Assessment of ischemic vascular damage.

This protocol describes a model convenient for acute experiments in anesthetized rats, performed by selective occlusion of the superior mesenteric artery. Such a model provides a means for assessing the role played by various pathophysiological mechanisms in the development of intestinal ischemic injury. It is especially suitable for studying different treatments, mainly pharmacological ones, to help cope with this problem in clinical practice. In the form of support protocols, this unit includes functional vascular and chemiluminescence studies, determination of vascular permeability and myeloperoxidase activity, transit time, and mortality.

Chemiluminescence response induced by mesenteric ischaemia/reperfusion: effect of antioxidative compounds ex vivo.

Ischaemia and reperfusion (I/R) play an important role in human pathophysiology as they occur in many clinical conditions and are associated with high morbidity and mortality. Interruption of blood supply rapidly damages metabolically active tissues. Restoration of blood flow after a period of ischaemia may further worsen cell injury due to an increased formation of free radicals. The aim of our work was to assess macroscopically the extent of intestinal pathological changes caused by mesenteric I/R, and to study free radical production by luminol enhanced chemiluminescence (CL) of ileal samples. In further experiments, the antioxidative activity of the drugs tested was evaluated spectrophotometrically by the use of the DPPH radical. We studied the potential protective ex vivo effect of the plant origin compound arbutin as well as of the pyridoindole stobadine and its derivative SMe1EC2. I/R induced pronounced haemorrhagic intestinal injury accompanied by increase of myeloperoxidase (MPO) and N-acetyl-ß-D-glucosaminidase (NAGA) activity. Compared to sham operated (control) rats, there was only a slight increase of CL response after I/R, probably in association with neutrophil increase, indicated by enhanced MPO activity. All compounds significantly reduced the peak values of CL responses of the ileal samples ex vivo, thus reducing the I/R induced increase of free radical production. The antioxidants studied showed a similar inhibitory effect on the CL response influenced by mesenteric I/R. If proved in vivo, these compounds would represent potentially useful therapeutic antioxidants.

Protective effect of melatonin in acetic acid induced colitis in rats.

Possible protective effects of exogenous melatonin on colonic inflammation were studied in rats. Colitis was induced by intracolonic (i.c.) instillation of 4% acetic acid (AA) and the resulting injury was assessed after 1 and 48 hr. Diffuse hyperemia and bleeding with erosions and ulcerations were observed in the colons of vehicle-treated rats. Melatonin administered in doses of 5 and 10 mg/kg reduced significantly the extent of gross mucosal damage after intraperitoneal as well as i.c. dosing. The inflammation induced increase in colonic wet weight was also reduced by melatonin treatment. In the early phase of colonic inflammation (60 min), melatonin partly prevented the decrease of reduced glutathione (GSH) content and limited lysosomal enzyme, N-acetyl-glucosaminidase and cathepsin D, activities induced by AA, with no changes in proteins or acid phosphatase activity. Increase of myeloperoxidase activity (MPO) caused by colonic inflammation was prevented by melatonin given i.c. As observed 48 hr after AA exposure, there was no difference between the effect of vehicle and melatonin on the content of GSH. Colitis did not influence the melatonin content of the colon. After administration of exogenous melatonin, plasma, pineal and gut melatonin tended to increase. The results indicate that melatonin participates in various defense mechanisms against colonic inflammatory processes by preserving the important endogenous antioxidant reserve of GSH, by preventing lysosomal enzyme disruption, by inhibiting enhanced MPO activity, thus reducing the extent of colonic damage, mainly in the early phase of colitis.

Ischaemia/reperfusion-induced organ injury in low dose streptozotocin diabetes.

OBJECTIVES: The influence of low dose streptozotocin diabetes on intestinal and vascular injury induced by mesenteric ischaemia/reperfusion (I/R) was studied in rats. The role of reactive oxygen species (ROS) in the exacerbation of ischaemic/postischaemic damage in diabetes was addressed. METHODS: Diabetes was induced by i.p. injection of 3 x 30 mg/kg streptozotocin and after 5 weeks male Wistar rats underwent 60 min ischaemia followed by 30 min reperfusion of the superior mesenteric artery (SMA). The extent of intestinal haemorrhagic injury was assessed macroscopically. Relaxation to acetylcholine of precontracted SMA rings was tested. Chemiluminescence (CL) enhanced by luminol of tissue samples excised from the ileum and SMA was measured. RESULTS: In diabetic rats I/R-induced intestinal injury was significantly more pronounced compared to non-diabetic rats (63.6% potentiation). Decreased endothelial-dependent relaxation of diabetic SMA was not further influenced by I/R. Diabetes itself did not change the CL response of SMA and there was a similar CL increase in the diabetic group with I/R as in the controls. In the intestinal samples CL response was suppressed and I/R only mildly increased CL in the diabetic group. CONCLUSIONS: Diabetes renders the intestinal tissue more vulnerable to the effects of I/R. Endothelial-dependent relaxation of diabetic SMA was not further worsened by I/R. CL responses showed a different involvement of ROS in diabetic intestinal versus vascular tissue.

Participation of reactive oxygen species in diabetes-induced endothelial dysfunction.

OBJECTIVES: In the present study, the relationship between diabetes-induced hyperglycemia, reactive oxygen species production and endothelium-mediated arterial function was examined. The effect of antioxidant on the reactive oxygen species induced damage was tested. METHODS: Diabetes was induced by streptozotocin (STZ), 3 x 30 mg/kg i.p., administered on three consecutive days. After 10 weeks of diabetes, the functional state of the endothelium of the aorta was tested, endothelemia evaluation was performed and systolic blood pressure was measured. Reactive oxygen species (ROS) formation in blood and the aorta was measured using luminol-enhanced chemiluminescence (CL). Levels of reduced glutathione (GSH) were determined in the aorta, kidney, and plasma. To study the involvement of hyperglycemia in functional impairment of the endothelium, aortal rings incubated in solution with high glucose concentration were tested in in vitro experiments. RESULTS: After 10 weeks of diabetes, endothelial injury was observed, exhibited by diminished endothelium-dependent relaxation of the aorta, increased endothelemia and by elevated systolic blood pressure. Using luminol-enhanced CL, a significant increase of ROS production was found in arterial tissue and blood. GSH levels were significantly increased in the kidney, while there were no GSH changes in plasma and the aorta. Incubation of aortic rings in solution with high glucose concentration led to impairment of endothelium-dependent relaxation. The synthetic antioxidant SMe1EC2 was able to restore reduced endothelium-mediated relaxation. CONCLUSIONS: Our results suggest an important role of hyperglycemia-induced ROS production in mediating endothelial dysfunction in experimental diabetes, confirmed by CL and the protective effect of the antioxidant SMe1EC2.