The effects of local ischemic preconditioning and topical hypothermia in renal ischemia/reperfusion injury in rats

Abstract Purpose: Topical hypothermia and local ischemic preconditioning have been shown to reduce renal ischemia-reperfusion (I/R) injury individually. We examined whether combination of both strategies lessens renal I/R injury. Methods: Post right nephrectomy, 40 male Wistar rats were randomly assigned to five experimental protocols performed in the left kidney: topical hypothermia without ischemia (TH), warm ischemia (IR), ischemic preconditioning followed by warm ischemia (IPC+IR), cold ischemia (TH+IR), and ischemic preconditioning followed by cold ischemia (IPC+TH+IR). Eight randomly assigned right kidneys constituted the control group. After 240 min of reperfusion, the left kidney was retrieved to evaluate histological changes, lipid peroxidation and antioxidant enzymes activity. Serum was collected to evaluate urea and creatinine. Results: IPC+TH+IR group revealed no difference to any other group subjected to ischemia in relation to histological changes, lipid peroxidation and antioxidant enzymes activity. Creatinine was lower in IPC+TH+IR group compared with IPC+IR, but showed no difference compared to TH+IR group. Conclusions: Combination of local ischemic preconditioning (IPC) and topical hypothermia conferred no protection in renal I/R injury. Moreover, local IPC solely followed by warm ischemia impaired renal function more than warm ischemia alone.

The renoprotective effect of oral Tadalafil pretreatment on ischemia/reperfusion injury in rats

Abstract Purpose: To evaluate the effect of tadalafil in renal ischemia/reperfusion (I/R) injury in rats Methods: Group I/R saline rats (n=6) were subjected to 45 minutes of left renal ischemia and treated with saline; the I/R tadalafil rats (n=6) received oral 10mg/kg tadalafil microemulsion one hour before ischemia. In both groups, 8 hours after ischemia, laboratory analysis were performed Results: Better tissue perfusion was lower in ischemic left/kidney than in right/kidney in saline group, suggesting reduced kidney clearance. Fluorescence in left/kidneys of tadalafil treated rats was lower than in right/kidneys (difference not significant). The fluorescence signal intensity in kidneys of tadafil treated rats was higher than in saline rats. TNF-α levels were significantly lower in I/R tadalafil group rats compared to I/R saline group (154±10.3 vs 391.3±12.3), as well as IL-1β (163.4±13.2 vs 279±11.5pg/dL), and IL-6 (122.9±8.1 vs 173.7±6.3 respectively; p=0.0001). Urea, creatinine and C-reactive protein were significantly lower in tadafil treated rats then in saline group Conclusion: Tadalafil therapy decreased the expression of circulating pro-inflammatory cytokines in a renal I/R rodent model, while improving kidney function proofs.

Study on the effect of black cumin (Nigella sativa Linn.) on experimental renal ischemia-reperfusion injury in rats

PURPOSE: To evaluate the effect of Black cumin (Nigella sativa Linn.) pre-treatment on renal ischemia/reperfusion (I/R) induced injury in the rats.METHODS: A total of 40 male Wistar rats were randomly allocated into five equal groups including Sham, I/R model and three I/R+ Black cumin (0.5, 1 and 2%)-treated groups. I/R groups' kidneys were subjected to 60 min of global ischemia at 37°C followed by 24 h of reperfusion. At the end of reperfusion period, the rats were euthanized. Superoxide dismutase, catalase and glutathione peroxidase activities as well as reduced glutathione and renal malondialdehyde contents were determined in renal tissues. Kidney function tests and histopathological examination were also performed.RESULTS: High serum creatinine, blood urea nitrogen and uric acid as well as malondialhehyde (MDA) levels, and low antioxidant enzyme activities were observed in I/R rats compared to the sham rats. Pre-treatment with Black cumin for three weeks prior to IR operation improved renal function and reduced I/R induced renal inflammation and oxidative injury. These biochemical observations were supported by histopathological test of kidney sections.CONCLUSION:Black cumin significantly prevented renal ischemia/reperfusion induced functional and histological injuries.

Effect of remote ischemic preconditioning in the expression of IL-6 and IL-10 in a rat model of liver ischemia-reperfusion injury

PURPOSE: To study the effect of remote ischemic preconditioning (RIPC) in ischemia-reperfusion (I/R) liver injury and in the expression of IL-6 and IL-10 in a rat model. METHODS: Thirty-six male rats were divided in three groups: Sham; I/R injury, a 45 minutes lobar liver ischemia and reperfusion; and RIPC, six cycles of four minutes of ischemia and four minutes of reperfusion on the right hindlimb followed by a 45 minutes lobar liver ischemia and reperfusion. Tissue and blood samples were collected after 1h and 3h of reperfusion for histopathological study, plasma cytokines and alanine aminotransferase (ALT) measurement. RESULTS: The histopathological study demonstrated a significant reduction in liver necrosis in the RIPC group (p<0,001). The ALT levels were also significant lower in the RIPC group (p<0.01). The cytokines assessment showed that IL-6 levels were increased in the RIPC group after 1h of reperfusion, in comparison to the I/R group (p<0.05). Interleukin-10 levels in RIPC groups did not differ significantly from I/R group. CONCLUSIONS: Remote ischemic preconditioning is effective in decreasing liver necrosis in a rat model of ischemia-reperfusion. The IL-6 expression is up-regulated and peaked at 60 min of reperfusion. There was no difference in IL-10 expression between the groups. .

