Ischemia reperfusion injury of the skeletal muscle after selective deafferentation.

The present study analyzes the effect of selective deafferentation on the reperfusion injury of the skeletal muscle when nociceptive sensory fibers of the left sciatic nerve are selectively damaged by capsaicin pretreatment in a rat model following tourniquet ischemia (ISC) applied for 30 min, 1 h, and 2 h on the left hind limb. The isometric tetanic contractile force of the extensor digitorum longus (EDL) muscle was measured after 1 h, and 1, 3, or 7 days of reperfusion. Contractile force of the damaged muscle was compared to the intact contralateral muscle. In another group, ISC was used without capsaicin pre-treatment. After 30 min of ISC, there was no difference between deafferented and non-pretreated groups. Following 1 h ISC, with the exception of 1 h reperfusion, the non-pretreated group produced stronger contractions than the deafferented group. After 2 h ISC, the contractile force of the deafferented muscle was significantly stronger compared to the non-deafferented muscle force at all reperfusion times. In conclusions, it was found that the absence of peptidergic sensory fibers after long-lasting (2 h) ischemia is beneficial in reperfusion injury, whereas the absence of vasodilator peptides has unfavorable effects if tissue damage is milder (after 1 h ischemia).

Effect of ischemia and reperfusion on lambda of the lumped constant of the [14C]deoxyglucose technique.

We measured the parameter lambda, which is the ratio of the distribution spaces of 2-deoxy-D-glucose (DG) and glucose in the brain, in a model of focal cerebral ischemia in the cat. lambda is the parameter in the lumped constant of the [14C]DG technique most susceptible to changes in ischemia. Cats were subjected to occlusion of the middle cerebral artery for a period of 2 h. During the last 60 min of occlusion, [14C]DG was infused in a programmed fashion so as to obtain a stable arterial blood [14C]DG concentration. The brain was funnel-frozen to preserve tissue metabolites and the frozen brain was sampled regionally (4 to 7-mg samples) for local concentrations of glucose, ATP, phosphocreatine (PCr), and lactate. In a separate series of cats, the infusion of [14C]DG was started after 2 h of occlusion and 3 h of recirculation. In both series, lambda declined slightly for increased levels of tissue glucose and increased appreciably as tissue glucose decreased. A similar relationship was observed between lambda and ATP and PCr, although the correlation was not as clear. Since lambda, and hence the lumped constant, increases in ischemia as well as in postischemic tissue, it is important to measure tissue glucose concentration if quantitative values of local cerebral glucose metabolism are desired in this condition.

Acute improvement in histological outcome by MK-801 following focal cerebral ischemia and reperfusion in the cat independent of blood flow changes.

The present study reports on the acute effects of MK-801 on the histopathological outcome and blood flow changes during focal cerebral ischemia and reperfusion. In addition, acute changes in the EEG and blood pressure are also reported. In 16 halothane-anesthetized cats, the left middle cerebral artery (MCA) was occluded for 2 h followed by 4 h of reperfusion. Thirty minutes after the onset of ischemia, eight animals were treated with 1 mg/kg of MK-801, while eight animals received saline. Blood flow from the peripheral MCA territory was measured with H2 clearance. There was a comparable reduction in blood flow (down to 20% of control) in the ischemic gyri of the two groups followed by a partial recovery after recirculation. There was a similar decrease in the EEG amplitude over the ischemic central MCA territory in the treated and the untreated group. Treatment with MK-801 induced a burst suppression in the EEG and a transient drop (11.4 +/- 6.5 mm Hg) in the mean arterial pressure. The volume of early ischemic damage decreased by one-third in the MK-801-treated group compared to the untreated one, both in the total hemisphere (from 29 +/- 10 to 20 +/- 5%) and in the hemispheric cortex (range 36 +/- 8 to 24 +/- 13%). A major fraction of this improvement was localized to the middle and posterior parietal (mainly perifocal) regions of the MCA territory. These results show that in our model, MK-801 improves histopathological outcome despite the lack of apparent effect on the cortical blood flow, and an adverse effect on the systemic blood pressure. This is the first report that describes data on a reproducible model of reperfusion after temporary occlusion of the MCA in a cat, extending the findings of the Glasgow group, who observed similar neuroprotection in models of permanent MCA occlusion.

Total antioxidant capacity of serum increased in early but not late period after intestinal ischemia in rats.

