Kukoamine A activates Akt/GSK-3ß signaling pathway to inhibit oxidative stress and relieve myocardial ischemia-reperfusion injury

Purpose: Myocardial ischemia/reperfusion (MI/R) injury refers to a pathological condition of treatment of myocardial infarction. Oxidative stress and inflammation are believed to be important mechanisms mediating MI/R injury. Kukoamine A (KuA), a sperm, is the main bioactive component extracted from the bark of goji berries. In this study, we wanted to investigate the possible effects of KuA on MI/R injury. Methods: In this experiment, all rats were divided into sham operation group, MI/R group, KuA 10 mg + MI/R group, KuA 20 mg + MI/R group. After 120 min of ischemia/reperfusion treatment, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximal rates of rising and fall of left ventricular pressure (±dp/dtmax), and ischemic area were detected. Serum samples of rats in each group were collected. The enzyme activities of catalase (CAT), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), levels of malondialdehyde (MDA), CK muscle/brain (CK-MB), tumor necrosis factor (TNF), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were detected using enzyme-linked immunosorbent assay (ELISA). The apoptosis of myocardium in each group was detected according to the instructions of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expressions of mammalian target of glycogen synthase kinase-3ß (GSH-3ß) and protein kinase B (Akt) mRNA level in myocardial tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR). Results: MI/R rats showed a significant increase in oxidative stress and inflammation. In addition, we showed that KuA significantly improved the myocardial function such as LVSP, left ventricular ejection fraction, +dp/dt, and -dp/dt. Here, it attenuated dose-dependent histological damage in ischemia-reperfused myocardium, which is associated with the enzyme activities of SOD, GSH-PX, and levels of MDA, IL-6, TNF-α, L-1ß. Conclusions: KuA inhibited gene expression of Akt/GSK-3ß, inflammation, oxidative stress and improved MR/I injury. Taken together, our results allowed us to better understand the pharmacological activity of KuA against MR/I injury.

Remote ischemic conditioning improves rat brain antioxidant defense in a time-dependent mechanism

ABSTRACT Purpose To clarify the best protocol for performing remote ischemic conditioning and to minimize the consequences of ischemia and reperfusion syndrome in brain, the present study aimed to evaluate different time protocols and the relation of the organs and the antioxidant effects of this technique. Methods The rat's left femoral artery was clamped with a microvascular clamp in times that ranged from 1 to 5 minutes, according to the corresponding group. After the cycles of remote ischemic conditioning and a reperfusion of 20 minutes, the brain and the left gastrocnemius were collected. The samples were used to measure glutathione peroxidase, glutathione reductase and catalase levels. Results In the gastrocnemius, the 4-minute protocol increased the catalase concentration compared to the 1-minute protocol, but the latter increased both glutathione peroxidase and glutathione reductase compared to the former. On the other hand, the brain demonstrated higher catalase and glutathione peroxidase in 5-minute group, and the 3-minute group reached higher values of glutathione reductase. Conclusions Remote ischemic conditioning increases brain antioxidant capacity in a time-dependent way, while muscle presents higher protection on 1-minute cycles and tends to decrease its defence with longer cycles of intermittent occlusions of the femoral artery.

Effects of icariside II on brain tissue oxidative stress and Nrf2/HO-1 expression in rats with cerebral ischemia-reperfusion injury

Purpose:To investigate the effects of icariside II on brain tissue oxidative stress and Nrf2/HO-1 expression in rats with cerebral ischemia-reperfusion injury (CIRI).Methods:One hundred SD rats were randomly divided into sham-operated, model, and 5, 10 and 20 mg/kg icariside II groups, 20 rats in each group. The middle cerebral artery occlusion model (ischemia for 2 h followed by reperfusion for 24 h) was established in the later 4 groups. In later 3 groups, at reperfusion beginning, the rats were intragastrically administrated with 5, 10 and 20 mg/kg icariside II, respectively. After 24 h of reperfusion, the neurological severity score, cerebral water content and cerebral infarction volume, brain tissue oxidative stress indexes and Nrf2 and HO-1 protein expressions were determined.Results:Compared with model group, in 20 mg/kg icariside II group the neurological severity score, cerebral water content and cerebral infarction volume, brain tissue ROS content and MDA level were significantly decreased (P<0.05), and the brain tissue SOD, GSH-Px and catalase levels and Nrf2 and HO-1 protein levels were significantly increased (P<0.05).Conclusion:Icariside II can alleviate the CIRI in rats through reducing brain tissue oxidative stress and improving Nrf2/HO-1 expression.(AU)