Renal biomarkers of male and female Wistar rats (Rattus norvegicus) undergoing renal ischemia and reperfusion

PURPOSE: To investigate biomarkers of acute renal injury in Wistar rats, subjected to left renal ischemia for 10 minutes, and then compare reperfusion at 24 hours, and at 5, 7, 14 and 21 days after the procedure. METHODS: Eight female and male rats between 60 and 81 days old were used in the Central Animal Facility of the UFMS. Assessed biomarkers included urine protein, urea, creatinine, glucose, sodium, potassium, urine alkaline phosphatase and gamma-glutamyl transferase activities, and protein-to-creatinine ratio; and in serum: urea, creatinine, sodium and potassium, fractional excretion of sodium, potassium, urine flow and creatinine clearance. RESULTS: Greater variance was observed in the parameters at 24 hours and at five days (p<0.05) after reperfusion. On the 21st day, these parameters approximated those obtained for the control group. CONCLUSIONS: Renal ischemia for 10 minutes was sufficient to raise urine levels of protein, glucose, fractional excretion of potassium, urea, creatinine clearance, urine activity of gamma-glutamyltransferase and alkaline phosphatase enzymes in the first 24 hours, up to five days after reperfusion, which may indicate risk of acute kidney injury, according to the RIFLE classification. .

Evaluation of the effects of ischemic preconditioning on the hematological parameters of rats subjected to intestinal ischemia and reperfusion

OBJECTIVES: Intestinal ischemia/reperfusion often leads to acute lung injury and multiple organ failure. Ischemic preconditioning is protective in nature and reduces tissue injuries in animal and human models. Although hematimetric parameters are widely used as diagnostic tools, there is no report of the influence of intestinal ischemia/reperfusion and ischemic preconditioning on such parameters. We evaluated the hematological changes during ischemia/reperfusion and preconditioning in rats. METHODS: Forty healthy rats were divided into four groups: control, laparotomy, intestinal ischemia/reperfusion and ischemic preconditioning. The intestinal ischemia/reperfusion group received 45 min of superior mesenteric artery occlusion, while the ischemic preconditioning group received 10 min of short ischemia and reperfusion before 45 min of prolonged occlusion. A cell counter was used to analyze blood obtained from rats before and after the surgical procedures and the hematological results were compared among the groups. RESULTS: The results showed significant differences in hematimetric parameters among the groups. The parameters that showed significant differences included lymphocyte, white blood cells and granulocyte counts; hematocrit; mean corpuscular hemoglobin concentration; red cell deviation width; platelet count; mean platelet volume; plateletcrit and platelet distribution width. CONCLUSION: The most remarkable parameters were those related to leukocytes and platelets. Some of the data, including the lymphocyte and granulocytes counts, suggest that ischemic preconditioning attenuates the effect of intestinal ischemia/reperfusion on circulating blood cells. Our work contributes to a better understanding of the hematological responses after intestinal ischemia/reperfusion and IPC, and the present findings may also be used as predictive values. .

Pentoxifylline associated to hypertonic saline solution attenuates inflammatory process and apoptosis after intestinal ischemia/reperfusion in rats

PURPOSE: To evaluate intestinal inflammatory and apoptotic processes after intestinal ischemia/reperfusion injury, modulated by pentoxifylline and hypertonic saline. METHODS: It was allocated into four groups (n=6), 24 male Wistar rats (200 to 250g) and submitted to intestinal ischemia for 40 min and reperfusion for 80 min: IR (did not receive any treatment); HS group (Hypertonic Saline, 4ml/kg-IV); PTX group (Pentoxifylline, 30mg/kg-IV); HS+PTX group (Hypertonic Saline and Pentoxifylline). All animals were heparinized (100U/kg). At the end of reperfusion, ileal fragments were removed and stained on hematoxylin-eosin and histochemical studies for COX-2, Bcl-2 and cleaved caspase-3. RESULTS: The values of sO2 were higher on treated groups at 40 minutes of reperfusion (p=0.0081) and 80 minutes of reperfusion (p=0.0072). Serum lactate values were lower on treated groups after 40 minutes of reperfusion (p=0.0003) and 80 minutes of reperfusion (p=0.0098). Morphologic tissue injuries showed higher grades on IR group versus other groups: HS (p=0.0006), PTX (p=0.0433) and HS+PTX (p=0.0040). The histochemical study showed lesser expression of COX-2 (p=0.0015) and Bcl-2 (p=0.0012) on HS+PTX group. A lower expression of cleaved caspase-3 was demonstrated in PTX (p=0.0090; PTXvsIR). CONCLUSION: The combined use of pentoxifylline and hypertonic saline offers best results on inflammatory and apoptotic inhibitory aspects after intestinal ischemia/reperfusion. .

Temporal relationship of serum markers and tissue damage during acute intestinal ischemia/reperfusion

OBJECTIVE: It is essential to identify a serological marker of injury in order to study the pathophysiology of intestinal ischemia reperfusion. In this work, we studied the evolution of several serological markers after intestinal ischemia reperfusion injury in rats. The markers of non-specific cell damage were aspartate aminotransferase, alanine aminotransaminase, and lactic dehydrogenase, the markers of inflammation were tumor necrosis factor alpha, interleukin-6, and interleukin-1 beta, and the markers of intestinal mucosal damage were intestinal fatty acid binding protein and D-lactate. We used Chiús classification to grade the histopathological damage. METHODS: We studied 35 Wistar rats divided into groups according to reperfusion time. The superior mesenteric artery was clamped for 30 minutes, and blood and biopsies were collected at 1, 3, 6, 12, 24, and 48 hours after reperfusion. We plotted the mean ± standard deviation and compared the baseline and maximum values for each marker using Student's t-test. RESULTS: The maximum values of interleukin-1 beta and lactic dehydrogenase were present before the maximal histopathological damage. The maximum tumor necrosis factor alpha and D-lactate expressions coincided with histopathological damage. Alanine aminotransaminase and aspartate aminotransferase had a maximum expression level that increased following the histopathological damage. The maximum expressions of interluken-6 and intestinal fatty acid binding protein were not significantly different from the Sham treated group. CONCLUSION: For the evaluation of injury secondary to acute intestinal ischemia reperfusion with a 30 minute ischemia period, we recommend performing histopathological grading, quantification of D-lactate, which is synthesized by intestinal bacteria and is considered an indicator of mucosal injury, and quantification of tumor necrosis ...