The ischemia of small intestine was induced in anesthetized Wistar rats by occluding the superior mesenteric artery for 45 min and then the reperfusion was set. Serum samples were obtained at the end of the ischemic period and also during early (1 to 4 h) and late postischemic period (1 to 4 d). The total antioxidant capacity (TRAP) of serum samples was evaluated using luminol enhanced chemiluminescence. The increased mobilization of phagocytic cells and the release of reactive oxygen species into the circulation was observed from the first and second hour of the postischemic period, respectively. Nevertheless, the activity of natural antioxidant mechanisms of serum was already elicited at the end of the ischemic period. Furthermore, the TRAP of serum increased with the increasing duration of early postischemic period. Among the antioxidants studied, urate and ascorbate concentrations exerted the highest correlation with TRAP, but 31.6% of the total antioxidant capacity remained for the activity of an unidentified antioxidant(s). After being exhausted, the TRAP of serum oscillated around the preoperation level at days 1-4 of the postischemic period. The increase in total antioxidant capacity of serum induced by oxidative stress was sufficient to prevent lipoperoxidation both in serum and intestinal tissue.

Leukocyte mobilization, chemiluminescence response, and antioxidative capacity of the blood in intestinal ischemia and reperfusion.

Intestinal ischemia and reperfusion elicits changes in leukocyte counts and increased production of reactive oxygen species (ROS). The purpose of this study was to investigate whether these changes were followed by and/or connected with changes in the extracellular antioxidative capacity in a rat superior mesenteric artery (SMA) occlusion/reperfusion model. The SMA was occluded for 45 min and then allowed to be reperfused. Changes of leukocyte, polymorphonuclear (PMN), and lymphocyte counts, chemiluminescence (CL) of whole blood samples as a marker of ROS production, and the total antioxidative capacity of the serum were quantified at the end of ischemia and in 1 h intervals during the postischemic period up to 4 h. The myeloperoxidase (MPO) activity in the serum and intestinal tissue samples was also determined. The MPO activity in the intestinal tissue samples was significantly elevated at the end of ischemia, and this elevation lasted for the whole postischemic period. The oxidative challenge to the body induced a fast mobilization of extracellular antioxidative mechanisms already at the end of ischemia, which was followed by a significant increase in PMN counts and whole blood CL starting at the 2nd hour after reperfusion. The increased CL activity of whole blood was attributed to the increase of the circulating PMNs. No significant changes were observed in leukocyte and lymphocyte counts. It is concluded that compensatory mechanisms of the oxidative-antioxidative balance of the body react very quickly if challenged.

Prolonged effects of MK-801 in the cat during focal cerebral ischemia and recovery: survival, EEG activity and histopathology.

Previously we reported an improvement in histological outcome in cats treated with MK-801 shortly after the induction of temporary middle cerebral artery occlusion, and examined after 2 h of ischemia followed by 4 h of reperfusion. This study investigates the prolonged effects of the same drug treatment. Focal cerebral ischemia was produced in 34 cats by temporary occlusion of the left middle cerebral artery for 2 h. Stroke severity was determined using the ratio of the EEG amplitude from the ipsilateral to that of the contralateral hemisphere. Thirty minutes after the onset of ischemia, cats were treated i.v. with either 1 mg/kg MK-801 or saline. Electrocortical activity of the animals who survived were followed for 6 days postocclusion at which point they were sacrificed for histopathological analysis. Twelve of the animals died during recovery, of which 4 were MK-801 treated, and 8 were saline controls. The EEG ratios in the non-surviving animals were more depressed than in the animals that survived, whereas the depression in the EEG amplitude in both the treated and the control surviving animals was equal. Among the survivors no reduction in infarct size with MK-801 treatment was observed. Thus treatment with MK-801 in the middle cerebral artery occlusion model in the cat leads to a significant increase in the rate of survival (P < 0.05), but no prolonged improvement in late histopathology, in contrast with acute histological findings using this model. MK-801 treatment may be shifting the stroke model towards the survival of animals with larger infarcts. Histological recovery during prolonged reperfusion may eliminate the early neuroprotective effects seen with MK-801 treatment.

Time course of leukocyte response and free radical release in an early reperfusion injury of the superior mesenteric artery.

