Morphofunctional characteristics of murine kidneys in single and fractionated irradiation in air and hypoxia (8% of O2)]

The protective effect of hypoxic gas mixture containing 8% of oxygen (HGM-8) has been studied in long terms after local single and fractional X-ray irradiation of the kidney in mice. Some criteria of injury shown that hypoxia protects the kidneys against irradiation, changes in irradiation dose constituting 1.25-1.33. When passing from single irradiation to five-fold daily one, the protective effect of hypoxia does not fall significantly.     

Influence of melatonin pretreatment and preconditioning by hypobaric hypoxia on the development of cortical photothrombotic ischemic lesion

Photothrombotic model of ischemia (PT) is based on free radical-mediated endothelial dysfunction followed by thrombosis. Free radicals are also involved in hypoxic preconditioning. We tested the sensitivity of PT to preconditioning with hypobaric hypoxia and to pretreatment with melatonin. In adult Wistar rats, after intravenous application of Rose Bengal, a stereo-tactically defined spot on the denuded skull was irradiated by a laser for 9 min. The first experimental group underwent hypobaric hypoxia three days before irradiation. In the second experimental group, melatonin was applied intraperitoneally one hour before irradiation. Three days after irradiation, animals were sacrificed, the brains perfused, and stained with TTC. Ischemic lesions were divided into grades (I, II, III). In the control group (where no manipulation preceded photothrombosis), most animals displayed deep damage involving the striatum (grade III). The group pre-exposed to hypoxia showed similar results. Only 28.57 % of the melatonin pretreated animals exhibited grade III lesions, and in 57.14 % no signs of lesions were detected. Pre-exposure to hypoxia was not protective in our model. Pretreatment with melatonin lead to a significant reduction of the number of large ischemic lesions. This result is probably caused by protection of endothelial cells by melatonin.     

Tissue-specific ceramide response in the chronically hypoxic rat model mimicking cyanotic heart disease

BACKGROUND AND OBJECTIVE: Acute hypoxia is associated with apoptosis and increase in ceramide levels in various organs. To assess the effect of chronic hypoxia on ceramide accumulation in the lungs and kidneys, we utilized an animal model mimicking cyanotic heart disease. METHODS: Rats were placed in a hypoxic environment at birth and oxygen levels were maintained at 10% in an air-tight Plexiglas chamber. Controls remained in room air. Animals were sacrificed and the lung and kidneys were harvested and weighed at 1 and 4 weeks, respectively. Ceramide levels were measured using a modified diacylglycerol kinase assay. RESULTS: Significant polycythemia developed in the hypoxic rats at 1 and 4 weeks. Indexed lung and kidney masses were significantly increased in the hypoxic animals as compared to controls at 1 and 4 weeks, respectively. The ceramide levels in the hypoxic lungs and kidneys were not significantly different from control groups at 1 and 4 weeks. [Ceramide/phosphate ratio in the kidneys was 1.28 +/- 0.17 (C) versus 1.18 +/- 0.12 (H) at 1 week; P = 0.39, and 1.46 +/- 0.08 (C) versus 1.33 +/- 0.15 (H) at 4 weeks (P = 0.44)] and [ceramide/phosphate ratio (pmol/nmol) in the lungs was 2.29 +/- 0.14 (C) versus 1.98 +/- 0.12 (H) at 1 week (P = 0.17), and 2.42 +/- 0.16 (C) versus 2.30 +/- 0.05 (H) at 4 weeks, P = 0.34]. CONCLUSION: The response of lungs and kidneys to chronic hypoxia includes increase in indexed mass and lack of ceramide accumulation. This is similar to the response previously reported in the chronically hypoxic brain and heart. Thus, various organs appear to have similar ceramide response pattern to chronic hypoxia.     

