Prevention of bacterial translocation using β-(1-3)-D-glucan in small bowel ischemia and reperfusion in rats

To investigate the role of β-(1-3)-D-glucan on 99mTc labelled Escherichia coli translocation and cytokines secretion in rats submitted to small bowel ischemia/reperfusion injury. Methods: Five groups (n=10 each) of Wistar rats were subjected to control(C), sham(S), group IR subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R), and group I/R+glucan subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R) and injected with 2mg/Kg intramuscular. Translocation of labelled bacteria to mesenteric lymph nodes, liver, spleen, lung and serum was determined using radioactivity/count and colony forming units/g(CFU/g). Serum TNFα, IL-1β, IL-6, IL-10 were measured by ELISA. Results: CFU/g and radioactivity/count were higher in I/R than in I/R+glucan rats. In C, S and S+glucan groups, bacteria and radioactivity/count were rarely detected. The I/R+glucan rats had enhancement of IL-10 and suppressed production of serum TNFα, IL-1β and, IL-6, compared to I/R untreated animals. Conclusion: The β-(1-3)-D-glucan modulated the production of pro-inflammatory and anti-inflammatory cytokines during bowel ischemia/reperfusion, and attenuated translocation of labelled bacteria

Prevention of bacterial translocation using β-(1-3)-D-glucan in small bowel ischemia and reperfusion in rats

To investigate the role of β-(1-3)-D-glucan on 99mTc labelled Escherichia coli translocation and cytokines secretion in rats submitted to small bowel ischemia/reperfusion injury. Methods: Five groups (n=10 each) of Wistar rats were subjected to control(C), sham(S), group IR subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R), and group I/R+glucan subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R) and injected with 2mg/Kg intramuscular. Translocation of labelled bacteria to mesenteric lymph nodes, liver, spleen, lung and serum was determined using radioactivity/count and colony forming units/g(CFU/g). Serum TNFα, IL-1β, IL-6, IL-10 were measured by ELISA. Results: CFU/g and radioactivity/count were higher in I/R than in I/R+glucan rats. In C, S and S+glucan groups, bacteria and radioactivity/count were rarely detected. The I/R+glucan rats had enhancement of IL-10 and suppressed production of serum TNFα, IL-1β and, IL-6, compared to I/R untreated animals. Conclusion: The β-(1-3)-D-glucan modulated the production of pro-inflammatory and anti-inflammatory cytokines during bowel ischemia/reperfusion, and attenuated translocation of labelled bacteria

Caffeic Acid Phenethyl Ester Effects in the Kidney During Ischemia and Reperfusion in Rats Anesthetized with Isoflurane

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Trimetazidine and N-acetylcysteine in attenuating hind-limb ischemia and reperfusion injuries: experimental study in rats

Aim. Lower-limb traumatic injury associated with ischemia and followed by reperfusion (I/R) is a common severe situation in muscle lesions due to trauma and hypoxia followed by local and systemic injuries induced by oxygen-derived free radical release during reperfusion. The aim of this study was to evaluate the attenuating effects of trimetazidine (TMZ) and N-acetylcysteine (NAC) in such situation.Methods. The muscles at the root of the right hind limb of Wistar rats were cross-sectioned, preserving femoral vessels and nerves and clamping the femoral artery for four hours. The clamp was then released and the femoral artery has been reperfused for 2 hours. Rats were randomly divided in groups of ten as follows: Group 1: sham I/R, treated with saline; Group 2: I/R, treated with saline; Group 3: sham I/R, treated with TMZ (7.5 mg/kg/dose); Group 4: sham I/R, treated with NAC (375 mg/kg/dose); Group 5: I/R treated with TMZ (7.5 mg/kg/dose); Group 6: I/R treated with NAC (375 mg/kg/dose). All rats received two intravenous bolus injections of the drugs, one before ischemia and one before reperfusion. Oxidative stress in plasma (MDA, total, oxidized and reduced glutathione), creatinephosphokinase (CPK), optical and electron microscopy and pelvic extremity circumference and volume were studied.Results. No statistical differences were found between the groups for MDA or total and reduced glutathione. Oxidized glutathione increased significantly in groups 5 and 2. Limb circumference as well as limb volume increased in all groups over time, mainly in groups 5, 2 and 1. CPK increased in all groups, being highest in groups 5, 6 and 2. Histological lesions were present in all but sham groups, being less severe in group 6. Soleus muscle analyses at electron microscopy exhibit some degree of alteration in all groups.Conclusion. This experimental model simulated severe limb trauma associated with ischemia and reperfusion, and, as such, it was aggressive, causing severe injury and local inflammatory reaction. The model did not show antioxidant action from NAC, and possible antioxidant action from TMZ was insufficient to attenuate tissue injuries. [Int Angiol 2009;28:412-7]