Ischemic pre and postconditioning in skeletal muscle injury produced by ischemia and reperfusion in rats

PURPOSE: To investigate the protective effects of ischemic pre and postconditioning, as well as the association of both methods, in skeletal muscle injury produced by ischemia and reperfusion in rats. METHODS: An experimental study was designed using 40 Wistar rats divided in four groups (n=10): Control - rats submitted to ischemia for 240 minutes (min) and reperfusion for 60 min; Ischemic preconditioning (Pre) - animals submitted to three cycles of clamping and releasing the aorta for five min before being submitted to the ischemia/reperfusion procedure; Ischemic postconditioning (Post) - rats submitted to three cycles of clamping and releasing the aorta for one min after the 240-minute ischemic phase; Ischemic pre and postconditioning (Pre-post) - animals submitted to the same procedures of Pre and Post groups. Skeletal muscle injury was evaluated by measuring serum levels of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine phosphokinase (CPK); and muscular levels of malondialdehyde (MDA) and glycogen. RESULTS: AST levels were significantly higher in Pre and Pre-post groups (P<.01). There were no differences in LDH and CPK levels. Muscular MDA levels were similar. Glycogen levels were significantly higher in Pre and Pre-post groups (P<.01). CONCLUSIONS: Both preconditioning and its association with postconditioning had a protective effect by avoiding glycogen depletion in skeletal muscle in rats submitted to ischemia and reperfusion. Association of pre and postconditioning did not show advantage compared to preconditioning alone. Postconditioning alone did not show protective effect.

Effect of hyperbaric oxygen therapy on liver function during intermittent ischemia / Efeito da oxigenoterapia hiperbárica sobre a função hepática na isquemia intermitente

PURPOSE: To analyze the effects of hyperbaric oxygen therapy on liver function in rats previously subjected to ischemia and reperfusion. METHODS: A randomly distribution of 23 Wistar rats was conducted into three groups: SHAM, animals subjected to surgical stress without restricting blood flow by clamping the hepatic pedicle, IR, rats underwent hepatic vascular occlusion intermittently for two complete cycles of 15 minutes of ischemia followed by 5 min of reperfusion, IR / HBO, rats underwent hepatic pedicle clamping and thereafter exposed to hyperbaric oxygen pressure of 2 absolute atmospheres for 60 minutes. We evaluated liver function through mitochondrial function, determined by the stages 3 and 4 of respiration, respiratory control ratio (RCR) and mitochondrial permeability transition (Swelling). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also quantified . We analyzed the results using the Mann-Whitney test and were considered significant all results with p <0.05. RESULTS: There were significant differences between the results of stage 3 in SHAM vs IR group ; of the stage 4 in the groups IR vs SHAM and SHAM vs IR /HBO; of the Respiratory Control Ratio (RCR) in the group IR vs IR / HBO ; of alanine aminotransferase in the groups IR vs SHAM , SHAM vs IR/HBO and IR vs IR / HBO; aspartate aminotransferase in the groups SHAM vs IR and SHAM vs IR / HBO. CONCLUSION: The whole analysis of the mitochondiral function indicators permits us to conclude that the hyperbaric oxygen therapy acted as a protective agent of the mitochondrial function, minimizing the ischemia-reperfusion injury of the hepatic parenchyma.

OBJETIVO: Analisar os efeitos da oxigenoterapia hiperbárica sobre a função hepática de ratos previamente submetidos à isquemia e reperfusão. MÉTODOS: Realizou-se a distribuição aleatória de 23 ratos Wistar em três grupos: SHAM, animais submetidos ao estresse cirúrgico sem restrição de fluxo sanguíneo por clampeamento do pedículo hepático; IR ratos submetidos a oclusão vascular hepática de maneira intermitente em dois ciclos completos de 15 minutos de isquemia, seguidos por 5 min de reperfusão; IR/HBO, ratos submetidos ao clampeamento do pedículo hepático e ,ulteriormente , expostos a oxigenoterapia hiperbárica a pressão de 2 atmosferas absolutas durante 60 minutos. Avaliou-se a função hepática através da função mitocondrial, determinada pelos estados 3 e 4 da respiração mitocondrial, razão de controle respiratório(RCR) e transição de permeabilidade mitocondrial (Swelling) .Quantificou-se também alanina aminotransferase e aspartato aminotransferase séricas. Analisou-se os resultados pelo teste de Mann-Whitney e foram considerados significativos os valores de p < 0,05. RESULTADOS:Houve diferença significativa nos valores do estado 3 no grupo SHAM vs IR; estado 4 nos grupos SHAM vs IR, SHAM vsIR/HBO; Razão de Controle Respiratório(RCR) no grupo IR vs IR/HBO; alanina aminotransferase nos grupos SHAM vs IR, SHAM vs IR/HBO, IR vs IR/HBO; aspartato aminotransferase nos grupos SHAM vs IR , SHAM vs IR/HBO. CONCLUSÃO: A análise dos parâmetros mitocondriais como um todo permitiu concluir que a oxigenoterapia hiperbárica atuou como agente protetor da função mitocondrial, minimizando os efeitos deletérios da lesão de isquemia e reperfusão do parênquima hepático.