Radioprotective effect of hypoxic hypoxia (8% O2) for different morphological elements of mouse kidney

The breathing of gas mixtures containing 8-9% O2 during irradiation of tumors has been tested at several cancer clinics (in Russia and abroad) with the purpose of decreasing the morbidity of normal issues, thus providing the possibility to increase the dose of radiation. Previous experiments have demonstrated a broad spectrum of dose modification factors (DMF) for different normal tissues as well as for different transplanted tumors, with in general larger protection of normal tissues. The present study was designed to assess the radioprotective effect for mouse kidney of breathing a gas mixture containing 8% O2 by morphometry of histological specimens. Both kidneys were locally irradiated using single fractions (11-19 Gy in air and 13-19 Gy in hypoxia) or 5 fractions separated by 24 h intervals (25-35 Gy in air and 30-40 Gy in hypoxia). Histological examination was performed 8 and 10 months after treatment. The DMF for glomeruli damage (glomerulosclerosis, ecstatic capillaries, hemorrhage) was in the range 1.25-1.29. Tubular damage showed a DMF of 1.28-1.37. Using the endpoint of development of interstitial tissue in the cortex a DMF of 1.32-1.37 was found after a single treatment, and 1.48 after fractionated irradiation. The radioprotective effect for arteriolar lesions was lower than measured using the above endpoints, namely 1.13-1.15 after single and 1.16-1.18 after fractionated irradiation. It was shown previously on groups of animals treated in the same manner that the DMF was between 1.24-1.26 when renal damage was assessed by hematocrit measurements, between 1.32-1.28 when it was evaluated by urination frequency, and 1.23-1.27 when kidney wet and dry weights were used as end-points. All these data witness that breathing 8% oxygen increases the tolerance of kidney function with a DMF above 1.2. The impact of low protection of arterioli on renal function in the late period after radiotherapy needs additional study.     

Effect of acute hypoxia on the renin and erythropoietin activities of the blood serum in rats

A comparative study of renin and erythropoietin content in the blood serum of rats with "endocrine" kidneys and changes in their activity following the action of specific erythropoietic stimulus (4-hour hypoxia) has been conducted. The presence of "endocrine" kidneys increased renin and erythropoietin activity in such animals. Acute hypoxia produced further increase in erythropoietin titre in the blood serum, with renin remaining at the same level. Possible differences in the mechanisms of renin and erythropoietin biogenesis in the kidneys are considered.     

The long-term effect of acute hypoxic hypoxia on shuttlebox avoidance learning in rats

Long-term influence was studied of the acute hypoxic hypoxia seance on rats behaviour in situation of elaboration of the conditioned reaction of active avoidance of electric shocks in shuttle chamber. It was found that in 2.5-3 months after the hypoxia seance, the experimental animals significantly differed from the intact controls by dynamics of CR elaboration (rats which had hypoxia were ahead of the control ones) and by distribution of the conditioned reactions latencies (for experimental animals this distribution was shifted to minor values). The character of these behavioural shifts coincided with that observed in the group of rats with local unilateral hippocampus lesion. The obtained results and numerous data presented in literature on the influence of the hippocampus lesion on animals shuttle avoidance learning, allow to conclude that the seance of hypoxic hypoxia leads to the disturbance of the hippocampus function. This conclusion conforms to the data on diffusive death of the hippocampal and neocortical neurones as a result of hypoxia action.     

Stressogenic exposure of local vibration to energetic metabolism of the heart, liver and kidney of rats

The purpose of the paper was to study the activity of energy producing system of the rabbit myocardium, liver and kidneys after exposure to local vibration during 7 days. The energy dependent reactions of native mitochondria were investigated by means of polarographic method using dark closed membrane electrode. The intensivity ofoxydative processes was assessed according to activity of the lymphocyte succinate dehydrogenase and catalase of the blood. The energy producing system of the tissues studied was shown to be involved in response reaction of the organism on vibration exposure. In that case we observed the formation of the second phase ofbioenergy hypoxia in the myocardium and kidneys and the first phase ofhypoxia in the liver.     