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

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.(AU)

Preconditioning with L-alanyl-L-glutamine in a Mongolian Gerbil model of acute cerebral ischemia/reperfusion injury / Pré-condicionamento com L-alanil-L-glutamina em modelo de isquemia/reperfusão cerebral aguda em Gerbils da Mongólia

PURPOSE: To investigate the effect of L-alanyl-L-glutamine (L-Ala-Gln) preconditioning in an acute cerebral ischemia/reperfusion (I/R) model in gerbils. METHODS: Thirty-six Mongolian gerbils (Meriones unguiculatus), (60-100g), were randomized in 2 groups (n=18) and preconditioned with saline 2.0 ml (Group-S) or 0.75g/Kg of L-Ala-Gln, (Group-G) administered into the femoral vein 30 minutes prior to I/R. Each group was divided into three subgroups (n=6). Anesthetized animals (urethane, 1.5g/Kg, i.p.) were submitted to bilateral occlusion of common carotid arteries during 15 minutes. Samples (brain tissue and arterial blood) were collected at the end of ischemia (T0) and after 30 (T30) and 60 minutes (T60) for glucose, lactate, myeloperoxidase (MPO), thiobarbituric acid reactive substances (TBARS), glutathione (GSH) assays and histopathological evaluation. RESULTS: Glucose and lactate levels were not different in studied groups. However glycemia increased significantly in saline groups at the end of the reperfusion period. TBARS levels were significantly different, comparing treated (Group-G) and control group after 30 minutes of reperfusion (p<0.05) in cerebral tissue. Pretreatment with L-Ala-Gln promoted a significant increase in cerebral GSH contents in Group-G at T30 (p<0.001) time-point compared with Group-S. At T30 and T60, increased levels of GSH occurred in both time-points. There were no group differences regarding MPO levels. Pyknosis, presence of red neurons and intracellular edema were significantly smaller in Group-G. CONCLUSION: Preconditioning with L-Ala-Gln in gerbils submitted to cerebral ischemia/reperfusion reduces oxidative stress and degeneration of the nucleus (pyknosis) and cell death (red neurons) in the cerebral tissue.(AU)

OBJETIVO: Investigar o efeito do pré-condicionamento com L-alanil-L-glutamina (L-Ala-Gln) em gerbils submetidos à isquemia/reperfusão (I/R) cerebral aguda. MÉTODOS: Trinta e seis gerbils (Meriones unguiculatus) (60-100g) foram divididos em dois grupos (n=18) e pré-condicionados com 2,0 ml de soro fisiológico (Grupo-S) ou 0.75g/kg de L-Ala-Gln, (Grupo-G), administrados na veia femoral 30 minutos antes da I / R. Cada grupo foi dividido em três subgrupos (n=6).Animais anestesiados com uretano, 1.5g/kg, ip, foram submetidos à oclusão bilateral das artérias carótidas comuns, durante 15 minutos. Amostras (tecido cerebral e sangue arterial) foram coletadas no final da isquemia (T0) e após 30 (T30) e 60 minutos (T60) para a aferição das concentrações de glicose, lactato, mieloperoxidase (MPO), substâncias reagentes ao ácido tiobarbitúrico (TBARS), glutationa (GSH) e avaliação histopatológica. RESULTADOS: As concentrações de glicose e lactato não foram diferentes nos grupos estudados; a glicemia aumentou significativamente no Grupo-S ao final da reperfusão. Concentrações de TBARS no tecido cerebral foram significativamente diferentes, comparando os Grupos G e S, no T30 (p <0,05). O pré-tratamento com L-Ala-Gln promoveu um aumento significativo de GSH cerebral no Grupo-G comparado ao Grupo-S no T30 (p <0,001). Houve aumento das concentrações de GSH no T30 e T60 no Grupo-G. Não houve diferenças quanto as concentrações de MPO. Picnose, presença de neurônios vermelhos e edema intracelular foram significativamente menores no Grupo-G. CONCLUSÃO: O pré-condicionamento com L-Ala-Gln em gerbils submetidos à isquemia/reperfusão cerebral reduz o estresse oxidativo, a degeneração nuclear (picnose) e morte celular (neurônios vermelhos) no tecido cerebral.(AU)

