and FA+LRIP all could improve behavioural score, and increase NO–mediated endothelium-dependent vasomotor responses, reduce infarction of rats subjected to IRI. Western blot and RT-PCR analyses showed that the Tyr-NO2 levels and the mRNA expression of NADPH oxidase catalytic subunit P22phox and XO were up-regulated in the ischemic brain, which was significantly inhibited by LRIP, FA and FA+LRIP. The mRNA expression of the rate-limiting enzyme in BH4 synthesis, GTPCH, was down-regulated in the ischemic brain, which could be significantly augmented by LRIP and FA+LRIP. It can be concluded that IRI induces eNOS uncoupling in the cerebral ischemic region and LRIP partially reverses the eNOS uncoupling induced by IRI.

Animals , Brain/blood supply , Brain/metabolism , Brain Ischemia/metabolism , Brain Ischemia/prevention & control , Extremities/blood supply , Ischemic Postconditioning/methods , Male , Nitric Oxide Synthase Type III/metabolism , Rats , Rats, Sprague-Dawley , Reperfusion Injury/metabolism , Reperfusion Injury/prevention & control
Int. j. morphol ; 30(3): 979-985, Sept. 2012. ilus
Article in English | LILACS | ID: lil-665512


NMDAR (N-methyl-D-aspartate receptor) is one subtype of ionotrophic glutamate receptor which is extensively distributed in the central nervous system (CNS). In the mammalian CNS, NMDAR serves prominent roles in the pathophysiologic process of cerebral ischemia. This study aimed to investigate the pattern of expression of protein and gene of the excitatory neurotransmitter NMDAR in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. 120 rats were randomly divided into 6 groups (20 animals each): control - no surgery; sham - simulation of surgery; ischemic - focal ischemia for 1 hour, without reperfusion; ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. Ten animals from each experimental group were used to establish the volume of infarct. Transient focal cerebral ischemia was obtained in rats by occlusion of the middle cerebral artery with an intraluminal suture. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the protein and gene NMDA were evaluated by immunohistochemistry and quantitative real-time PCR, respectively. Increases in the protein and gene NMDA receptor in the ischemics areas were observed and these increases were reduced by hypothermia and ketoprofen. The increase in the NMDA receptor protein and gene expression observed in the ischemic animals was reduced by neuroprotection (hypothermia and ketoprofen). The NMDA receptor increases in the ischemic area suggests that the NMDA mediated neuroexcitotoxicity plays an important role in cell death and that the neuroprotective effect of both, hypothermia and ketoprofen is directly involved with the NMDA...

NMDAR (N-metil-D-aspartato) es un tipo de receptor de glutamato ionotrópico y está ampliamente distribuido en el sistema nervioso central (SNC). En el SNC de mamíferos, NMDAR se destaca de manera importante en el proceso fisiopatológico de la isquemia cerebral. Este estudio tuvo como objetivo investigar el patrón de expresión de proteínas y genes para el NMDA neurotransmisor excitatorio experimental de la isquemia cerebral focal y el vacío en la neuroprotección con hipotermia y ketoprofeno. Se dividieron 120 ratas aleatoriamente en grupos de 6 animales cada uno (20): Control - sin cirugía; Sham - simulación de cirugía; isquémicas - isquemia focal durante 1 hora, sin reperfusión isquémica; hipotermia intra-isquémica; isquemia; previa aplicación de ketoprofeno intravenoso, e hipotermia isquémica y ketoprofeno. Diez animales de cada grupo experimental fueron utilizados para establecer el volumen de infarto.La isquemia cerebral focal transitoria fue obtenida en ratas mediante oclusión de la arteria cerebral media con una sutura intraluminal. El volumen de infarto fue medido mediante análisis morfométrico de las áreas de infarto definidas por cloruro de trifenil tetrazolio y patrones de expresión de la proteína y el gen de NMDA, fueron evaluados por inmunohistoquímica y PCR cuantitativa en tiempo real, respectivamente. Se observaron aumentos en la proteína y en el gen del receptor de NMDA en las áreas isquémicas y estos aumentos fueron reducidos por la hipotermia y ketoprofeno. El aumento de la proteína del receptor de NMDA y la expresión génica observada en los animales isquémicos fue reducido mediante hipotermia y ketoprofeno. Los aumentos del receptor de NMDA en el área isquémica sugiere que la neuro excitotoxicidad mediada por NMDA desempeña un papel importante en la muerte celular y que el efecto neuroprotector de ambos, hipotermia y ketoprofeno está directamente relacionado al NMDA...