Effects of pentoxifylline and n-acetylcysteine on injuries caused by ischemia and reperfusion splanchnic organs in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Experimental ischemia and reperfusion in equine small colon

Sob anestesia geral, com constante controle sobre a pressão arterial e a saturação de oxigênio da hemoglobina arterial, realizou-se celiotomia em 12 eqüinos. No cólon menor exposto foram demarcados três segmentos de 25cm, separados entre si por igual distância. Dois desses segmentos foram submetidos à isquemia arteriovenosa completa por 90 (grupo A) ou 180 minutos (grupo B). O terceiro segmento foi o grupo-controle. Amostras para histopatologia foram colhidas ao final dos períodos de isquemia e após 90 e 180 minutos de reperfusão no grupo A e após 90 minutos de reperfusão no grupo B. No controle, colheram-se amostras no início e final do procedimento. Avaliaram-se as lesões produzidas na mucosa e na submucosa pelos métodos semiquantitativos-escores para desprendimento de epitélio, edema, hemorragia e infiltrado de neutrófilos, e pelos quantitativos-porcentagem de perda de mucosa (PM) e razão cripta:interstício (C:I). As lesões isquêmicas foram mais intensas no grupo B do que no A para PM, C:I, desprendimento de epitélio e edema de mucosa. As amostras obtidas após a reperfusão revelaram que houve agravamento na PM, C:I, desprendimento de epitélio e edema de submucosa em ambos os grupos. Concluiu-se que a reperfusão agravou as lesões isquêmicas no cólon menor e que o modelo proposto é viável para produção dessas lesões.

Ischemia and reperfusion in skin flaps: effects of mannitol and vitamin C in reducing necrosis area in a rat experimental model

OBJETIVO: Neste trabalho foi padronizado modelo experimental de isquemia e reperfusão em retalho cutâneo em ratos no qual estudou-se possibilidade de uma solução antioxidante, composta por Ringer lactato, vitamina C e manitol de reduzir a área de necrose. MÉTODOS: O modelo consistiu de levantamento de retalho cutâneo axial de 6,0 x 3,0cm, submetido à isquemia de 8 horas e reperfusão de 7 dias. Os animais foram divididos em quatro grupos: grupos S1, S2 (10 animais cada), C e T (14 animais cada). Nos grupos S1 e S2 todos os procedimentos dos demais grupos foram efetuados, exceto a isquemia e reperfusão: S1 recebeu apenas Ringer lactato e S2 a solução antioxidante. Os grupos C e T foram submetidos à isquemia. O grupo C recebeu somente Ringer lactato e o grupo T a solução antioxidante. No 7(0) dia de pós-operatório as áreas de necrose e pele viável do retalho foram delineadas em decalque de acetato, os quais foram por sua vez analisados em sistema computadorizado KS-300 (Carl Zeiss). RESULTADOS: A análise estatística mostrou que não houve diferenças significativas entre o grupo tratado e controle quanto à área de necrose. CONCLUSÃO: Concluiu-se que o modelo experimental é consistente, determinando área de necrose limitada e uniforme nos animais não tratados e que as drogas usadas, nessa posologia e modo de aplicação, não foram efetivas em diminuir a área de necrose no modelo experimental em questão.

