Hyperbaric oxygenation and the genic expression related to oxidative stress in the heart of mice during intestinal ischemia and reperfusion

Abstract Purpose: To investigate the effects of hyperbaric oxygenation (HBO) on intestinal ischemia and reperfusion (IR) injury, we evaluated the expression of 84 genes related to oxidative stress and the antioxidant response in mouse hearts. Methods: Four groups were subjected to 60 minutes of intestinal ischemia followed by 60 minutes of reperfusion: IRG, ischemia and reperfusion group without HBO; HBO-IG, which received HBO during ischemia; HBO-RG, which received HBO during reperfusion; and HBO-IRG, which received HBO during ischemia and reperfusion. The control group (CG) underwent anesthesia and laparotomy and was observed for 120 minutes. The (RT-qPCR) method was applied. Genes with expression levels three times below or above the threshold cycle were considered significantly hypoexpressed or hyperexpressed, respectively (Student's t-test p<0.05). Results: Eight genes (9.52%) were hyperexpressed in the IRG. When the HBO groups were compared to the IRG, we found a decrease in the expression of eight genes in the HBO-IG, five genes in the HBO-RG, and seven genes in the HBO-IRG. Conclusion: The reduction in the expression of genes related to oxidative stress and antioxidant defense following HBO in mouse hearts resulting from intestinal IR injury was more favorable during the ischemic period than during the reperfusion period.

Gene expression profile of oxidative stress in the lung of inbred mice after intestinal ischemia/reperfusion injury

To determine the gene expression profile associated with oxidative stress and antioxidant defense in the lung tissue of mice subjected to intestinal ischemia and reperfusion. METHODS: Twelve male, inbred mice (C57BL/6) were randomly assigned to one of two groups. The control group (CG) underwent anesthesia and laparotomy and was observed for 120 minutes; the ischemia/reperfusion group (IRG) was subjected to anesthesia, laparotomy, and ischemia of the small intestine for 60 minutes and to 60 minutes of reperfusion. A pool of six mice from each group was subjected to a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to analyze the oxidative stress and antioxidant defense genes. All genes that were up-regulated or down-regulated greater than three-fold, based on the algorithm 2.

Expressao genica associada ao estresse oxidativo no coracao de camundongo apos isquemia intestinal / Gene expression related to oxidative stress in the heart of mice after intestinal ischemia

FUNDAMENTO: O fenômeno da isquemia e reperfusão intestinal é um evento frequente na clínica e está associado a repercussões deletérias em órgãos a distância, em especial ao coração. OBJETIVO: Investigar a expressão gênica do estresse oxidativo e defesa antioxidante no coração de camundongos isogênicos, submetidos a isquemia e reperfusão intestinal (IR). MÉTODOS: Doze camundongos (C57BL/6) foram distribuídos em dois grupos: Grupo IR (GIR) com 60 min de oclusão da artéria mesentérica superior, seguidos de 60 min de reperfusão. Grupo Controle (GC) submetidos a anestesia e a laparotomia sem o procedimento de IR observados por 120 min. As amostras de intestino e coração foram processadas pelo método (RT-qPCR / Reverse transcriptase - quantitative Polymerase Chain Reaction) para determinar a expressão gênica de 84 genes relacionados ao estresse oxidativo ("t" de Student, p < 0,05). RESULTADOS: Observou-se no tecido intestinal (GIR) uma expressão significantemente aumentada em 65 (74,71%) genes em relação ao tecido normal (GC), e 37 (44,04%) genes estiveram hiperexpressos (maior que três vezes o limiar permitido pelo algoritmo). No tocante aos efeitos da I/R intestinal a distância no tecido cardíaco verificou-se a expressão significantemente aumentada de 28 genes (33,33%), mas somente oito genes (9,52%) se hiperexpressaram três vezes acima do limiar. Quatro (7,14%) desses oito genes se expressaram simultaneamente nos tecidos intestinal e cardíaco. No GIR notaram-se cardiomiócitos com núcleos de menor tamanho, picnóticos, ricos em heterocromatina e raros nucléolos, indicando sofrimento cardíaco. CONCLUSÃO: A I/R intestinal promoveu a hiperexpressão estatisticamente significante de oito genes associados ao ...