Animals , Rats , Brain Ischemia/metabolism , Brain Ischemia/pathology , Receptors, N-Methyl-D-Aspartate/metabolism , Ketoprofen/metabolism , Neuroprotective Agents/metabolism , Gene Expression , Hypothermia , Immunohistochemistry , Real-Time Polymerase Chain Reaction , Receptors, N-Methyl-D-Aspartate/genetics
Article in English | WPRIM | ID: wpr-14964


Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a dose- and time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.

Animals , Mice , Rats , Blood-Brain Barrier/metabolism , Brain Ischemia/metabolism , CA1 Region, Hippocampal/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Gerbillinae , Intracellular Signaling Peptides and Proteins/administration & dosage , Lipid Peroxidation , Malondialdehyde/metabolism , Neuroprotective Agents/administration & dosage , Oncogene Proteins/administration & dosage , Oxidative Stress , Prosencephalon/drug effects , Recombinant Fusion Proteins/administration & dosage , tat Gene Products, Human Immunodeficiency Virus/administration & dosage
Indian J Biochem Biophys ; 2011 Apr; 48(2): 73-81
Article in English | IMSEAR | ID: sea-135303


Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. PPAR-alpha is involved in wound healing, stimulation of lipid and folic acid catabolism, inflammation control, inhibition of ureagenesis and peroxisome proliferation. The PPAR/ is involved wound healing, cell proliferation, embryo implantation, adipocyte differentiation, myelination alteration and apoptosis. The PPAR is involved in fat, lipid and calorie utilization, sugar control, inflammation control and macrophage (MQ) matutation. Homocysteine (Hcy) binds to nuclear peroxisome proliferator activated receptor. Increase in PPAR expression decreases the level of nitrotyrosine and increases endothelial nitric oxide concentration, decreases metalloproteinase activity and expression as well as elastinolysis and reverses Hcy-mediated vascular dysfunction. The PPAR initially recognized as a regulator of adipocyte development has become a potential therapeutic target for the treatment of diverse disorders. In addition, the activation of PPAR receptor ameliorates neurodegenerative disease. This review focuses on the recent knowledge of PPAR in neuroprotection and deals with the mechanism of neuroprotection of central nervous system disorder by PPAR.

Animals , Brain Ischemia/metabolism , Brain Ischemia/pathology , Brain Ischemia/therapy , Cell Death , Central Nervous System/cytology , Central Nervous System/metabolism , Central Nervous System/pathology , Cytoprotection , Humans , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Neurodegenerative Diseases/therapy , Neurons/cytology , Neurons/pathology , Neuroprotective Agents/metabolism , PPAR gamma/metabolism
Arq. bras. neurocir ; 29(2): 58-63, jun. 2010.
Article in Portuguese | LILACS | ID: lil-583496


Procede-se a uma revisão das alterações bioquímicas e hemodinâmicas causadas pela isquemia do tecido nervoso. Trata-se de uma visão introdutória que aborda a fisiopatologia e as repercussões da terapêutica sobre a hemodinâmica encefálica.

This is a review on the biochemical and hemodynamic alterations caused by the isquemia in the neural tissue. This text introduce the pathophysiology and the therapeutic effects of this ischemic alterations.

Hemodynamics , Intracranial Hypertension , Brain Ischemia/classification , Brain Ischemia/complications , Brain Ischemia/physiopathology , Brain Ischemia/metabolism , Brain Ischemia/therapy , Acidosis