Protective effect of N-acetylcysteine on kidney as a remote organ after skeletal muscle ischemia-reperfusion / Efeito protetor da N-acetilcisteína no rim como um órgão remoto músculo esquelético após isquemia-reperfusão

PURPOSE: To investigate whether N-acetylcysteine has a protective effect against renal injury as a remote organ after skeletal muscle ischemia-reperfusion in rats. METHODS: Twenty Wistar male rats were divided randomly into two experimental groups: group ischemia-reperfusion (group I) and group ischemia-reperfusion + N-acetylcysteine (group II). After ketamine and xylazine anesthesia, femoral artery was exposed. All animals were undergone 2h of ischemia by occlusion femoral artery and 24h of reperfusion. Rats that were treated with N-acetylcysteine given IV at a dose of 150 mg/kg-¹, immediately before reperfusion. After 24h of reperfusion, the blood samples were collected and submitted for evaluation of plasmatic urea, creatinine values and then rats were euthanized and left kidney harvested for histopathological analysis under light microscopy. RESULTS: The urea (35±7.84 mg.dL-1), creatinine (1.46±0.47 mg.dL-1) values were significantly lower in group II (P=0.000). Renal histopathologic study in group I showed extensive distal and proximal tubular cells necrosis and sloughing of epithelial cells into the tubular lumen, cast formation in tubule and glomerul, glomerul fibrosis and hemorrhage. Histopathologically, there was a significant difference (p=0.037) between two groups. CONCLUSION: The N-acetylcysteine was able to decrease renal injury induced by skeletal muscle ischemia reperfusion in rats.

OBJETIVO: Investigar se a N-acetilcisteína tem um efeito protetor contra a lesão renal como um órgão remoto músculo esquelético após isquemia-reperfusão em ratos. MÉTODOS: Vinte ratos Wistar machos foram distribuídos aleatoriamente em dois grupos experimentais: grupo isquemia-reperfusão (grupo I) e grupo isquemia-reperfusão N-acetilcisteína (grupo II). Após a anestesia de ketamina e xilazina, a artéria femoral foi exposta. Todos os animais foram submetidos a 2h de isquemia pela oclusão da artéria femoral e 24h de reperfusão. Os ratos que foram tratados com N-acetilcisteína administrados IV na dose de 150 mgkg-1, imediatamente antes da reperfusão. Após 24h de reperfusão, as amostras de sangue foram coletadas e submetidas para avaliação de uréia, creatinina e, em seguida, os ratos foram sacrificados e rim esquerdo retirados para estudo histopatológico em microscopia de luz. RESULTADOS: A uréia (35 ± 7,84 mg.dL-1), creatinina (1,46 ± 0,47 mg.dL-1) os valores foram significativamente menores no grupo II (p=0,000). Estudo histopatológico renal do grupo I mostrou extensa necrose distal e proximal, células tubular e descamação das células epiteliais para o lúmen tubular, formação de elenco no túbulo e glomerulo, fibrose glomerular e hemorragia. Histopatologicamente houve uma diferença significativa (p=0,037) entre os dois grupos. CONCLUSÃO: A N-acetilcisteína foi capaz de diminuir a lesão renal induzida por reperfusão de isquemia do músculo esquelético em ratos.

L-arginine in the ischemic phase protects against liver ischemia-reperfusion injury / A L-arginina durante a fase isquêmica protege o fígado das lesões de isquemia e reperfusão

PURPOSE: To investigate the effects of intravenous L-arginine (LG) infusion on liver morphology, function and proinflammatory response of cytokines during the early phase of ischemia-reperfusion injury (IRI). METHODS: Thirty rabbits were subjected to 60 minutes of hepatic ischemia and 120 minutes of reperfusion. An intravenous injection of saline or L-arginine was administered five minutes before the ischemia and five minutes before initiating the reperfusion and at the 55th and 115th minutes after the ischemia. Samples were collected for histological analysis of the liver and measurements of the serum AST, ALT and LDH and the cytokines IL-6 and TNF-alpha. RESULTS: It was observed a significant reduction of sinusoidal congestion, cytoplasmic vacuolization, infiltration of polymorphonuclear leukocyte, nuclear pyknosis, necrosis and steatosis in liver tissue, as well as AST, ALT and LDH after injection of LG in the ischemia (p <0.001). Lower levels of IL-6 and TNF-alpha were associated with LG infusion during ischemia. Higher levels these proteins were observed in animals receiving LG during reperfusion. CONCLUSION: L-arginine protects the liver against ischemia/reperfusion injury, mainly when is administered during the ischemic phase.

OBJETIVO: Investigar os efeitos da infusão endovenosa da L-arginina (LG) na morfologia, função e resposta de citocinas pró-inflamatórias do fígado durante a fase precoce da lesão de isquemia e reperfusão (IRI). MÉTODOS: Trinta coelhos foram submetidos a 60 minutos de isquemia hepática e 120 minutos de reperfusão. Foi administrada injecção intravenosa de solução salina ou L-arginina aos cinco minutos antes de iniciar a isquemia e cinco minutos antes de iniciar a reperfusão e aos 55 e 115 minutos após o início da isquemia. Realizou-se análise histológica do fígado e dosagens séricas de AST, ALT, LDH, citocinas IL-6 e TNF-alfa. RESULTADOS: Ocorreu redução significante da congestão sinusoidal, vacuolização citoplasmática, infiltração de leucócitos polimorfonucleares, picnose nuclear, necrose e esteatose no tecido hepático, assim como nos níveis de AST, ALT e LDH após a injeção da LG na isquemia (p<0,001). Níveis mais baixos de IL-6 e TNF-alfa foram associados com a infusão LG durante a isquemia. Níveis mais elevados dessas proteínas foram observados nos animais que receberam LG durante a reperfusão. CONCLUSÃO: A L-arginina protegeu o fígado contra a lesão de isquemia e reperfusão principalmente quando administrada durante a fase de isquemia.