Activation of glycogen synthase in myocardium induced by intermittent hypoxia is much lower in fasted than in fed rats

Obstructive sleep apnea is characterized by intermittent obstruction of the upper airway, which leads to intermittent hypoxia. Myocardial glycogen is a major energy resource for heart during hypoxia. Previous studies have demonstrated that intermittent hypoxia rapidly degrades myocardial glycogen and activates glycogen synthase (GS). However, the underlying mechanisms remain undefined. Because sleep apnea/intermittent hypoxia usually happens at night, whether intermittent hypoxia leads to GS activation in the postabsorptive state is not known. In the present study, male adult rats were studied after either an overnight fast or ad libitum feeding with or without intermittent ventilatory arrest (3 90-s periods at 10-min intervals). Hearts were quickly excised and freeze-clamped. Intermittent hypoxia induced a significant decrease in myocardial glycogen content in fed rats and stimulated GS in both fasted and fed rats. However, the portion of GS in the active form increased by approximately 38% in fasted rats compared with a larger, approximately 130% increase in fed rats. The basal G-6-P content was comparable in fasted and fed animals and increased approximately threefold after hypoxia. The basal phosphorylation states of Akt and GSK-3beta and the activity of protein phosphatase 1 (PP1) were comparable between fasted and fed control rats. Hypoxia significantly increased Akt phosphorylation and PP1 activity only in fed rats. In contrast, hypoxia did not induce significant change in GSK-3beta phosphorylation in either fasted or fed rats. We conclude that hypoxia activates GS in fed rat myocardium through a combination of rapid glycogenolysis, elevated local G-6-P content, and increased PP1 activity, and fasting attenuates this action independent of local G-6-P content.     

Role of adenosine in hypoxic ventilatory depression

The role of adenosine in the ventilatory depression induced by hypoxia was studied in 82 spontaneously breathing urethan-anesthetized 4-day-old rabbit pups. Respiration was monitored with a pneumotachograph. The animals were exposed to hypoxia (6% O2 in N2) for 30 min or until the occurrence of terminal apnea. In all animals hypoxia produced an initial increase in ventilation followed by a decrease. In the control group 52% of the animals became apneic after 7 min of hypoxic exposure. By contrast, pretreatment with dipyridamole (10 or 20 mg/kg), an adenosine uptake blocker, significantly shortened the time needed to reach apnea. Thus at 7 min of hypoxia 93% of the animals that received dipyridamole became apneic. On the other hand, administration of adenosine antagonists 8-p-sulfophenyltheophylline (5 or 8 mg/kg) and aminophylline (10 or 25 mg/kg) significantly prolonged the time required to produce apnea. Only 20% of the animals that received these antagonists became apneic at 7 min of hypoxia. These results suggest that adenosine is potentially involved in the ventilatory depression produced by hypoxia in neonatal rabbit pups.     

Application of autologous hematopoietic cell therapy to a nonhuman primate model of heterogeneous high-dose irradiation

We developed a model of heterogeneous irradiation in a nonhuman primate to test the feasibility of autologous hematopoietic cell therapy for the treatment of radiation accident victims. Animals were irradiated either with 8 Gy to the body with the right arm shielded to obtain 3.4 Gy irradiation or with 10 Gy total body and 4.4 Gy to the arm. Bone marrow mononuclear cells were harvested either before irradiation or after irradiation from an underexposed area of the arm and were expanded in previously defined culture conditions. We showed that hematopoietic cells harvested after irradiation were able to expand and to engraft when reinjected 7 days after irradiation. Recovery was observed in all 8-Gy-irradiated animals, and evidence for a partial recovery was observed in 10-Gy-irradiated animals. However, in 10-Gy-irradiated animals, digestive disease was observed from day 16 and resulted in the death of two animals. Immunohistological examinations showed damage to the intestine, lungs, liver and kidneys and suggested radiation damage to endothelial cells. Overall, our results provide evidence that such an in vivo model of heterogeneous irradiation may be representative of accidental radiation exposures and may help to define the efficacy of therapeutic interventions such as autologous cell therapy in radiation accident victims.     