BACKGROUND: Intestinal ischemia-reperfusion is a frequent clinical event associated to injury in distant organs, especially the heart. OBJECTIVE: To investigate the gene expression of oxidative stress and antioxidant defense in the heart of inbred mice subjected to intestinal ischemia and reperfusion (IR). METHODS: Twelve mice (C57BL / 6) were assigned to: IR Group (GIR) with 60 minutes of superior mesenteric artery occlusion followed by 60 minutes of reperfusion; Control Group (CG) which underwent anesthesia and laparotomy without IR procedure and was observed for 120 minutes. Intestine and heart samples were processed using the RT-qPCR / Reverse transcriptase-quantitative Polymerase Chain Reaction method for the gene expression of 84 genes related to oxidative stress and oxidative defense (Student's "t" test, p < 0.05). RESULTS: The intestinal tissue (GIR) was noted to have an up-regulation of 65 genes (74.71%) in comparison to normal tissue (CG), and 37 genes (44.04%) were hyper-expressed (greater than three times the threshold allowed by the algorithm). Regarding the remote effects of intestinal I/R in cardiac tissue an up-regulation of 28 genes (33.33%) was seen, but only eight genes (9.52%) were hyper-expressed three times above threshold. Four (7.14%) of these eight genes were expressed in both intestinal and cardiac tissues. Cardiomyocytes with smaller and pyknotic nuclei, rich in heterochromatin with rare nucleoli, indicating cardiac distress, were observed in the GIR. CONCLUSION: Intestinal I/R caused a statistically significant over expression of 8 genes associated with oxidative stress in remote myocardial tissue. .

Expression of oxidative stress and antioxidant defense genes in the kidney of inbred mice after intestinal ischemia and reperfusion

PURPOSE: To determine the gene expressions profile related to the oxidative stress and the antioxidant response in the kidneys of mice subjected to intestinal ischemia and reperfusion. METHODS: Twelve inbred mice (C57BL/6) were randomly assigned to one of two groups: the control group (CG) underwent anesthesia and was observed for 120 min and the ischemia/reperfusion group (IRG), animals were anesthetized and subjected to laparotomy and ischemia for 60 minutes followed by 60 minutes of reperfusion. The expressions of 84 genes from the kidney were determined by the Reverse Transcription qualitative Polymerase Chain Reaction (RT-qPCR). All genes that were up regulated by more than threefold using the algorithm [2(ΔΔCt)] were considered statically significant (p<0.05). RESULTS: In the IRG group 29 (34.52%) of 84 genes, were up regulated by more than threefold. The genes that were differentially up regulated in the glutathione peroxidase cluster (10 genes): were Gpx2 and Gpx7. The genes that were up regulated in the peroxidase cluster (16 genes) were following: Duox1, Epx, Lpo, Mpo, Ptgs2, Rag2, Serpinb1b, Tmod1 and Tpo. The genes that up regulated in the reactive oxygen species cluster (16 genes): Il19, Il22, Nos2, Nox1, Noxa1, Noxo1, Recql4 and Sod2. The genes that were up regulated in the oxidative stress cluster (22 genes) were: Mpp4, Nudt15, Upc3 and Xpa. The genes that were up regulated in the oxygen carriers cluster (12 genes) were: Hbq1, Mb, Ngb, Slc38a1 and Xirp1. The peroxiredoxins genes (10) showed no consistent differential regulation. CONCLUSION: The genes related to oxidative stress and antioxidant defense showed increased expression in renal tissue trigged intestinal ischemia and reperfusion.

Oxidative stress gene expression profile in inbred mouse after ischemia/reperfusion small bowel injury / Perfil da expressão gênica do estresse oxidativo em camundongos isogênicos após lesão de isquemia e reperfusão intestinal

PURPOSE: To determine the profile of gene expressions associated with oxidative stress and thereby contribute to establish parameters about the role of enzyme clusters related to the ischemia/reperfusion intestinal injury. METHODS: Twelve male inbred mice (C57BL/6) were randomly assigned: Control Group (CG) submitted to anesthesia, laparotomy and observed by 120min; Ischemia/reperfusion Group (IRG) submitted to anesthesia, laparotomy, 60min of small bowel ischemia and 60min of reperfusion. A pool of six samples was submitted to the qPCR-RT protocol (six clusters) for mouse oxidative stress and antioxidant defense pathways. RESULTS: On the 84 genes investigated, 64 (76.2%) had statistic significant expression and 20 (23.8%) showed no statistical difference to the control group. From these 64 significantly expressed genes, 60 (93.7%) were up-regulated and 04 (6.3%) were down-regulated. From the group with no statistical significantly expression, 12 genes were up-regulated and 8 genes were down-regulated. Surprisingly, 37 (44.04%) showed a higher than threefold up-regulation and then arbitrarily the values was considered as a very significant. Thus, 37 genes (44.04%) were expressed very significantly up-regulated. The remained 47 (55.9%) genes were up-regulated less than three folds (35 genes - 41.6%) or down-regulated less than three folds (12 genes - 14.3%). CONCLUSION: The intestinal ischemia and reperfusion promote a global hyper-expression profile of six different clusters genes related to antioxidant defense and oxidative stress.