Preconditioning of the response to ischemia/ reperfusion-induced plasma leakage in hamster cheek pouch microcirculation

OBJECTIVE: Ischemic preconditioning and some drugs can protect tissues from injury by preserving microcirculation. This study evaluated vascular permeability in a hamster cheek pouch preparation using either short ischemic periods or bradykinin as preconditioning stimuli followed by 30 min of ischemia/reperfusion. METHOD: Sixty-six male hamsters were divided into 11 groups: five combinations of different ischemic frequencies and durations (one, three or five shorts periods of ischemia, separated by one or five minutes) with 10 min intervals between the ischemic periods, followed by 30 min ischemia/reperfusion; three or five 1 min ischemic periods with 10 min intervals between them followed by the topical application of histamine (2 µM); bradykinin (400 nM) followed by 30 min of ischemia/reperfusion; and three control groups (30 min of ischemia/reperfusion or histamine or bradykinin by themselves). Macromolecular permeability was assessed by injection of fluorescein-labeled dextran (FITC-dextran, MW= 150 kDa; 250 mg/Kg body weight), and the number of leaks/cm2 was counted using an intravital microscope and fluorescent light in the cheek pouch. RESULTS: Plasma leakage (number of leaks/cm²) was significantly reduced by preconditioning with three and five 1 min ischemic periods, one and three 5 min ischemic periods and by bradykinin. Histamine-induced macromolecular permeability was also reduced after three periods of 5 min of ischemia. CONCLUSION: Short ischemic periods and bradykinin can function as preconditioning stimuli of the ischemia/reperfusion response in the hamster cheek pouch microcirculation. Short ischemic periods also reduced histamineinduced macromolecular permeability.

Lovastatin protects mithochondrial and renal function in kidney ischemia-reperfusion in rats / Lovastatina protege a função renal e mitocondrial na isquemia/reperfusão renal em ratos

PURPOSE: To investigate the effect of lovastatin on renal ischemia followed by reperfusion. METHODS: Thirty one Wistar rats submitted to left renal ischemia for 60 minutes followed by contralateral nephrectomy were divided into two groups: A (n =17, control, no treatment), and B (n=14, lovastatin 15 mg/kg/day p.o. ten days before ischemia). The animals were sacrificed at the end of ischemia, after 24 hours and at seven days after reperfusion. Survival, serum urea and creatinine levels and renal mitochondrial function were evaluated. RESULTS: Mortality was 29.4% in group A and 0.7% in group B. Urea and creatinine levels were increased in both groups, but the values were significantly lower in group B. Mitochondrial function showed decoupling in 83.4% of group A, as opposed to 38.4/% of group B. CONCLUSIONS: The result shows a protective action of renal function by lovastatin administered before ischemia/reperfusion. Since most of the mitochondrial fraction presented membranes with the ability to maintain ATP production in group B, stabilization of the mitochondrial membrane should be considered as part of the protective action of lovastatin on renal function in ischemia/reperfusion.

OBJETIVO: Investigar a ação da lovastatina na isquemia renal seguida de reperfusão. MÉTODOS: Trinta e um ratos Wistar submetidos à isquemia renal esquerda durante 60 minutos, seguida da nefrectomia contralateral, foram distribuídos em dois grupos: A (n=17, controle, sem tratamento) e B (n=14, recebendo 15 mg/Kg/dia de lovastatina via oral), durante os dez dias que antecederam a isquemia. Os animais foram mortos ao final da isquemia, e com 24 horas e sete dias após a reperfusão. Foram avaliadas a sobrevida, os valores séricos de uréia e creatinina e a função mitocondrial renal. RESULTADOS: A mortalidade foi 29,4% no grupo A e 0,7% no grupo B. Os níveis de uréia e creatinina elevaram-se nos dois grupos, mas foram significativamente menores no grupo B. No grupo A a função mitocondrial renal ficou desacoplada em 83,4% dos ensaios, enquanto que no grupo B isto ocorreu em apenas 38,4% dos ensaios. CONCLUSÕES: Os resultados mostram que a administração de lovastatina antes do episódio de isquemia protege a função renal. No grupo B, como a maior parte da fração mitocondrial isolada apresentou função acoplada à produção de ATP, deve-se também considerar a estabilização da membrana mitocondrial como parte da ação protetora da lovastatina na função renal durante isquemia e reperfusão.

Prevention of renal ischemia/reperfusion injury in rats using acetylcysteine after anesthesia with isoflurane / Prevenção de lesão de isquemia/reperfusão em ratos com acetilcisteína após anestesia com isoflurano

PURPOSE: To evaluate the effect of N-acetylcysteine, as a renoprotective agent, when administered early after anesthesia induction, against ischemia/reperfusion injury in rats anesthetized with isoflurane. METHODS: Eighteen male Wistar rats weighing > 300g were anesthetized with isoflurane. The internal jugular vein and the left carotid artery were dissected and cannulated. The animals were randomly divided into GAcetyl, receiving intravenous N-acetylcysteine, 300mg/kg, and GIsot, isotonic saline. After 30 minutes, right nephrectomy was performed and the left renal artery was clamped during 45 minutes. The animals were sacrificed after 48 hours and blood samples were taken after anesthetic induction and upon sacrificing of the animals to evaluate blood creatinine. The kidneys were sent for histological analysis. RESULTS: The variation in serum creatinine was 2.33mg/dL ± 2.21 in GAcetyl and 4.38mg/dL ± 2.13 in GIsot (p=0.074). Two animals presented intense tubular necrosis in GAcetyl, compared to 5 in GIsot. Only GAcetyl presented animals free of tubular necrosis (two) and tubular degeneration (one). CONCLUSION: After renal ischemia/reperfusion, the rats which were given N-acetylcysteine presented less variation in serum creatinine and milder kidney injuries than the control group.