Sialic acid and neuraminidase activity in rat kidneys 6 months after uninephrectomy

Sialic acid and neuraminidase activity were determined in the cortex of the remnant kidneys of six uninephrectomized rats. As controls served either the kidneys removed at operation or age-matched kidneys from eight sham operated rats. Six months after uninephrectomy the kidneys became hypertrophied and their mean weight was about 40% higher than age-matched kidneys. Blood urea and creatinine and protein levels in 24-hr urine collections were significantly higher in the experimental animals as compared to those of the same animals before nephrectomy and to sham operated rats, indicating a marked impairment of kidney function. The mean concentration of sialic acid in the cortex of hypertrophied kidneys was not statistically different from either that of the removed or that of the age-matched kidneys. Neuraminidase activity expressed as either per gram fresh tissue or per milligram protein, was not different in the removed and in the hypertrophied kidney. The activity, however, in the latter was significantly lower than in the age-matched kidneys. Whether this finding can be associated with the impairment of kidney function in rats 6 months after uninephrectomy remains to be studied.     

Effect of hypoxia on DNA fragmentation in different brain regions of the newborn piglet

DNA fragmentation has been studied in different regions of the newborn piglet brain following different times of normobaric hypoxia (5% O(2), 95% N(2)). After 1 hr of hypoxia, fragmented DNA was observed in cerebellum, cortex, hippocampus, and striatum but not in hypothalamus. More fragmentation occurred in these areas of the brain when the animals were kept under hypoxia for times up to 8 hr 45 min. When the animals were submitted to hypoxia for two and a half hours, integrity of DNA was recovered respectively after 3 hr of exposure to the ambient atmosphere in hippocampus and striatum, but 4 hr of recovery were necessary for cerebellum and cortex. These results are discussed in terms of the consequences of neonatal hypoxia and apnea for newborn infants and economical impact for farm animals.     

Histopathological changes in gerbil liver and kidney after aluminum subchronic intoxication

Young gerbil livers and kidneys were analyzed by means of light and electron microscope to assess the histopathological changes caused by prolonged systemic aluminum (Al) administration. The experimental group was injected with AlCl3 i.p. for 5 weeks, while litter mates received PBS as sham-injected controls or served as untouched controls. Mortality occurred in 33% of experimental and 12.5% of sham-injected groups. The animals were perfused intracardially with 1% glutaraldehyde plus 1% paraformaldehyde and samples of liver and kidneys were processed for aluminum and iron histochemistry and conventional light- and transmission electron microscopy. White deposits composed of cellular debris appeared on the surface of liver and kidneys and in the mesentery as a consequence of Al treatment. Adherences of Glisson capsule to the diaphragm, as well as scattered small foci of hepatocyte necrosis with non-caseificant microgranulomas and mild portal inflammation, developed in the experimental group. Sham-injected animals also exhibited these granulomas but to a lesser degree. Al deposits were found in experimental animal granulomas and inside macrophages cytoplasm scattered throughout the liver. Iron deposition appeared in pericentral hepatocytes of experimental animals, in granulomas and in portal spaces of the three groups of animals. Ultrastructurally, hepatocytes of experimental animals showed mitochondria hyalinization, disintegration of endoplasmic reticulum and clustering of ribosomes. Phagolysosomes appeared larger and occurred more frequently in both hepatocytes and Kupffer cells of experimental animals. In 2 out of the 6 experimental animals studied, tubular atrophy was present in the renal cortical region, the kidneys of the remaining animals appearing normal. Al and iron were found very occasionally in the kidney parenchyma of experimental animals, while isolated mesangial cells showed iron deposits in a few glomeruli of both experimental and the two groups of control animals.     

Alterations in jejunal transport and (Na+-K+)-ATPase in an experimental model of hypoxia in rats