The sequence of changes in circulating immune cells and in free radical production was studied during the small intestine reperfusion. Rat small intestine ischemia/reperfusion was induced by a 45 min superior mesenteric artery occlusion followed by a 4-hour reperfusion. Samples of peripheral blood were collected every 20 min during reperfusion. While the number of polymorphonuclear leukocytes increased significantly both in the sham-operated controls and the experimental group (about 400 per cent at the end of reperfusion), a decrease in lymphocyte counts to 60 per cent was observed in the experimental group only. Although there were no changes in the counts of total T lymphocytes, a significant reduction in B cell counts was observed. Flow-cytometrical measurements showed no changes in the Tc subpopulation, while the Th subpopulation increased in the experimental group only. Free radical generation in blood (luminometric measurements) increased gradually and reached an eight-fold level by the end of reperfusion in both groups. Thus, it has been shown that the increase in free radical production is mainly due to the increased number of polymorphonuclear leukocytes mobilized already at the initial stages of reperfusion. The reduction in B lymphocyte population is probably due to homing mechanisms

Transient metabolic and vascular volume changes following rapid blood pressure alterations which precede the autoregulatory vasodilation of cerebrocortical vessels.

It has been shown by surface fluoro-reflectometry that stepwise decrease of arterial blood pressure causes a biphasic cerebrocortical vascular volume response. After the arterial blood pressure decrease the vascular volume first decreased and later increased. In both parts of the biphasic reflectance change, the cerebrocortical NAD-NADH redox state shifted considerably towards reduction and there was no reoxidation after the onset of cortical vasodilatation. Since a very rapid NADH reduction occurred during the first 30 secs. of the arterial hypotension in parallel with the vascular volume decrease, it is suggested that in the transient phase of arterial hypotension cerebral hypoxia may occur. Furthermore it is suggested that anaerobic tissue metabolites or some unknown NAD-NADH dependent process might dilate the cerebrocortical arterial network during the autoregulatory adjustment of CBF. The participation of the sympathetico-adrenal system in transient brain hypoxia caused by bleeding is a possibility since both the early vasoconstriction and the steep NADH reduction were prevented by the administration of phenoxybenzamine (1 mg/kg) before bleeding.

The first histological demonstration of pancreatic oxidative stress in human acute pancreatitis.

Necrotizing acute pancreatitis is associated with an inflammatory explosion involving numerous pro-inflammatory mediator cascades and oxidative stress. Acinar oxygen free radical production aggravates pancreatic tissue damage, and promotes cellular adhesion molecule upregulation resulting in leukocyte adherence and activation. The cerium capture oxygen free radical histochemistry combined with reflectance confocal laser scanning microscopy allows the "in situ" histological demonstration of oxygen free radical formation in live tissues. Here we present a case report, where oxidative stress is demonstrated on a histological level for the first time in human acute pancreatitis. A 44-year-old male patient suffering from acute exacerbation of his chronic pancreatitis developed a pancreato-pleural fistula with amylase-rich left pleural exudate causing respiratory compromise. Subsequent to an urgent thoracic decompression a distal pancreatectomy and splenectomy was performed with the closure of abdomino-thoracic fistula. The postoperative course was uneventful, except for a transient pancreatico-cutaneous fistula, which healed after conservative treatment. To carry out cerium capture oxygen free radical histochemistry the resected pancreas specimen was readily perfused with cerium-chloride solution through the arteries on the resection surface. Frozen sections were cut, E-, P-selectin, ICAM and VCAM were labeled by immunofluorescence. The tumor-free margin of an identically treated pancreas carcinoma specimen served as a control. Intrapancreatic oxidative stress and cellular adhesion molecule expression were detected by confocal laser scanning microscopy. Numerous pancreatic acini and neighboring capillaries showed oxygen free radical-derived cerium-perhy-droxide depositions corresponding to strong local oxidative stress. Acinar cytoplasmic reflectance signals suggested xanthine-oxidase as a source of oxygen free radicals. These areas presented considerably increased endothelial P-selectin expression with adherent, oxygen free radical-producing polymorphonuclear leukocytes displaying pericellular cerium-reflectance. Modest ICAM upregulation was noted, E-selectin and VCAM expression was negligible. The control pancreas specimen showed minimal oxidative stress with weak, focal P-selectin expression. The development of deleterious pancreatic oxidative stress was based on indirect evidence in human acute pancreatitis. To the best of our knowledge this is the first report demonstrating persistent intrapancreatic oxidative stress histologically in human acute pancreatitis. We have noted P-selectin overexpression with a preponderance in the areas of acinar oxidative stress.