OBJETIVO: Avaliar o efeito da N-acetilcisteína na proteção renal contra lesão de isquemia/reperfusão, quando administrada logo após a indução anestésica, em ratos anestesiados com isoflurano. MÉTODOS: Dezoito ratos Wistar machos pesando mais que 300g foram anestesiados com isoflurano. A jugular interna direita e a carótida esquerda foram dissecadas e canuladas. Os animais foram distribuídos aleatoriamente em GAcetil, recebendo N-acetilcisteína por via intravenosa, 300mg/kg, e GIsot, solução salina. Foi realizada nefrectomia direita e clampeamento da artéria renal esquerda por 45 min. Os animais foram sacrificados após 48h, sendo colhidas amostras sanguíneas após a indução anestésica e ao sacrifício dos mesmos para avaliar a creatinina sérica. Realizou-se histologia renal. RESULTADOS: A variação da creatinina foi 2,33mg/dL ± 2,21 no GAcetil e 4,38mg/dL ± 2,13 no GIsot (p=0,074). Dois animais apresentaram necrose tubular intensa no GAcetil, comparados a cinco no GIsot. Apenas GAcetil apresentou animais livres de necrose tubular (dois) e degeneração tubular (um). CONCLUSÃO: Após isquemia/reperfusão renais, os ratos aos quais se administrou N-acetilcisteína apresentaram menor variação na creatinina sérica e lesões renais mais leves que o grupo controle.

Effect of N-acetylcysteine in liver ischemia-reperfusion injury after 30% hepatectomy in mice / Efeito da N-acetilcisteína na lesão hepática por isquemia e reperfusão após 30% de hepatectomia em camundongos

PURPOSE: Evaluate the effect of N-acetylcysteine in liver remnant after hepatectomy associated to ischemia-reperfusion injury in mice. METHODS: Male adult BALB/c mice, weighing 20-22g were used. Animals were anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg); received N-acetylcysteine (150 mg/kg, H-IR-NAC group) or vehicle (H-IR group). Surgical procedures were performed under 10X magnification. Partial hepatectomy (30%) was followed by ischemia-reperfusion injury (30 minutes of ischemia and 60 minutes of reperfusion). Blood sample and liver tissue were removed before animal was euthanized. AST and ALT were evaluated in blood samples and histomorphological analyses were performed in remnant liver. Groups were compared by Mann-Whitney test, and it was considered significant when p<0.05. RESULTS: Biochemical evaluations showed reduced levels of ALT in NAC group (H-IR-NAC=376±127U/l vs H-IR=636±39U/l, p=0.023). AST was similar (p=0.456). H-IR group showed hepatic tissue with preserved architecture, large area of steatosis, vascular congestion and rare mitogenic activity. NAC group showed hepatic tissue with small area of steatosis, vascular congestion and elevated mitogenic activity, evidenced by increased binuclear cells (H-IR-NAC=15.88±0.52 vs H-IR=7.4±0.37, p<0.001). CONCLUSION: N-acetylcysteine promotes enzymatic and morphological protection against hepatectomy and ischemia-reperfusion injury.

OBJETIVO: Investigar se a N-acetilcisteína promove proteção do remanescente hepático após ressecção associada à isquemia e reperfusão do fígado em camundongos. MÉTODOS: Foram utilizados 12 camundongos BALB/c, machos, pesando entre 20-22g. Os animais foram anestesiados com quetamina (70mg/kg) e xilazina (10mg/kg); receberam a N-acetilcisteína (150mg/kg, grupo H-IR-NAC) ou controle (grupo H-IR). Os procedimentos cirúrgicos ocorreram na magnificação de 10X. A lesão por isquemia e reperfusão (30 minutos de isquemia e 60 minutos de reperfusão) foi precedida pela hepatectomia de 30%. Foram utilizados como parâmetro de avaliação: a bioquímica sangüínea (AST e ALT) e a histologia do fígado (coloração de hematoxilina-eosina). Para avaliação estatística empregou-se o teste de Mann-Whitney e o nível de significância foi 5%. RESULTADOS: Na avaliação bioquímica houve redução no nível de ALT no grupo tratado (H-IR-NAC=376±127 U/l vs H-IR=636±39 U/l, p=0,023). AST foi similar (p=0,456). Na histologia, o grupo H-IR apresentou um tecido hepático com arquitetura preservada, com grandes áreas de infiltração gordurosa, presença de congestão vascular e de alguma atividade mitótica; o grupo com a N-acetilcisteína apresentou menor infiltração gordurosa e congestão vascular, maior atividade mitótica, evidenciada pela quantidade elevada de células binucleadas (H-IR-NAC=15,88±0,52 vs H-IR=7,4±0,37, p<0,001). CONCLUSÃO: A N-acetilcisteína promove proteção ao fígado, do ponto de vista morfológico e enzimático, após hepatectomia associada à isquemia e reperfusão.