OBJETIVO: Determinar o perfil de expressão dos genes associados com estresse oxidativo e contribuir para estabelecer parâmetros sobre o papel das familias de enzimas relacionadas com a lesão de isquemia / reperfusão intestinal. MÉTODOS: Doze camundongos machos isogênicos (C57BL/6) foram distribuídos aleatoriamente: Grupo Controle (CG) submetido à laparotomia anestesia, e observado por 120min; Grupo isquemia/reperfusão (IRG) submetido à anestesia, laparotomia, 60min de isquemia do intestino delgado e 60min de reperfusão. Um pool dos seis camundongos de cada grupo foi submetido ao protocolo de qPCR-RT (seis famílias) para o estresse oxidativo e defesa antioxidante. RESULTADOS: Dos 84 genes investigados, 64 (76,2%) tiveram expressão estatística significante e 20 (23,8%) não apresentaram diferença estatística com o grupo controle. Dos 64 genes expressos de forma significante, 60 (93,7%) foram hiper-expressos e 04 (6,3%) foram hipo-expressos. Do grupo sem expressão estatisticamente significante, 12 genes foram hiper e 8 genes foram hipo-expressos. Surpreendentemente, 37 (44,04%) apresentaram expressão três maior que o limiar de normalidade e arbitrariamente os valores foram considerados como altamente significantes. Assim, 37 genes (44,04%) foram hiper-expressos de modo muito significante. Nos demais, 47 (55,9%) dos genes foram hiper-expressos menos de três vezes (35 genes - 41,6%) ou hipo-expressos menos de três vezes(12 genes - 14,3%). CONCLUSÃO: A isquemia e reperfusão intestinal promoveu um perfil de hiper-expressão global das seis familias de genes relacionados com estresse oxidativo antioxidante e defesa antioxidante.

The role of Fas ligand protein in the oxidative stress induced by azoxymethane on crypt colon of rats / O papel da proteína ligante Fas no estresse oxidativo induzido pelo azoximetano em criptas do colo de ratos

PURPOSE: To study the protein Fas ligand (FasL) on the expression of apoptosis, using a model of oxidative stress induced by azoxymethane (AOM), in the crypt of colon in rats. METHODS: Wistar rats (n=14) were assigned into two groups: control (n=7) and AOM (n=7). A single subcutaneous administration of AOM (5mg/kg) or saline solution was performed at the beginning of third week and after three hours samples of proximal colon were collected. The expression of FasL was quantified (Software ImageLab) in percentage of areas in the top, base and all crypt. Results were expressed as mean ± sd (Shapiro-Wilks test and t Student test) (p < 0.05). RESULTS: In the animals of CG there was no significant difference between the FasL expression of the top (10.75±3.33) and basal (11.14±3.53) colon crypt (p=0.34293740). In the animals of AOM there was no significant difference between the FasL expression of the top (8.86±4.19) and basal (8.99±4.08) colon crypt (p=0.78486003). In the animals of CG (10.95±3.43) and AOM (8.92±4.13) there was a significant difference of the FasL expression (p=0.026466821). A significantly decrease on the FasL expression was observed in the animals of CG (10.75±3.33) and AOM (8.86±4.19) in the top crypt (p=0.00003755*). A significant decrease was also observed in the animals of CG (11.14±3.53) and AOM (8.99±4.08) in the basal colon crypt (p=0.00000381**). CONCLUSION: Azoxymethane induce the oxidative stress and the significantly decrease of FasL expression, although there is no significant difference between basal and top of colon crypt linked to consumption-activation of Fas ligand.