Hypoxia was induced by exposing rats to an atmosphere of 93% N2, 7% O2 for 4-48 hr. The animals became hypoxic as indicated by a decreased blood PaO2 (mean +/- SEM: 48 +/- 10 mm Hg). Hypoxia was accompanied by metabolic acidosis (pH 7.22 +/- 0.02) and decreased serum bicarbonate levels (9.0 +/- 4.0 meq/liter). Hypoxic rats also showed evidence of tissue hypoxia; liver tryptophan oxygenase levels were increased to 21 +/- 2 nmole/min/mg protein. In the hypoxic animals there was decreased jejunal mucosal (Na+-K+)-ATPase activity and an inhibition of active intestinal transport of sodium, glucose, 3-O-methylglucose, galactose, tyrosine, phenylalanine, and glycine as determined by in vivo perfusion studies. Jejunal fructose transport, which has a large passive component, was unaffected by hypoxia. The electrolyte, carbohydrate, and amino acid transport alterations produced by hypoxia were seen in the absence of an effect on jejunal cell number, DNA synthesis, or cell turnover. There was also no evidence of histological or ultrastructural damage. Furthermore, studies with a luminal macromolecular tracer, horseradish peroxidase, indicated that the jejunal lumen-to-blood barrier to macromolecules was also unaltered in these hypoxic animals. In vitro local oxygenation of the jejunum, by bubbling of 95% O2:5% CO2, markedly improved sodium and glucose (but not 3-O-methylglucose) absorption in hypoxic rats and control rats. The (Na+-K+)-ATPase activity of the jejunal mucosa of hypoxic rats was significantly enhanced by the local bubbling of 95% O2:5% CO2. Overall, our data indicate that during relatively mild conditions of hypoxia there is an inhibition of jejunal (Na+-K+)-ATPase activity and related transport processes that is prevented by in situ oxygenation.     

Delayed effect of prenatal exposure to hypoxia on the susceptibility of rats to electric seizures

We studied the delayed effects of prenatal exposure to hypoxia on the susceptibility of rats to seizures. The later was estimated using graded electroshock. The experiments were performed in two groups of 1.5-year-old male Wistar rats. The experimental group consisted of the animals that were exposed to hypoxia on day 14 of prenatal development, and the control group consisted of the animals that developed under the normal conditions. In the rats subjected to prenatal hypoxia, seizure episodes induced by weak currents in the range of 10–40 mA and their average duration were more pronounced as compared to the control animals.     

Effects of hypoxia on lung mechanics in the newborn cat

The present study was designed to investigate the effects of hypoxia on lung mechanics in the newborn cat and to determine if vagal efferent innervation to the airways is involved in the response. We studied 11 animals, aged 2-7 days, anesthetized with a mixture of chloralose-urethane administered intraperitoneally. A tracheal cannula was inserted just below the larynx and following paralysis (pancuronium bromide), mechanical ventilation was initiated. A pneumothorax was created by a midline thoracotomy and an end-expiratory load was applied to maintain functional residual capacity. Animals were placed in a flow plethysmograph from which measurements of transpulmonary pressure, flow, and volume, mean inspiratory resistance, and dynamic compliance of the lung were calculated. The experimental protocol consisted of a series of 8-min trials, each preceded by a controlled volume history. The hypoxia challenge was composed of 1 min of ventilation with 40% O2, followed by 5 min exposure to 10% O2 and 2 min of recovery. In the majority of animals (7 out of 11), hypoxia had no effect on lung mechanics compared with control trials. Four animals responded to hypoxia with an increase in resistance and a decrease in compliance. Resistance remained elevated throughout the hypoxia with an average maximal increase of 47.2 +/- 22.2% (SD). Dynamic compliance was significantly decreased at the 2nd, 3rd, and 4th min only of hypoxia. Bilateral vagotomy abolished the response in the four animals and hypoxia had no effect on mechanics postvagotomy. Our data suggest that in most cases changes in lung mechanics do not play a causal role in the biphasic ventilatory response to hypoxia seen in the newborn.     

The ultrastructural picture of cerebral cortex of rat after hypoxia. I. Findings after inhalation of 5% O2 and 95% N2

The ultrastructure of some elements in the motor region of the cerebral cortex of the rat were studied after hypoxia. The experimental animals, after receiving intraperitoneal chloral hydrate anaesthesia, were placed in a chamber with a controlled supply of a mixture of 95% N2 and 5% O2. After 2- and 3-hour exposure to hypoxic conditions the animals were processed for electron optic study. Edematous mitochondria pith partial or total destruction of the mitochondrial matrix were observed. Some mitochondria were changed into large vacuolar formations. The granular endoplasmic reticulum of neurocytes was dilated and in the broad dilatation structures of lamellar shape were sporadically found. The Golgi complex contained vacuoles of different sizes and long cisterns. Hydrated astrocytes were visualized in the neuropil and perivascular astrocyte processes displayed edematous changes. In the group of animals exposed to hypoxia for 3 hours but processed only 24 hours after termination of hypoxia the same changes were observed yet their extent was considerably diminished. This finding indicates that changes induced by hypoxia tend to return to normal conditions.     

