Biomedical subjects
Publications and source records attributed to E Jennische.
A histochemical method combining an intravital dye-exclusion test and a staining for calcium, Alizarin red S, was used for the evaluation of irreversible ischemic injury in rat kidney. The method was found to allow an easy and rapid evaluation of the extent of irreversible post-ischemic injury, and selective damage to the S3-segments of proximal tubules could be demonstrated.
The regeneration of skeletal muscle cells was investigated in the extensor digitorum longus muscle (EDL) of rats subjected to hindlimb tourniquet ischaemia preceded by glycogen depletion. In this experimental model a distinct population of 'white' muscle cells are preferentially damaged, while there is minor damage to microvessels. A rapid regenerative response occurred after injury. Mitotic figures were obvious in satellite cells of damaged as well as of uninjured muscle cells within 48 h after the ischaemic insult. All debris of injured cells was phagocytized and removed within 72 h and replaced by clusters of myoblasts. Small immature muscle cells could be demonstrated within 96 h after injury. During the entire regeneration process the basic architecture of the muscle was preserved and surviving muscle cells were present among the regenerating ones. Histochemical investigations 3 weeks after injury revealed an increased number of 'red' muscle cells in the postischaemic EDL compared to the contralateral undamaged muscle. It is concluded that the described experimental model may be useful for studies on skeletal muscle regeneration.
The early development of post-ischemic cell injury was investigated in rat livers. Rats were subjected to 90 minutes of liver ischemia, followed by periods of re-perfusion from 10 minutes to 3 hours. The ischemia-induced injury was quantified by using a double staining method. An intravital dye exclusion test with Evans blue was combined with a histochemical stain for calcium, Alizarin red S (ARS). It was found that the markers identified two populations of injured cells, positive for Evans blue (EBA) and ARS respectively. The number of injured cells increased successively during the re-perfusion period. The overlapping between the two populations was small during the early post-ischemic phase but increased with increasing re-perfusion time. Treatment with ruthenium red, a blocker of mitochondrial calcium uptake, during the re-perfusion period significantly reduced the number of ARS-positive cells, while the number of EBA-positive cells was not affected. It is suggested that the two markers used identify cell populations, which are injured by different mechanisms operating in the post-ischemic phase. These mechanisms may or may not be dependent on calcium.
The effect of preischaemic glycogen-depletion on the development of skeletal muscle injury was investigated in rats subjected to tourniquet hind-limb ischaemia. Glycogen depletion was performed by direct electrical stimulation of the extensor digitorum longus (EDL) muscle during ischaemic conditions. The metabolite load during the subsequent 2.5 h of ischaemia was modified by allowing, or not allowing, a short reperfusion period after termination of the electrical stimulation. The extent of injury was evaluated morphologically after 5 or 15 h of reperfusion, by the combination of an intravital dye exclusion test and a histochemical staining, demonstrating calcium-precipitates-Alizarin red S. Minimal damage was found in animals subjected to ischaemia without preceding glycogen depletion. In both groups of preischaemically glycogen-depleted animals, significant irreversible injury occurred. The injury was significantly less in animals in which a wash-out period was allowed after termination of stimulation. Fast-glycogenolytic fibres (FG) were most sensitive to the ischaemic insult during both experimental conditions, while slow-oxidative fibres (SO) were spared. Fast-oxidative-glycogenolytic fibres (FOG) showed an intermediate response. The injury seemed to be established after 5 h of reperfusion, indicating that cells react with an all-or-nothing response in the present model.
The effect of energy substrate depletion and of high lactic acid (LA) load on the development of irreversible cell injury was evaluated in the lateral gastrocnemius muscle of rabbits subjected to 4 hr of tourniquet hindlimb ischemia. Three groups of animals were studied. Group I, high ATP-ischemia, these animals were subjected to 4 hr of ischemia; group II, low ATP--low LA ischemia, in this group the gastrocnemius muscle was electrically stimulated for 5 min during ischemic conditions to reduce the glycogen store, a short reperfusion period was allowed after the stimulation in order to wash out the built up LA, and the muscle was then subjected to 4 hr of ischemia; group III, low ATP--high LA ischemia, in this group glycogen was depleted as in group II, but no reperfusion period was allowed before the 4 hr period of ischemia. In group I, ATP levels were well preserved during the ischemic period, whereas in the substrate-deprived groups (II and III) a rapid depletion of ATP and phosphocreatine (CP) occurred. The LA was twice as high in the "high LA" group (III) as in the "low LA" group (II) during the ischemic period. The extent of injury was evaluated after 24 hr of reperfusion by measuring ATP and CP content, and contractile force and by light microscopy. No or minor cell damage was found in group I. In group III--high LA--no recovery was obtained in any of the variables used for evaluation. In group II--Low LA--there was a certain recovery. ATP and CP increased to about 35% and contractile force to 25% of control. Morphologically about 20% of the muscle cells appeared to be unaffected by the ischemic insult. It is concluded that reduction of the glycogen available for ATP resynthesis during the ischemic period drastically reduces the ability of skeletal muscle to withstand prolonged ischemia. A high LA load seems to amplify the deleterious effects of a low initial substrate level.