OBJETIVO: Avaliar o marcador de apoptose Fas ligante (FasL) em um modelo de estresse oxidativo induzido pelo azoximetano (AOM) na cripta de cólon em ratos. MÉTODOS: 14 ratos Wistar foram distribuídos em dois grupos: controle (n=7) e AOM (n=7). A AOM (5mg/kg) ou solução salina foi aplicada via subcutânea e a coleta de amostras de colo proximal ocorreu 3 horas após. A FasL foi quantificada pelo percentual de áreas no topo, base e toda a cripta. Os resultados foram submetidos aos testes de Shapiro-Wilks e t de Student (p < 0,05). RESULTADOS: No grupo GC, não houve diferença significativa entre a expressão da FasL no topo (10,75 ± 3,33) e base (11,14 ± 3,53) da cripta (p=0,34293740). No grupo AOM não houve diferença significativa entre a expressão da FasL no topo (8,86 ± 4,19) e base (8,99 ± 4,08) e cripta (p=0,78486003). No grupo GC (10,95 ± 3,43) e AOM (8,92 ± 4,13), houve uma diferença significativa da expressão da FasL (p=0,026466821). Redução significativa na expressão da FasL ocorreu nos em GC (10,75 ± 3,33) e AOM (8,86 ± 4,19) no topo da cripta (p = 0,00003755*). Foi observada diminuição significativa em GC (11,14 ± 3,53) e AOM (8,99 ± 4,08) na base da cripta (p=0,00000381**). CONCLUSÃO: Azoximetano induziu o estresse oxidativo identificado pela diminuição significativa da expressão da FasL, embora não haja diferença significativa entre a base e parte superior da cripta associada à ativação de consumo do FasL.

Apoptotic effects of inositol hexaphosphate on biomarker Itpr3 in induced colon rat carcinogenesis

PURPOSE: To study the effect of the modulation of inositol hexaphosphate (IP6) in the biological immunohistochemistry expression of cellular signaling marker apoptosis, in model of carcinogenesis of colon induced by azoxymethane (AOM). METHODS: Wistar rats (N=112) distributed in 4 groups (n=28): Control; B, AOM (5 mg kg-1, 2x, to break week 3); C, IP6 (in water 1 percent, six weeks); D, IP6+AOM. Weekly euthanasia (n=7), from week three. Immunohistochemistry of ascendant colon with biological marker inositol 1,4,5 triphosphate receptor type III (Itpr3). Quantification of the immune-expression with use of computer-assisted image processing. Analysis statistics of the means between groups, weeks in groups, groups in weeks, and established significance when p<0.05. RESULTS: One proved significant difference between groups in the expression of Itpr3, p<0.0001; with Itpr3 reduction of BxD group, p<0.001. CONCLUSION: Inositol hexaphosphate promotes modulation of biological markers with reduction of Itpr3 in carcinogenesis of colon.

OBJETIVO: Estudar os efeitos da modulação do inositol hexafosfato (IP6) na expressão imunoistoquímica de marcador biológico de sinalização celular de apoptose, em modelo de carcinogênese induzida pelo azoximetano (AOM). MÉTODOS: Ratos Wistar (N=112) distribuídos em 4 grupos (n=28): A, controle; B, AOM (5 mg Kg-1, 2x, a partir semana 3); C, IP6 (em água a 1 por cento, seis semanas); D, IP6+AOM. Eutanásia semanal (n=7), a partir de semana três. Imunoistoquímica de colo ascendente com marcador biológico inositol 1,4,5 trisphosphate receptor type III (Itpr3). Quantificação da imunoexpressão com uso de processamento imagem assistida computador. Análise estatística da expressão média entre grupos, semanas em grupos e grupos em semanas, e estabelecido significância quando p<0.05. RESULTADOS: Evidenciou-se diferença significante entre grupos na expressão de Itpr3, p<0.0001; com diminuição Itpr3 de grupo BxD, p<0.001. CONCLUSÃO: O inositol hexafostato promove a modulação de marcador biológico com diminuição Itpr3 em carcinogênese de colo.

The effects of pentoxifylline into the kidneys of rats in a model of unilateral hindlimb ischemia/reperfusion injury