Prevention of apoptosis reperfusion renal injury by calcium channel blockers.

Apoptosis has been shown in the literature to be the form of cell death occurring in renal tubular epithelial cells during the reperfusion phase after brief periods of renal ischaemia. In the present study apoptosis was examined in the rat kidney in the first 24 hours after 30 min ischaemia and apoptosis was influenced by administration of the Ca channel blockers Verapamil, Bepridil, Nifedipin and Sensit. Apoptosis was observed in the renal tubular cells two hours after the start of reperfusion and reached in maximum at hour 6. All above mentioned Ca channel blockers decreased the occurrence and degree of apoptosis.

Myocardial collagen degradation: morphological and biochemical correlation.

An attempt was made to prove the phenomenon of myocardial collagen breakdown in a norepinephrine (NE) induced model of reversible cardiopathy in anesthetized rabbits. NE (0.3 mg/kg in 90 min) was infused to 22 animals. Histologically it was found that immediately and during the first two days after the catecholamine administration the myocardial collagenous network became disrupted and it partly disappeared. Collagenase and peptidase activities were measured from homogenates of the same hearts. A significant increase of these activities was observed and that could be correlated with the intracellular matrix destruction.

Hydrogen peroxide via thromboxane A2 receptors mediates myogenic response of small skeletal muscle veins in rats.

UNLABELLED: We aimed to test two hypotheses: (1) isolated small veins develop substantial myogenic tone in response to elevation of intraluminal pressure, (2) H2O2 contributes to the mediation of myogenic response via activation of arachidonic acid (AA) cascade and release constrictor prostaglandins. METHODS: Small veins were isolated from gracilis muscle of male rats, then cannulated. Changes of diameter to increases in intraluminal pressure, H2O2 and arachidonic acid in the presence and absence of various inhibitors were measured by videomicroscope and microangiometer. At the end of experiments the passive diameter were obtained in Ca2+ -free PSS solution. RESULTS: Isolated rat gracilis muscle small veins developed a substantial myogenic tone in response to increases in intraluminal pressure (1-12 mmHg). Calculated maximum myogenic tone was 70 ± 5% at 10 mmHg. Presence of catalase or indomethacin or SQ 29,548 reduced significantly the pressure-induced myogenic response. Also, H2O2 (10-9-10-5 M) and arachidonic acid (10-7-10-4 M) elicited concentration dependent constrictions, which were inhibited by the presence of indomethacin or SQ 29,548. CONCLUSION: We propose that both myogenic response and pressure-induced release of H2O2 play important roles in regulating the vasomotor function of venules both in physiological and pathological conditions.

Bioassay-comparison of the antioxidant efficacy of hydrogen sulfide and superoxide dismutase in isolated arteries and veins.

Recent studies suggest that hydrogen sulfide (H2S) exhibits potent antioxidant capacity and improves vascular and tissue functions. Thus we aimed to compare the antioxidant efficacy of H2S to that of superoxide dismutase (SOD).Isometric force of isolated rat carotid arteries and gracilis veins was measured with a myograph. The vasomotor effect of the superoxide-generator pyrogallol (10-5M) was obtained in control conditions, and then in the presence of SOD (120 U/ml) or H2S (10-5M or 10-4M), respectively. Spectrophotometric measurements were performed to detect the effect of SOD and H2S on the auto-oxidation of pyrogallol.Pyrogallol increased the isometric force of carotid arteries (9.7 ± 0.8 mN), which was abolished by SOD (5.3 ± 0.8 mN), was not affected by 10-5M H2S (9.1 ± 0.5 mN), whereas 10-4M H2S slightly, but significantly reduced it (8.1 ± 0.7 mN). Pyrogallol significantly increased the isometric force of gracilis veins (1.3 ± 0.2 mN), which was abolished by SOD (0.9 ± 0.2 mN), whereas 10-5M (1.3 ± 0.2 mN), or 10-4M H2S (1.2 ± 0.2 mN) did not affect it. Pyrogallol-induced superoxide production was measured by a spectrophotometer (A420 = 0.19 ± 0.0). SOD reduced absorbance (A420 = 0.02 ± 0.0), whereas 10-5M H2S did not (A420 = 0.18 ± 0.0) and 10-4M H2S slightly reduced it (A420 = 0.15 ± 0.0).These data suggest that H2S is a less effective vascular antioxidant than SOD. We propose that the previously described beneficial effects of H2S are unlikely to be related to its direct effect on superoxide.