Dynamics of hypoxia, proliferation and apoptosis after irradiation in a murine tumor model

Proliferation and hypoxia affect the efficacy of radiotherapy, but radiation by itself also affects the tumor microenvironment. The purpose of this study was to analyze temporal and spatial changes in hypoxia, proliferation and apoptosis after irradiation (20 Gy) in cells of a murine adenocarcinoma tumor line (C38). The hypoxia marker pimonidazole was injected 1 h before irradiation to label cells that were hypoxic at the time of irradiation. The second hypoxia marker, CCI-103F, and the proliferation marker BrdUrd were given at 4, 8 and 28 h after irradiation. Apoptosis was detected by means of activated caspase 3 staining. After immunohistochemical staining, the tumor sections were scanned and analyzed with a semiautomatic image analysis system. The hypoxic fraction decreased from 22% in unirradiated tumors to 8% at both 8 h and 28 h after treatment (P < 0.01). Radiation did not significantly affect the fraction of perfused vessels, which was 95% in unirradiated tumors and 90% after treatment. At 8 h after irradiation, minimum values for the BrdUrd labeling index (LI) and maximum levels of apoptosis were detected. At 28 h after treatment, the BrdUrd labeling and density of apoptotic cells had returned to pretreatment levels. At this time, the cell density had decreased to 55% of the initial value and a proportion of the cells that were hypoxic at the time of irradiation (pimonidazole-stained) were proliferating (BrdUrd-labeled). These data indicate an increase in tumor oxygenation after irradiation. In addition, a decreased tumor cell density without a significant change in tumor blood perfusion (Hoechst labeling) was observed. Therefore, it is likely that in this tumor model the decrease in tumor cell hypoxia was caused by reduced oxygen consumption.     

Increased number of cardiac adrenergic receptors following local heart irradiation

The effect of local X irradiation on cardiac alpha and beta receptors was studied in Wistar rats. Animals were given local heart irradiation with single doses of 15 or 20 Gy and were examined after a range of latency times of 7 to 400 days. Using the radioactive ligands [3H]CGP-12177 and [3H]prazosin, the maximal binding capacity was determined from saturation experiments. At 7 days after 20 Gy the maximal binding capacity of both alpha and beta receptors was reduced to below the level of untreated control animals. Subsequently it rose continually to a maximum of 160% of the control level for beta receptors and 130% for alpha receptors at 400 days postirradiation. The antagonist affinity as judged from the dissociation constant for [3H]CGP 12177 and [3H]prazosin did not change significantly. A similar effect was observed after 15 Gy. An increase in adrenergic receptors may represent an important pathogenetic link between early morphological and late functional changes in the pathogenesis of radiation-induced heart disease.     

The role of alpha-adrenergic receptors in carbon monoxide hypoxia

The importance of alpha-adrenergic receptors in the cardiac output and peripheral circulatory responses to carbon monoxide (CO) hypoxia was studied in anesthetized dogs. Phenoxybenzamine (3 mg/kg i.v.) was injected to block alpha-receptor activity and the data obtained were then compared with those from a previous study of CO hypoxia in unblocked animals. Values for cardiac output, hindlimb blood flow, vascular resistance, and oxygen uptake were obtained prior to and at 30 and 60 min of CO hypoxia which reduced arterial oxygen content by approximately 50%. alpha-Adrenergic blockade resulted in a lower (p less than 0.05) control value for cardiac output than observed in unblocked animals, but no differences were present between the two groups at 30 or 60 min of CO hypoxia. Similarly, limb blood flow was lower (p less than 0.05) during the control period in the alpha-blocked group but rose to the same level as that in the unblocked animals at 60 min of COH. No change in limb blood flow occurred during CO hypoxia in the unblocked group. These findings demonstrated that during CO hypoxia alpha-receptor mediated venoconstriction does not contribute to the cardiac output response and alpha-receptor mediated vasoconstriction probably does prevent a rise in hindlimb skeletal muscle blood flow.