The effect of cholera toxin (CT)-challenge on histochemically demonstrable activities of adenylatecyclase and alkaline phosphatase was investigated in rat small intestine, using an intestinal loop model. CT-challenge increased the activities of adenylatecyclase and alkaline phosphatases within 15 minutes, and the changes were confined to enterocytes in the upper third parts of the villi. There was no change in the staining of the crypt cells. There was an increased basal activity of both adenylatecyclase and alkaline phosphatases in animals desensitized to cholera toxin by multiple peroral exposures. CT-challenge in the desensitized rats did not further increase the enzyme activity. It is concluded that desensitization to secretagogues induces profound alterations in the cell systems responsible for fluid secretion.
To study the effects of ATP-MgCl2 on impaired protein synthesis and hepatic cell membrane potential (HCMP) in the postischemic liver, ATP-MgCl2 (50 or 150 mumole/kg) or saline was administered intravenously following 90 min of liver ischemia in rats. Protein synthesis was measured by determining rate of leucine incorporation into proteins in incubated liver slices. HCMP was registered in vivo with glass microelectrodes. No effects of the two doses of ATP-MgCl2 on protein synthesis or HCMP in the postischemic liver were found. The results indicate that beneficial effects of ATP-MgCl2 previously reported following liver ischemia are not primarily caused by direct energy provision to hepatocytes.
The effect of the free radical scavenger glutathione (GSH) on the early post-ischemic liver cell death was studied on liver tissue of the rat. Animals with different pre-ischemic liver GSH contents were subjected to a 90 min period of ischemia, followed by a 3 h period of reperfusion. Cell death was evaluated morphologically by estimating intracellular calcium, using the stain Alizarin red S (ARS), and by dye-exclusion test using Evans blue. The extent of post-ischemic injury was also assessed by registration of the membrane potential (MP) in liver cells. Four groups of animals were studied: 1) fed rats. 2) fed rats pretreated with diethylmaleate. 3) rats fasted for 48 h. 4) fasted rats pretreated with cobalt-chloride. It was found that the early post-ischemic cell death was more extensive in rats with low initial GSH content (group 2 and 3), than in rats with high GSH content (group 1 and 4). It is suggested that GSH is protective against post-ischemic injury, probably by reducing lipid peroxidation.
The extent of post-ischemic calcification in skeletal muscle was studied in rats subjected to 5.5 h of ischemia followed by periods of reperfusion ranging from 30 min to 16 h. Sections from the tibialis posterior and soleus muscle were stained with alizarin red S (ARS) and were inspected with light microscopy. The number of ARS-positive cells increased during the first 5 h of reperfusion. The extent of calcification varied between fibers and the pattern of calcification appeared to be dependent on fiber type. In some fast glycogenolytic fibers (FG) central calcifications, probably representing mitochondria, were observed. In fast oxidative glycogenolytic (FOG) and slow oxidative (SO) fibers the most common pattern was an ARS-positive granulation, probably representing calcification of sarcoplasmic reticulum and/or mitochondria. The granulation was more extensive in FOG than in SO fibers. In some areas extracellular ARS-positive material was observed. In such areas, which probably represent areas of no-reflow, no calcification of muscle fibers was observed.
The effect of the scavenger compound disulfiram (Antabus) on postischemic cell injury was investigated. Rats were subjected to 90 min of liver ischemia and 3 h of reperfusion. The extent of cell injury was evaluated morphologically by a dye exclusion test and a histochemical stain for calcium. It was found that pretreatment with disulfiram significantly reduced the number of injured cells compared to untreated controls.
The effect of ischemia on the hepatic cell membrane potential (MP) was studied in vivo in fed and fasted rats. Liver ischemia was induced for 1 h by clamping all afferent vessels to the left lateral and medial lobes and was followed by a 3 h period of reflow. The liver content of ATP, glucose, lactate and glycogen was repeatedly measured and related to the MP changes. The initial ATP content was 40% higher and the glycogen content 10 times higher in the fed animals, while the MP was similar in both groups. During the ischemic period the ATP content as well as the MP decreased in a similar manner in both groups of animals. During reflow the ATP content increased to about 50% of the initial value in both groups of animals. The MP was normalized more rapidly in fed animals than in fasted ones, indicating a better capability for recovery after ischemic injury in fed animals.
The relation between the lactate content and the membrane potential was investigated in the gastrocnemius and soleus muscle of the cat during and after a 4h period of ischemia. The skeletal muscle content of ATP and glucose was also measured. No change occurred in the ATP content of the gastrocnemius muscle during ischemia and increased above initial values after reflow in both muscles. The lactate content increased, and the membrane potential decreased linearly in both muscles during the ischemic period. The final lactate accumulation was higher and the decrease in membrane potential was less in the gastrocnemius than in the soleus. After release of the tourniquet both variables returned to normal or near normal values within 1.5 h in both muscles. A significant correlation was found between the lactate content and the membrane potential in both muscles during the entire experimental period. It is suggested that the depolarisation occurring in skeletal muscle during hypoxia is partly caused by changes in intracellular pH.