Effect of intraarterial somatostatin infusion on SMA blood flow.

Experiments were carried out on anaesthetized cats to study the effect of somatostatin on the mesenteric circulation. Intraarterial infusions of somatostatin were applied into the superior mesenteric artery. The effect of atropine, propranolol and phentolamine were investigated. Catecholamine release or uptake of the mesenteric vascular bed during somatostatin infusions were also measured. We found dose dependent vasodilatory effect of somatostatin on the mesenteric vasculature that was not influenced by atropine and by the adrenoreceptors or by denervation. A direct effect of somatostatin on the vascular smooth muscle membrane is assumed.

In vivo determination of transport and metabolism of deoxyglucose in intestinal tissues.

Experiments were carried out on 11 anesthetized cats (Na-pentobarbital). Uptake of a glucose analogue (2-deoxy-D-glucose) by intestinal mucosa and muscularis from arterial plasma was studied in order to determine net rate of transport and phosphorylation of the material. The theoretical basis for calculating the rate constants of forward (kx1) and reverse (kx2) transport between plasma and tissue and also phosphorylation (kx3) and dephosphorylation (kx4) of 14C labeled deoxy-glucose (DG) was determined. The method can also be used for estimating tissue glucose uptake. The rate constants were found to be: kx1 = 0.669 and 0.873; kx2 = 2.285 and 4.656; kx3 = 0.057 and 0.067; kx4 = 0.091 and 0.097 [s-1] in the mucosa and muscularis, respectively. Glucose utilisation of intestinal mucosa was 2.69 and that of muscularis 2.46 mg/(min X 100 g) tissue, respectively. Arterial glucose concentration was constant during the studies, however, it showed a variation from 120 to 250 mg/100 ml of plasma from animal to animal. Tissue glucose uptake or any of the rate constants were not influenced by the plasma level over this range.

Sequence of morphological alterations in a small intestinal ischaemia/reperfusion model of the anesthetized rat. A light microscopy study.

Previous studies have shown serious mucosal damage and destruction to be associated with intestinal ischaemia/reperfusion. As both destruction and concomitant regeneration can be observed together in this potentially lethal condition we have studied the development and sequence of events by evaluating morphological changes of the small intestine in an ischaemia/reperfusion model in anaesthetized rats. Forty-five minutes of ischaemia was followed by 4 hours of reperfusion. Tissue samples of the small intestine were examined by light microscopy in normal and semithin sections. Samples were collected at the end of ischaemia, at 10 min, and at 1, 2, 3 and 4 hours of reperfusion, respectively. Survival was assessed in a parallel group of anaesthetized rats. The morphological changes were described and they were analysed by a semi-quantitative method using five different markers of histological alteration. The mortality rate of a control survival group was 100%. Mucosal destruction at the end of ischaemia and during reperfusion was diffuse and steadily increased as a function of reperfusion time. At the same time the epithelium showed intensive regenerative growth which covered the denuded mucosal surface by the third hour of reperfusion. A secondary epithelial desquamation followed this process and was accompanied by heavy inflammatory cell infiltration. The infiltration may be the cause of the secondary epithelial injury.

Acute effect of scalding injury on blood flow in muscle and subcutaneous tissue in the paw of the anaesthetized dog.

Blood flow in muscle and subcutaneous tissue was studied before and after a scalding injury of the dog paw. The hydrogen clearance technique was used in anaesthetized dogs. As scalding of the paw resulted in an increased blood pressure and muscular movements, animals were given a muscle relaxant and ventilated artificially. Before scalding injury the blood flow was 29.8 and 19.7 ml per min per 100 g in muscular and subcutaneous tissue, respectively. After immersion of the paw for 10 sec in water of 100 degrees C the blood flow transiently increased and later decreased. No major changes were found in the contralateral paw. The decrease was most pronounced (50%) in subcutaneous tissue. In the acute situation the impeded circulation in the burn oedema may protect the organism from potentially harmful substances formed in the injured tissue.