Effect of glutamine on the total antioxidant system of rats subjected to renal ischemia and reperfusion / Efeito da glutamina no sistema antioxidante total de ratos submetidos a isquemia e reperfusão renal

PURPOSE: To evaluate the protective effects of glutamine administered before renal ischemia-reperfusion on plasma antioxidant protection, and lung and renal tissue injury. METHODS: 33 rats underwent right nephrectomy. On the eighth postoperative day, animals were randomized into three groups (n=11): glutamine, control and sham. Each group of animals received, by gavage, a particular diet for 7 days. On day 14 following nephrectomy, the animals were subjected to left renal ischemia-reperfusion. After this, blood samples were collected and the animals were killed. At necropsy the kidney and lung were removed for histology. RESULTS: The levels of total antioxidant capacity were higher in the glutamine group and control group compared with the sham group. The levels of glutathione peroxidase in both the sham and glutamine groups were higher when compared with the control group (p<0.05). The level of superoxide dismutase in the sham group was clearly higher than that in the glutamine and control groups. Histological examination showed no differences between the three groups. CONCLUSION: Prior intake of glutamine improves total antioxidant capacity and increases glutathione peroxidase levels in rats subjected to renal ischemia-reperfusion.

OBJETIVO: Avaliar os efeitos na proteção antioxidante plasmática e na lesão tecidual renal e pulmonar da glutamina oral administrada precedendo a isquemia/ reperfusão renal. MÉTODOS: Trinta e três ratos foram submetidos à nefrectomia à direita. No oitavo dia de pós-operatório, os animais foram randomizados em três grupos (n=11): glutamina, controle e sham. Cada grupo de animal recebeu por gavagem uma dieta distinta por sete dias. Ao final do 14º dia da nefrectomia procedeu-se a isquemia renal esquerda e posterior reperfusão. A seguir procedeu-se a coleta de sangue, eutanásia e retirada do rim e pulmões para análise histológica. RESULTADOS: Os níveis de capacidade antioxidante total foram maiores no grupo glutamina e grupo controle em relação ao grupo sham. Os níveis de glutationa peroxidase nos grupos sham e glutamina foram mais elevados quando comparados com o grupo controle (p<0,05). A dosagem de superóxido dismutase foi maior no grupo sham quando comparado com os grupos glutamina e controle. Não houve diferença na análise histológica do rim e pulmão entre os grupos. CONCLUSÃO: O uso de glutamina antecedendo a isquemia reperfusão renal melhora os níveis da capacidade antioxidante total e eleva a glutationa peroxidase em ratos submetidos a isquemia-reperfusão renal.

Effect of short-term ornithine alpha-ketoglutarate pretreatment on intestinal ischemia-reperfusion in rats / Efeitos do pré-tratamento em curto prazo com ornitina alfa-cetoglutarato na isquemia-reperfusão intestinal em ratos

PURPOSE: To investigate the effects of preventive enteral administration of ornithine alpha-ketoglutarate (OKG) in an ischemia-reperfusion rat model. METHODS: Sixty rats were randomized into five groups (G1-G5, n = 12). Each group was divided into two subgroups (n = 6) and treated with calcium carbonate (CaCa) or OKG by gavage. Thirty minutes later, the animals were anesthetized with xylazine 15mg + ketamine 1mg ip and subjected to laparotomy. G1-G3 rats served as controls. Rats in groups G4 and G5 were subjected to ischemia for 30 minutes. Ischemia was achieved by clamping the small intestine and its mesentery, delimiting a segment of bowel 5 cm long and 5 cm apart from the ileocecal valve. In addition, G5 rats underwent reperfusion for 30 minutes. Blood samples were collected at the end of the laparotomy (G1), after 30 minutes (G2, G4) and 60 minutes (G3, G5) to determine concentrations of metabolites (pyruvate, lactate), creatine phosphokinase (CPK), thiobarbituric acid reactive substances (TBARS) and glutathione (GSH). RESULTS: There was a significant decrease in tissue pyruvate and lactate and plasma CPK levels in OKG-treated rats at the end of reperfusion period. GSH levels did not change significantly in ischemia and reperfusion groups. However, TBARS levels increased significantly (p<0.05) in tissue samples in OKG-treated rats subjected to ischemia for 30 minutes. CONCLUSION: Short-term pretreatment with OKG before induction of I/R decreases tissue damage, increases pyruvate utilization for energy production in the Krebs cycle and does not attenuate the oxidative stress in this animal model.

OBJETIVO: Investigar os efeitos da administração enteral preventiva de ornitina alfa-cetoglutarato (OKG) em modelo de isquemia-reperfusão no rato. MÉTODOS: Sessenta ratos foram randomizados em cinco grupos (G1-G5, n=12). Cada grupo foi redistribuído em dois subgrupos (n=6) e tratado com carbonato de cálcio (CaCa) ou OKG por gavagem. Trinta minutos mais tarde, os animais foram anestesiados com xilazina 1mg+cetamina 15mg i.p. e submetidos à laparotomia. Os ratos dos grupos G4-G5 foram submetidos à isquemia por 30 minutos. A isquemia foi obtida por pinçamento do intestino delgado, delimitando um segmento com 5 cm de comprimento e distando 5 cm da válvula ileocecal. O grupo G5 foi submetido à reperfusão por 30 minutos. Amostras de sangue foram coletadas no final da laparotomia (G1), após 30 minutos (G2, G4) e 60 minutos (G3, G5) para determinação das concentrações de metabolitos (piruvato, lactato), creatinofosfoquinase (CPK), substâncias reativas ao ácido tiobarbitúrico (TBARS) e glutationa (GSH). RESULTADOS: Observou-se redução significante (p<0,05) das concentrações de piruvato e lactato, teciduais e CPK plasmático em ratos tratados com OKG, no final do período de reperfusão. Não houve alteração significante nos níveis plasmáticos e teciduais de GSH. Entretanto os níveis de TBARS aumentaram significativamente (p<0,05) em amostras de tecido de ratos tratados com OKG submetido à isquemia por 30 minutos. CONCLUSÃO: o pré-tratamento em curto prazo com OKG antes da indução da I/R diminui a lesão tecidual, aumenta a utilização de piruvato para produção de energia no ciclo de Krebs, mas não atenua o estresse oxidativo neste modelo animal.