Effects of pentoxifylline and n-acetylcysteine on injuries caused by ischemia and reperfusion of splanchnic organs in rats

Aim. Occlusion and reperfusion of splanchnic arteries cause local and systemic changes due to the release of cytotoxic substances and the interaction between neutrophils and endothelial cells. This study evaluated the role of pentoxifylline (PTX) and n-acetylcysteine (NAC) in the reduction of ischemia, reperfusion shock and associated intestinal injury. Methods. Sixty rats were divided into 6 groups of 10 animals. Rats in three groups underwent mesenteric ischemia for 30 minutes followed by 120 minutes of reperfusion, and were treated with saline (SAL-5 mL/kg/ h), pentoxifylline (PTX-50 mg/kg) or n-acetylcysteine (NAC-430 mg/kg/h). The other 3 groups underwent sham ischemia and reperfusion (I/R) and received the same treatments. Hemodynamic, biochemical and histological parameters were evaluated. Results. No significant hemodynamic or intestinal histological changes were seen in any sham group. No histological changes were found in the lung or liver of animals in the different groups. There was a progressive decrease in mean arterial blood pressure, from mean of 111.53 mmHg (30 minutes of ischemia) to 44.30±19.91 mmHg in SAL-I/R. 34.52±17.22 mmHg in PTX-I/R and 33.81±8.39 mmHg in NAC-I/R (P<0.05). In all I/R groups, there was a progressive decrease in: aortic blood flow, from median baseline of 19.00 mL/min to 2.50±5.25 mL/min in SAL-I/ R; 2.95±6.40 mL/min in PTX-I/R and 3.35±3.40 mL/min in NAC-I/R (P<0.05); in the heart rate, from mean baseline of 311.74 bpm to 233.33±83.88 bpm in SAL-I/R, 243.20±73.25 bpm in PTX-I/R and 244.92±76.05 bpm in NAC-I/R (P<0.05); and esophageal temperature, from mean baseline of 33.68°C to 30.53±2.05°C in SAL-I/R, 30.69±2.21°C in PTX-I/R and 31.43±1.03°C in NAC-I/R (P<0.05). In the other hand, there was an attenuation of mucosal damage in the small intestine of the animals receiving PTX, and only in the ileum of the animals receiving NAC. No changes were found in ileum or plasma malondialdehyde levels in any group. Conclusion. PTX was more efficient in reducing histological lesions than NAC, but neither treatment prevented hemodynamic changes during splanchnic organs I/R.

Evaluation of Renal Protection From High Doses of Melatonin in an Experimental Model of Renal Ischemia and Reperfusion in Hyperglycemic Rats

Background. Melatonin is a free radical scavenger with important actions in the study of renal ischemia and reperfusion (I/R). This study evaluated possible renal protection of high doses of melatonin in an experimental model of I/R in which rats were submitted to acute hyperglycemia under anesthesia with isoflurane.Method. Forty-four male Wistar rats, weighing more than 300 g, were randomly divided into 5 groups: G1, sham (n = 10); G2, melatonin (n = 10; 50 mg.kg(-1)); G3, hyperglycemia (n = 9; glucose 2.5 g.kg(-1)); G4, hyperglycemia/melatonin (n = 10; 2.5 g.kg(-1) glucose + melatonin 50 mg.kg(-1)); and G5, I/R (n = 5). In all groups, anesthesia was induced with 4% isoflurane and maintained with 1.5% to 2.0% isoflurane. Intraperitoneal injection of melatonin (G1, G4), glucose (G3, G4), or saline (G1, G5) was performed 40 minutes before left renal ischemia. Serum plasma values for creatinine and glucose were determined at baseline (M1), immediately following reperfusion (M2), and 24 hours after completion of the experiment (M3). Histological analysis was performed to evaluate tubular necrosis (0-5).Results. Serum glucose was higher at M2 in the groups supplemented with glucose, hyperglycemia (356.00 +/- 107.83), and hyperglycemia/melatonin (445.3 +/- 148.32). Creatinine values were higher at T3 (P = .0001) for I/R (3.6 +/- 0.37), hyperglycemia/melatonin (3.9 +/- 0.46), and hyperglycemia (3.71 +/- 0.69) and lower in the sham (0.79 +/- 0.16) and melatonin (2.01 +/- 1.01) groups, P < .05. Histology showed no necrosis injury in the G1, lesion grade 2 in the G2, and severe acute tubular necrosis in the G3: (grade 4), G4: (grade 5) and G5: (grade 4) groups (P < .0001).Discussion. Melatonin protected the kidneys submitted to I/R in rats without hyperglycemia; however, this did not occur when the I/R lesion was associated with hyperglycemia.Conclusions. Due to its antioxidant and antiapoptotic action, melatonin was able to mitigate, but not prevent acute tubular necrosis in rats with hyperglycemia under anesthesia by isoflurane.