PURPOSE: To study the role of pentoxifylline (PTX) on remote kidney injury caused by muscle ischemia of left hindlimb of rats. METHODS: After xylazine and ketamine anesthesia, the left hindlimb of rats (n=66) were submitted to 6 hours ischemia (clamping the left common iliac artery). Three groups were used: sham group (SG, n=6), early group (EG, n=30) with reperfusion after 4 hours and late group (LG, n=30) with reperfusion after 24 hours. The saline solution (EG1, n=10 and LG1, n=10) or PTX (40mg.Kg-1) was administered in the reperfusion beginning (EG2, n=10/LG2, n=10) or divided in two doses in the ischemia beginning and reperfusion beginning (EG3, n=10/LG3, n=10). The plasmatic creatinokinase, urea, creatinine, sodium and potassium values were measure and histological samples from left kidney were prepared and H&E stained for scored cellular necrosis and degeneration of kidney tubules and thickness glomerulus determination. The apoptosis index was determined by immunohistochemical expression of the caspase-3. The tests of Mann-Whitney and Kruskal-Wallis (p < 0.05) were applied. RESULTS: The urea (90.5 ± 30.96 mg.dL-1), creatinine (2.28 ± 0.54 mg.dL-1), potassium (16 ± 3.66 mmol.dL-1) and mesangium thickness (0.97 ± 0.42 µm) values were significantly higher in group LG3. There was no significantly difference of caspase 3 expression between EG2 (16.35 ± 1.65 percent) and LG3 (15.57 ± 2.54 percent), and both were significantly worse than SG (9.8 ± 1.98 percent). CONCLUSIONS: The PTX has some protecting effect on remote kidney injury due to hindlimb ischemia/reperfusion injury only in the early phase of reperfusion.

OBJETIVO: Estudar o papel da pentoxifilina (PTX) nas lesões à distância no rim causadas pela isquemia no membro posterior esquerdo de ratos. MÉTODOS: Sob anestesia com xilazina e quetamina, o membro posterior de ratos (n=66) foi submetido a 6 horas de isquemia pelo clampeamento da artéria ilíaca comum esquerda. Foram estudados três grupos: grupo simulado (SG, n=6), grupo precoce (EG, n=30) após quatro horas de reperfusão e grupo tardio (LG, n=30) após 24 de reperfusão. A solução salina (EG1, n=10 e LG1, n=10) ou a PTX (40mg.Kg-1) foram administradas no início da reperfusão(EG2, n=10/LG2, n=10) ou divididas em duas aplicações no início da isquemia e no início da reperfusão (EG3, n=10/LG3, n=10). Foram medidos os valores plasmáticos da creatinofosfoquinase, uréia, creatinina, sódio e potássio. Amostras do rim esquerdo foram preparadas e coradas em HE para realizar o escore de necrose de células tubulares renais ou presença de obstrução tubular renal na área do córtex renal e da presença do espessamento do mesângio glomerular. O índice de apoptose foi determinado pela expressão imunoistoquímica da caspase-3. Foram aplicados os testes de Mann-Whitney e Kruskal-Wallis (p < 0.05). RESULTADOS: a dosagem de uréia (90,5 ± 30,96 mg.dL-1), creatinina (2,28 ± 0,54 mg.dL-1), potássio (16 ± 3,66 mmol.dL-1) e a espessura do mesângio(0,97 ± 0,42 µm) foram significantemente maiores nos animais do grupo LG3. Não houve diferença significante na expressão da caspase-3 entre os grupos EG2 (16,35 ± 1,65 por cento) e LG3 (15,57 ± 2,54 por cento) e ambos foram significantemente piores que o grupo SG (9,8 ± 1,98 por cento). CONCLUSÃO: A PTX oferece algum efeito protetor nas lesões à distância nos rins de animais submetidos à lesão de isquemia e reperfusão de membro posterior, no período de até quatro horas após a reperfusão.

Isquemia e reperfusão de músculo sóleo de ratos sob ação da pentoxífilina / Ischemia and reperfusion of the soleus muscle of rats with pentoxifylline

Contexto: A reperfusão de músculo esquelético piora as lesões ja presentes no período de isquemia, pois a produção de espécies reativas de oxigênio, associadas à intensa participação de neutrófilos, amplia a reação inflamatória que induz alterações teciduais. Objetivo: Avaliar as alterações morfológicas e imuno-histoquímicas de músculo esquelético (sóleo) de ratos submetidos a isquemia e reperfusão com pentoxifilina. Métodos: Sessenta ratos foram submetidos a isquemia do membro pélvico, por 6 horas, pelo clampeamento da artéria ilíaca comum esquerda. Após isquemia, os animais do grupo A(n igual a 30) foram observados por 4 horas, e os do grupo B(n igual a 30), por 24 horas. Seis animais constituiram o grupo simulado. Administrou-se pentoxifilina apenas no período de reperfusão em A2(n igual 100) e B2(n igual 10) e nos períodos de isquemia e reperfusão em A3(n igual 10) e B3(n igual 10). O músculo sóleo foi avaliado por análise histológica (dissociação de fibras, infiltrado leucocitário, necrose) e imuno histoquímica (apoptose pela extensão da caspase-3). Foram...