The relationship between the extracellular potassium concentration (K+ e.c.) and the resting membrane potential (MP) of the gastrocnemius muscle was studied in the anesthetized rabbit during a 3-h period of hindleg ischemia and a subsequent 1.5-h period of resumed flow. The K+ e.c. was measured on the skeletal muscle surface, using a potassium selective electrode. The MP was recorded with conventional microelectrodes. Small biopsies were taken and analysed for content of ATP and lactate. The lactate content increased fourfold during the ischemic period, but returned to normal values after reflow. No significant changes occurred in the ATP content during the period of the experiment. K+ e.c. increased from 3.6 +/- 0.2 to 16.1 +/- 0.7 mmol/l, and the MP decreased from -90.2 +/- 0.6 to -58.5 +/- 1.9 mV during the 3-h period of ischemia. After release of the tourniquet there was an initial rapid decrease in K+ e.c. to 7.8 +/- 0.9 mmol/l during the first 15 min of resumed flow, followed by a slower decrease. The MP increased linearly during the first hour of resumed flow and both variables returned to near normal values 1.5 h after releasing the tourniquet.
The effect of chlorpromazine (CPZ) on net fluid transport in the small intestine of the rat was studied, using a ligated loop model. Given intramuscularly at 5 mg/kg, CPZ induced a 50% increase of net fluid absorption in jejunum, but had no effect in ileum. Similarly, the drug decreased net fluid accumulation induced by dibutyryl-cyclic AMP in jejunum, but did not influence net fluid accumulation in ileum. In contrast, net fluid accumulation induced by cholera toxin was decreased by CPZ in both jejunum and ileum. Inhibition of cyclic AMP (cAMP)-which probably mediates the hypersecretion by the toxin-was also noted. These results suggest that CPZ reverses fluid transport during cholera in two ways: by a cAMP-independent enhancement of fluid absorption in jejunum and by inhibition of the toxin-induced cAMP-production in jejunum as well as ileum. Each of these processes may be independently affected by pharmacological substances and subjected to neural and hormonal regulation.
The effect of membrane-stabilizing and sedative drugs on enterotoxic diarrhoea was studied in mice. Fluid secretion was induced in ligated loops of the small intestine by challenge with cholera toxin (CT), heat-labile enterotoxin (LT) from Escherichia coli or dibuturyl-cyclic AMP (dB-cAMP). Chlorpromazine, melperone, diazepam, mebumal, ketamine and ethanol all inhibited CT-induced hypersecretion. ED50 being 1.5, 4, 4, 35, 70 and 1500 mg/kg, respectively. The drugs also blocked CT-stimulation of adenylate cyclase, which mediates the action of CT. Concomitant with the antisecretory effect a sedative effect was induced, as judged by the motility and righting reflex of the animals. Hypersecretion by E. coli and LT was also totally blocked by chlorpromazine, diazepam, ketamine and ethanol. In contrast, the secretion by dB-cAMP which bypasses adenylate cyclase in its action, was not affected by diazepam and was only partly reversed by chlorpromazine, ketamine and ethanol. The reversal of dB-cAMP-secretion is probably due to enhanced absorption, rather than inhibition of adenylate-cyclase. The use of membrane-stabilizing drugs may represent a new principle for pharmacological treatment of diarrhoea.
Hepatic cell membrane potential (HCMP) and protein synthesis were studied in rats during and after a period (60 min) of liver ischemia. ATP-MgCl2 (5 mumol . 100 g bw-1) or saline was administered slowly intravenously before or after the period of liver ischemia. No beneficial effect of ATP-MgCl2 on the impaired HCMP or protein synthesis was observed. On the contrary, the post-ischemic restitution of these cellular metabolic variables was slower in animals receiving ATP-MgCl2 after the period of liver ischemia as compared with saline-treated rats. At the end of the ischemic period, ATP, glucose and lactate in liver tissue were the same in animals receiving ATP-MgCl2 or saline before ischemia. These results suggest that the beneficial effects previously reported after administration of ATP-MgCl2 in conditions with reduced hepatic blood flow are not primarily due to an improved metabolic situation in the liver.
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The relationship between central haemodynamics and vascular and metabolic parameters in skeletal muscle was studied in dogs subjected to controlled haemorrhagic hypotension and treated with cumulative doses of methylprednisolone (4-32 mg x kg-1), (or saline in the control group), followed by phenoxybenzamine. There were no significant haemodynamic or metabolic changes between the groups during the injections of steroid or saline. The alpha-adrenergic receptor blockade caused, as found earlier, a pronounced vasodilation in the steroid group, which was parallelled by an increase in muscle blood flow in the same order of magnitude. There was no clearcut relationship between metabolic and vascular effects in the groups. Despite the significant difference in blood flow between the groups after phenoxybenzamine, only small and insignificant differences were seen in muscle metabolites, with the exception of muscle lactate which showed higher values in the steroid group. The study provides further evidence in support of the hypothesis that the haemodynamic effects of the combination of methylprednisolone and phenoxybenzamine are of neurogenic origin.