Effects of cilostazol in kidney and skeletal striated muscle of Wistar rats submitted to acute ischemia and reperfusion of hind limbs

PURPOSE: To investigate the effect of cilostazol, in kidney and skeletal muscle of rats submitted to acute ischemia and reperfusion. METHODS: Fourty three animals were randomized and divided into two groups. Group I received a solution of cilostazol (10 mg/Kg) and group II received saline solution 0.9% (SS) by orogastric tube after ligature of the abdominal aorta. After four hours of ischemia the animals were divided into four subgroups: group IA (Cilostazol): two hours of reperfusion. Group IIA (SS): two hours of reperfusion. Group IB (Cilostazol): six hours of reperfusion. Group IIB (SS) six hours of reperfusion. After reperfusion, a left nephrectomy was performed and removal of the muscles of the hind limb. The histological parameters were studied. In kidney cylinders of myoglobin, vacuolar degeneration and acute tubular necrosis. In muscle interstitial edema, inflammatory infiltrate, hypereosinophilia fiber, cariopicnose and necrosis. Apoptosis was assessed by immunohistochemistry for cleaved caspase-3 and TUNEL. RESULTS: There was no statistically significant difference between groups. CONCLUSION: Cilostazol had no protective effect on the kidney and the skeletal striated muscle in rats submitted to acute ischemia and reperfusion in this model.

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway

OBJECTIVES: The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/reperfusion conditions following cilostazol administration. METHODS: Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence. RESULTS: Acetylcholine- and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/Cilostazol/Reperfusion groups. CONCLUSION: Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endothelium-dependent vascular reactivity under ischemia/reperfusion conditions.

Effects of ischemia and reperfusion on subpopulations of rat enteric neurons expressing the P2X7 receptor

BACKGROUND: Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death. AIM: To analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of rat ileum. METHODS: The ileal artery was occluded for 35 min and animals were euthanized 6, 24, and 72 h, and 1 week later. Immunohistochemistry was performed with antibodies against the P2X7 receptor as well as nitric oxide synthase (NOS), calbindin, calretinin, choline acetyltransferase (ChAT), or the pan-neuronal marker anti-HuC/D. RESULTS: Double immunolabeling demonstrated that 100% of NOS-, calbindin-, calretinin-, and ChAT-immunoreactive neurons in all groups expressed the P2X7 receptor. Following I/R, neuronal density decreased by 22.6% in P2X7 receptor-immunoreactive neurons, and decreased by 46.7, 38, 39.8, 21.7, and 20% in NOS-, calbindin-, calretinin-, ChAT-, and HuC/D-immunoreactive neurons, respectively, at 6, 24, and 72 h and 1 week following injury compared to the control and sham groups. We also observed a 14% increase in the neuronal cell body profile area of the NOS-immunoreactive neurons at 6 and 24 h post-I/R and a 14% increase in ChAT-immunoreactive neurons at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12% at 6 h post-I/R and increased by 8% at 24 h post-I/R. CONCLUSIONS: This work demonstrates that I/R is associated with a significant loss of different subpopulations of neurons in the myenteric plexus accompanied by morphological changes, all of which may underlie conditions related to intestinal motility disorder