Transient middle cerebral artery occlusion in rats as an experimental model of brain ischemia / Modelo experimental de isquemia cerebral em ratos por obliteração temporária da artéria cerebral média

PURPOSE: To assess a rat model of cerebral ischemia induced by occlusion of the middle cerebral artery and its effect on the area of cerebral infarction. METHODS: Brain ischemia was induced in 52 male Wistar rats by introduction of a 3-0 nylon suture into the middle cerebral artery for either 90 (n=28) or 120 (n=24) minutes. Ischemic injury volume was determined by TTC staining, digital photography and analysis with the Image J software. Statistical analysis employed Student's t test and the Mann-Whitney U test. RESULTS: The groups were similar in terms of weight (p=0.59). The length of thread inserted was 14.7 mm in the 90 min group and 20.2 mm in the 120 min group (p=0.37). Ischemic injury was detected in 11 animals (39 percent) after 90 min and 11 (45 percent) after 120 min (p=0.77). In animals exhibiting injury, filament length was 16.1±11 mm (90 min) vs. 21.9±7.4 mm (120 min) (p=0.15). The mean infarction zone volume was greater after 120 (259.2 mm³) than after 90 min (162.9 mm³) (p=0.04). The neurological deficit score for the 90 and 120 min groups was 2.0 and 2.4, respectively (p=0.84). CONCLUSION: The experimental model induced significant ischemic cerebral injury in both groups.(AU)

OBJETIVO: Avaliar o modelo de isquemia cerebral por oclusão da artéria cerebral média, mediante introdução de fio intraluminal por 90 e 120 minutos, e seu efeito sobre a área de infarto cerebral em ratos. MÉTODOS: 52 ratos machos Wistar foram submetidos à isquemia cerebral por introdução de fio de nylon 3-0 na artéria cerebral média por 90 ou 120 minutos. O volume da lesão isquêmica foi determinado pelo corante TTC, fotografia digital e utilização do programa ImageJ. Na análise estatística, foi utilizado o teste t- student e o U de Mann-Whitney. RESULTADOS: O comprimento do fio introduzido foi de 14,7 mm no grupo 90 minutos e 20,2 mm no grupo 120 minutos. Lesão isquêmica foi detectada em 11 animais (39 por cento) no grupo que de 90 minutos e 11 (45 por cento) do grupo de 120 minutos. Nos animais que apresentaram lesão, o comprimento do fio foi de 16,1±11 mm (90 minutos) e 21,9±7,4 mm (120 minutos). O volume médio da área de infarto foi maior no grupo 120 minutos do que no grupo 90 minutos. O escore de déficit neurológico foi de 2,0 no grupo 90 minutos e de 2,4 no grupo 120 minutos. CONCLUSÃO: O modelo experimental estudado induz lesão isquêmica cerebral significativa em ambos os grupos.(AU)

Avaliação de dois modelos experimentais de isquemia e reperfusão cerebral em ratos com oclusão temporária carotídea associada ou não à oclusão vertebral

PURPOSE: To evaluate reproducibility of two experimental brain ischemia and reperfusion models. METHODS: Sixty rats were randomly distributed to three experimental groups: I - (20) temporary clipping of the left carotid artery; II - (20) cauterization of vertebral arteries and temporary clipping of the left carotid artery; "Sham" - (20) simulation group, with neither ischemia nor reperfusion. All animals had permanent occlusion of right carotid artery and the three groups were subdivided into two reperfusion periods: A - 60 minutes e B - 120 minutes. Parameters verified were: mean systemic arterial blood pressure and carotid blood flow measurement; brain malondialdehyde measurement by TBARS assay and histological evaluation of the brain hemisphere submitted to ischemia and reperfusion. A supplementary brain angiography study was also conducted on 5 additional animals. RESULTS: There was no significant difference in brain malondialdehyde dose and in frequency and severity of histological brain alterations among the three groups. In groups GI and GII, mean arterial blood pressure was significantly higher during the ischemia period. Blood flow in the periods before and after clipping increased in Groups IA and IIB, decreased in Group IB and did not alter in Group IIA. Supplementary study angiographs showed blood supply to brain through collateral circulation. CONCLUSION: The ischemia and reperfusion models studied did not show consistent change in brain injury markers as far as lipoperoxide production and histological damage are concerned.