Cardioprotective effects of exogenous and endogenous hydrocortisone in the rabbit model of ischemia-reperfusion / Efectos cardioprotectores de la hidrocortisona exógena y endógena en el modelo isquemia-reperfusión de conejo

Reducing the infarct size in acute myocardial infarction is one of the most important goals driving new drug research and development. During the last two decades, many clinical studies have found cardioprotective effects of corticosteroids, but their exact role in ischemic preconditioning remains questionable. The aim of the present study was to determine the protective effects of hydrocortisone sodium succinate on myocardial preconditioning in rabbit hearts. Twenty-four male New Zealand rabbits were divided randomly & equally in four groups: 1) control, 2) Infarct, 3) Ischemic preconditioning (IP) and 4) Hydrocortisone (HYD). The HYD group received 50mg/kg Hydrocortisone 45min before major ischemia. Serum levels of cardiac troponin-T(cTNT) and cortisole were measured before and after the protocols. Triphenyl-tetrazolium chloride staining was used to determine the infarcted area. In the present study, exogenous hydrocortisone decreased infarct size by 53 percent in comparison to the infarct group. Serum level of cortisole was increased in the IP and HYD groups, and was significant in the HYD group (p<0.01). An increasing trend in cortisole level was associated with a decreasing trend in infarct size and cTNT in the IP and HYD groups (p>0.01). In conclusion, we showed that hydrocortisone has cardioprotective effects when injected before the onset of myocardial infarction. In addition, we have proposed for the first time that endogenous hydrocortisone may play a role in ischemic preconditioning phenomena.

La reducción del tamaño del infarto en el infarto agudo de miocardio es una de las metas más importantes que impulsan la investigación y el desarrollo de nuevos fármacos. Durante las dos últimas décadas, muchos estudios clínicos han encontrado efectos cardioprotectores de los corticosteroides, pero su papel exacto en el preacondicionamiento isquémico sigue siendo cuestionable. El objetivo del presente estudio fue determinar los efectos protectores de succinato sódico de hidrocortisona en el preacondicionamiento del miocardio en el corazón de conejo. Veinticuatro conejos neozelandeses machos fueron divididos al azar en cuatro grupos : 1) control, 2) infarto, 3) preacondicionamiento isquémico (PI) y 4) Hidrocortisona (HYD). El grupo HYD recibió 50 mg/kg de hidrocortisona 45 minutos antes de la isquemia mayor. Los niveles séricos de troponina cardíaca T (cTNT) y cortisol se midieron antes y después de los protocolos. Se utilizó la tinción cloruro de trifenil-tetrazolio para determinar el área infartada. En el presente estudio, la hidrocortisona exógena disminuyó el tamaño del infarto en un 53 por ciento en comparación con el grupo de infarto. Los niveles séricos de cortisol se incrementaron en los grupos IP y HYD, siendo significativa en el grupo de HYD (p <0,01). Un aumento en el nivel cortisol se asoció con la disminución del tamaño del infarto y la cTNT en los grupos IP y HYD (p> 0,01). En conclusión, hemos demostrado la hidrocortisona tiene efectos cardioprotectores cuando se inyecta antes de la aparición del infarto al miocardio. Además, hemos propuesto, por primera vez que la hidrocortisona endógena puede jugar un papel en los fenómenos de preacondicionamiento isquémico.

Blood carbonyl protein measurement as a specific oxidative stress biomarker after intestinal reperfusion in rats / Dosagem da proteína carbonilada sanguínea como biomarcador específico do estresse oxidativo após reperfusão intestinal em ratos

PURPOSE: An experimental study was performed to investigate the use of protein carbonyl group as a specific biological marker for oxidative stress in a rat model of intestinal ischaemia-reperfusion. METHODS: Twenty four male Wistar rats were randomly distributed into three groups with eight animals each: Group 1 - Control group; Group 2 - Sham; Group 3 - Intestinal ischaemia by clamping ileal branches of the superior mesenteric artery for one hour, followed by another hour of reperfusion. Blood samples were taken in order to analyze the protein carbonyl level by Slot blotting assay. RESULTS: In group 3 a significant increase of protein carbonyl level was observed if compared to the homogenous levels of groups 1 and 2. CONCLUSION: From the results it may be concluded that the protein carbonylation may be used as a specific marker for measuring oxidative stress in rat intestinal reperfusion model.

OBJETIVO: Realizou-se um estudo experimental com a finalidade de investigar o uso da proteína carbonilada como um marcador biológico específico do estresse oxidativo em um modelo de isquemia e reperfusão intestinal, em ratos. MÉTODOS: Vinte e quarto ratos da linhagem Wistar, machos foram distribuídos, aleatoriamente, em três grupos compostos por oito animais cada: Grupo 1 - Controle; Grupo 2 - Simulação e Grupo 3 - Submetido à isquemia, mediante clampeamento de ramos ileais da artéria mesentérica superior por uma hora, seguida de reperfusão, por igual período. Amostras sanguíneas obtidas foram utilizadas para analise dos níveis de proteína carbonilada, através do método Slot blotting. RESULTADOS: No grupo 3 houve uma elevação significante da concentração de proteína carbonilada sérica se comparada aos níveis sanguíneos homogêneos encontrados nos grupos 1 e 2. CONCLUSÃO: Fundamentado nos resultados é possível concluir que, a carbonilação protéica pode ser utilizada como um marcador específico para a mensuração do estresse oxidativo em modelos de reperfusão intestinal, em ratos.