Alterations in nitric oxide synthase isoforms in acute lower limb ischemia and reperfusion

Alterations in nitric oxide synthase (NOS) are implicated in ischemia and ischemia-reperfusion injury. Changes in the 3 NOS isoforms in human skeletal muscle subjected to acute ischemia and reperfusion were studied. Muscle biopsies were taken from patients undergoing total knee replacement. Distribution of the specific NOS isoforms within muscle sections was studied using immunohistochemistry. NOS mRNA levels were measured using real-time reverse transcription-polymerase chain reaction and protein levels studied using Western blotting. NOS activity was also assessed using the citrulline assay. All 3 NOS isoforms were found in muscle sections associated with muscle fibers and microvessels. In muscle subjected to acute ischemia and reperfusion, NOS I/neuronal NOS mRNA and protein were elevated during reperfusion. NOS III/endothelial NOS was also upregulated at the protein level during reperfusion. No changes in NOS II/inducible NOS expression or NOS activity occurred. In conclusion, alterations in NOS I and III (neuronal NOS and endothelial NOS) at different levels occurred after acute ischemia and reperfusion in human skeletal muscle; however, this did not result in increased NOS activity. In the development of therapeutic agents based on manipulation of the NO pathway, targeting the appropriate NOS isoenzymes may be important.

Prevention of bacterial translocation using β-(1-3)-D-glucan in small bowel ischemia and reperfusion in rats

To investigate the role of β-(1-3)-D-glucan on 99mTc labelled Escherichia coli translocation and cytokines secretion in rats submitted to small bowel ischemia/reperfusion injury. Methods: Five groups (n=10 each) of Wistar rats were subjected to control(C), sham(S), group IR subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R), and group I/R+glucan subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R) and injected with 2mg/Kg intramuscular. Translocation of labelled bacteria to mesenteric lymph nodes, liver, spleen, lung and serum was determined using radioactivity/count and colony forming units/g(CFU/g). Serum TNFα, IL-1β, IL-6, IL-10 were measured by ELISA. Results: CFU/g and radioactivity/count were higher in I/R than in I/R+glucan rats. In C, S and S+glucan groups, bacteria and radioactivity/count were rarely detected. The I/R+glucan rats had enhancement of IL-10 and suppressed production of serum TNFα, IL-1β and, IL-6, compared to I/R untreated animals. Conclusion: The β-(1-3)-D-glucan modulated the production of pro-inflammatory and anti-inflammatory cytokines during bowel ischemia/reperfusion, and attenuated translocation of labelled bacteria

Prevention of bacterial translocation using β-(1-3)-D-glucan in small bowel ischemia and reperfusion in rats

To investigate the role of β-(1-3)-D-glucan on 99mTc labelled Escherichia coli translocation and cytokines secretion in rats submitted to small bowel ischemia/reperfusion injury. Methods: Five groups (n=10 each) of Wistar rats were subjected to control(C), sham(S), group IR subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R), and group I/R+glucan subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R) and injected with 2mg/Kg intramuscular. Translocation of labelled bacteria to mesenteric lymph nodes, liver, spleen, lung and serum was determined using radioactivity/count and colony forming units/g(CFU/g). Serum TNFα, IL-1β, IL-6, IL-10 were measured by ELISA. Results: CFU/g and radioactivity/count were higher in I/R than in I/R+glucan rats. In C, S and S+glucan groups, bacteria and radioactivity/count were rarely detected. The I/R+glucan rats had enhancement of IL-10 and suppressed production of serum TNFα, IL-1β and, IL-6, compared to I/R untreated animals. Conclusion: The β-(1-3)-D-glucan modulated the production of pro-inflammatory and anti-inflammatory cytokines during bowel ischemia/reperfusion, and attenuated translocation of labelled bacteria