OBJETIVO: Avaliar a reprodutibilidade de dois modelos experimentais de isquemia e reperfusão cerebral. MÉTODOS: 60 ratos foram distribuídos, aleatoriamente, em três grupos experimentais, com 20 animais cada: I - pinçamento temporário de artéria carótida esquerda; II - cauterização prévia das artérias vertebrais e pinçamento temporário da artéria carótida esquerda; simulado - sem isquemia nem reperfusão. Todos os animais tiveram oclusão definitiva de artéria carótida direita e os três grupos foram subdivididos em dois períodos de reperfusão: A - 60 minutos e B - 120 minutos. Os parâmetros verificados foram: medidas de pressão arterial média sistêmica e fluxo sangüíneo carotídeo; medida de malondialdeído cerebral através do teste TBARS e avaliação histológica do hemisfério cerebral submetido à isquemia e reperfusão. Foi feito também um estudo complementar com angiografia cerebral em 5 animais adicionais. RESULTADOS: Não houve diferenças significativas nas dosagens de malondialdeído cerebral e na freqüência e gravidade das alterações histológicas cerebrais entre os três grupos. Nos grupos GI e GII, a PAM foi significantemente maior no período de isquemia. O fluxo sangüíneo entre os períodos pré e pós-pinçamento aumentou nos grupos IA e IIB, diminuiu no grupo IB e no grupo IIA manteve-se inalterado. As angiografias do estudo complementar mostraram aporte sangüíneo para cérebro através de circulação colateral. CONCLUSÃO: Os modelos de isquemia e reperfusão estudados não demonstraram alterações consistentes de marcadores de lesão cerebral, seja quanto à produção de lipoperóxidos ou de lesões histológicas.

Recruitment of neutrophils across the blood-brain barrier: the role of posttraumatic hepatic ischemia

PURPOSE: To study the effects of total hepatic ischemia, and reperfusion on the accumulation of neutrophils in the brain of rats submitted to normovolemic conditions as well as to controlled hemorrhagic shock state. METHODS: Thirty two adult male Wistar rats, were divided into four groups: the Control group, was submitted to the standard procedures for a period of 60 min of observation; Shock group, was submitted to controlled hemorrhagic shock (mean arterial blood pressure=40mmHg, 20min) followed by volemic resuscitation (lactated Ringer's solution + blood, 3:1) and reperfusion for 60min; Pringle group, was submitted to total hepatic ischemia for 15min and reperfusion for 60min. The total group was submitted to controlled hemorrhagic shock for 20min followed by volemic resuscitation (lactated Ringer's solution + blood, 3:1), total hepatic ischemia for 15min and reperfusion for 60min. Measurements of serum lactate and base excess were used to characterize the hemorrhagic shock state with low tissue perfusion. The counting of neutrophils on the brain was performed after the euthanasia of animals. RESULTS: The values for the counting of neutrophils on the brain indicate that did not occur difference among studied groups (p=0.196) (Control 0.12± 0.11, Shock 0.12± 0.13, Pringle 0.02± 0.04, Total 0.14± 0.16). CONCLUSION: Hemorrhagic shock associated to total hepatic ischemia for 15 minutes, followed by 60 minutes of reperfusion, did not causes significant neutrophils accumulation in the brain of rats.

OBJETIVO: estudar o efeito da isquemia e reperfusão hepática total sobre acúmulo de neutrófilos no cérebro de ratos, em condições de normalidade e submetidos ao estado de choque hemorrágico controlado. MÉTODOS: Foram utilizados 32 ratos Wistar, machos, distribuídos em quatro grupos de oito animais cada: Grupo Controle, submetido aos procedimentos padrões com um período de 60 minutos de observação; Grupo Choque, submetido a choque hemorrágico controlado (PAM=40mmHg, 20 min) seguido de reposição volêmica (Ringer lactato + sangue, 3:1) e reperfusão (60 min); Grupo Pringle, submetido à isquemia hepática total (15 min) e reperfusão (60 min); Grupo Total submetido a choque hemorrágico controlado (15 min) seguido de reposição volêmica (Ringer lactato + sangue, 3:1) mais isquemia hepática total (15 min) e reperfusão (60 min). A dosagem do lactato arterial e déficit de base foram utilizados para caracterizar o estado de choque hemorrágico com baixa perfusão tecidual. Após a eutanásia dos animais, procedeu-se à contagem de neutrófilos no cérebro. RESULTADOS: a contagem de neutrófilos mostrou que não houve diferença estatística entre os grupos (p=0.196). Grupo Controle 0.12± 0.11, Choque 0.12± 0.13, Pringle 0.02± 0.04 e Total 0.14± 0.16. CONCLUSÃO: Em ratos submetidos a estado de choque hemorrágico controlado associado à isquemia hepática total de 15 minutos, seguido de 60 minutos de reperfusão, não ocorreu acúmulo significativo de neutrófilos no cérebro.

Glial reaction in the hippocampus after global cardiogenic ischemia

Many experimental surgerical procedures have been perfomed in the analyse of the phenomenon of brain trophism and plasticity, however undesirable intercorrence can occour leading to specific changes in the results that should be taken into attention. To study this issue we have promoted a transient cardiogenic interruption of the blood flow together with a transient occlusion of the bilateral common carotid arteries (2VO) in rats and analysed the state of activation of astrocyte and microglia by means of the glial fibrillary acidic protein (GFAP) and OX42 immunohistochemistry, respectively. Rats were submitted to incomplete global cerebral ischemia (IGCI) by occlusion of the bilateral carotid arteries for 30 minutes. During the IGCI surgical, some rats received a higher dose of the chloral hydrate anaesthesia which promoted a cardiogenic interruption of the blood flow (CIBF) for a period of 10 minutes followed by and prompt reperfusion. During that period, animals were submited to a cardiac massage and ventilated. Sham operation were made in control animals. Rats were killed and their brains processed 14 days after the surgery. The animals that have received a IGCI showed a slight astroglial and microglial reaction in all subfields of the hippocampal formation, however the animal submitted to CIBF showed a massive infiltration of the reactive astrocyte and microglia in CA1 subfield. This results demonstrated that a transient occlusion of the bilateral common carotid arteries leads to activation of glial cells in the hippocampus, however this response can be remarkable changed in animal developing a transient systemic hypoperfusion during surgery. Thus, an accurated monitoration of the hemodinamic condition of the animal has to be done in experimental models of brain ischemia and the results have to be analysed in view of this aspect.

Muitos procedimentos experimentais são desenvolvidos para analisar o fenômeno do trofismo e plasticidade cerebral. Entretanto, eventos indesejáveis durante os procedimentos cirúrgicos podem ocorrer promovendo mudanças específicas nos resultados que devem ser levadas em consideração. Para estudar possibilidade, interrupção cardiogênica transitória do fluxo sangüíneo junto com a oclussão bilateral transitória das artérias caróridas comum (2VO) foi realizada em ratos e o estado de ativação de astrócitos e microglia foi analisado através da imunohistoquímica da proteína fibrilar ácida glial (GFAP) and OX42, respectivamente. Os ratos foram submetidos à isquemia cerebral global incompleta (IGCI) pela oclusão bilateral das artérias caróditas por 20 minutos. Durante o procedimento cirurgico da IGCI, alguns ratos receberam uma alta dose de anestésico de hidrato de cloral que promoveu uma interrupção cardiogênica do fluxo sangüíneo (CIBF) por um período de 10 minutos. Durante este período os ratos foram submetidos a massagem cardíaca e ventilados. Uma operação simulada foi realizada nos ratos controles. Os ratos foram mortos 14 dias após a cirurgia e seus cérebros processados para a imunohistoquímica. Os animais que receberam uma IGCI apresentaram uma leve reação astroglial e microglial em todos os sub-campos da formação hipocampal, entretanto os animais submetidos à CIBF mostraram uma infiltração massiça de astrócito e microglia reativos no sub-campo CA1. Estes resultados demonstram que oclusão bilateral transitória das artérias carótidas comum ativam as células gliais no hipocampo, entretanto esta resposta pode ser mudada substancialmente nos animais desenvolvendo hipoperfusão sistêmica durante o procedimento cirúrgico. Então, monitoramente acurado das condições hemodinâmicas do animal deve ser feito em modelos de isquemia cerebral e os resultados devem ser analisados em vista deste aspecto.