Anna Nowakowska

Perinatal asphyxia, hyperthermia and hyperferremia as factors inducing behavioural disturbances in adulthood : A rat model

Alertness was studied in adult male Wistar rats after neonatal critical anoxia applied under three different thermal conditions: (i) at physiological neonatal body temperature of 33 degrees C, (ii) at body temperature elevated to 37 degrees C, and (iii) at body temperature elevated to 39 degrees C (both during anoxia and for 2 h postanoxia). To elucidate the effect of iron-dependent postanoxic oxidative damage to the brain, half of the group (iii) was injected with deferoxamine, a chelator of iron. Postanoxic behavioural disturbances were recorded in open-field, elevated plus-maze, and sudden silence tests when the rats reached the age of 4 month. Moreover, spontaneous motor activity of the rats was recorded radiotelemetrically in their home-cages. Both open-field stress-induced and spontaneous motor activity were reduced in rats subjected to neonatal anoxia under hyperthermic conditions. In contrast, these rats were hyperactive in the plus-maze test. Both the plus-maze and sudden silence tests revealed that these rats show reduced alertness to external stimuli signalling potential dangers. The behavioural disturbances were prevented by the body temperature of 33 degrees C and by postanoxic administration of deferoxamine. These data support the conclusion that permanent postanoxic behavioural disturbances are due to iron-dependent oxidative damage to the brain, which can be prevented by the reduced neonatal body temperature.

Effect of temperature on postanoxic, potentially neurotoxic changes of plasma pH and free iron level in newborn rats.

In asphyxiated newborns, iron, released from heme and ferritin and deposited in the brain, contributes to neurodegeneration. Because hypothermia provides neuroprotection, newborn mammals, showing reduced body temperature, might avoid iron-mediated neurotoxicity. However, hypothermia leads to acidosis, which induces hyperferremia. Therefore, we decided to study the effects of body temperature on plasma pH and iron levels in newborn rats exposed to a critical anoxia. Rectal temperature was kept at 33 degrees C (typical of neonates), reduced by 2 degrees C, or elevated to a level typical of healthy (37 degrees C) or febrile (39 degrees C) adults. Arterial blood samples were collected at 0, 10, 20, 30, and 120 min postanoxia. Control samples were obtained from normoxic, temperature-matched neonates. Anoxia tolerance time decreased progressively at rectal temperatures exceeding 33 degrees C. Neither pH nor plasma iron were significantly affected by anoxia at 33 degrees C. Although hypothermia (31 degrees C) resulted in acidosis in normoxic rats, both pH and iron levels were hardly influenced by anoxia. However, acidosis and hyperferremia, proportional to body temperature, developed at 37 and 39 degrees C. In conclusion, reduced body temperature is likely to protect asphyxiated newborns against iron-mediated brain injury.

Stress-induced behaviour in juvenile rats: effects of neonatal asphyxia, body temperature and chelation of iron.

Newborn mammals, showing reduced normal body temperature, might be protected against iron-mediated, delayed neurotoxicity of perinatal asphyxia. Therefore, we investigated the effects of (1) neonatal body temperature and neonatal critical anoxia as well as (2) postanoxic chelation of iron with deferoxamine, on open-field stress-induced behaviour in juvenile rats. The third aim of this study was to compare (after the above-mentioned treatments) circadian changes in spontaneous motor activity and body temperature in juvenile rats permanently protected from any stress. Neonatal anoxia at body temperature adjusted (both during anoxia and 2 h reoxygenation) to a level typical of healthy (37 degrees C) or febrile (39 degrees C) adults led to the stress-induced hyperactivity in juvenile (5-45 days old) rats. Both normal neonatal body temperature of 33 degrees C and chelation of iron prevented the hyperactivity in rats. Neither neonatal body temperature nor neonatal anoxia affected spontaneous motor activity or body temperature of juvenile rats, recorded in their home-cages with implantable transmitters. Circadian rhythmicity was also undisturbed. Presented data support the hypothesis that physiologically reduced neonatal body temperature can provide a protection against iron-mediated postanoxic disturbances of behavioural stress responses in juvenile rats.

Effect of winter torpor upon antioxidative defence in Helix pomatia

Arousal of land snails from torpor is inseparably connected with an increase in oxygen consumption leading to oxidative stress. Therefore, activity of antioxidant defence system (antioxidant enzymes and reduced glutathione) and degree of oxidative damage (concentration of malondialdehyde as an index of lipid peroxidation) in the snail Helix pomatia L., 1758 were tested to check whether torpid snails are able to activate their antioxidative defence against oxidative damage prior to arousal from winter torpor. Snails, which were collected from their natural habitats, were tested at the beginning, in the middle part, and at the end of winter torpor. Active snails collected in autumn and spring were taken as control groups. Snails were immediately killed and their foot, hepatopancreas, and kidney were used for the biochemical assays. Winter torpor induced significant changes in activities of the crucial antioxidant substances. The lowest activities were observed at the beginning of torpor, whereas activity of...

Defence against oxidative stress in two species of land snails (Helix pomatia and Helix aspersa) subjected to estivation.

During summer, land snails are exposed to estivation/arousal cycles that imposes oxidative stress, but they exhibit different patterns of antioxidant defence. To test the ability of two related species, Helix pomatia and Helix aspersa, to modulate their antioxidant defence mechanism during estivation/arousal cycles, we examined activities of catalase and glutathione-related enzymes and concentrations of glutathione and thiobarbituric acid reactive substances (TBARS; as products of lipid peroxidation). In both species, estivation evoked changes in activity of total and selenium-dependent glutathione peroxidase (GPx), but did not affect activity of catalase, glutathione reductase, and glutathione transferase, and had no effect on concentration of glutathione. Activity of catalase in estivating snails, instead of the expected increase, showed a tendency to diminish. Extremely low activities of catalase in the foot were usually associated with extremely high activities of both forms of GPx. In conclusion, maintenance of relatively high activities of the antioxidant enzymes and accumulation of glutathione, resulting in a low and stable concentration of TBARS, plays an important role in scavenging oxygen free radicals from the organism of both species.

Deferoxamine prevents cerebral glutathione and vitamin E depletions in asphyxiated neonatal rats: role of body temperature

Abstract Hypoxic-ischaemic brain injury involves increased oxidative stress. In asphyxiated newborns iron deposited in the brain catalyses formation of reactive oxygen species. Glutathione (GSH) and vitamin E are key factors protecting cells against such agents. Our previous investigation has demonstrated that newborn rats, showing physiological low body temperature as well as their hyperthermic counterparts injected with deferoxamine (DF) are protected against iron-mediated, delayed neurotoxicity of perinatal asphyxia. Therefore, we decided to study the effects of body temperature and DF on the antioxidant status of the brain in rats exposed neonatally to critical anoxia. Two-day-old newborn rats were exposed to anoxia in 100% nitrogen atmosphere for 10 min. Rectal temperature was kept at 33 °C (physiological to rat neonates), or elevated to the level typical of healthy adult rats (37 °C), or of febrile adult rats (39 °C). Half of the rats exposed to anoxia under extremely hyperthermic conditions (39 °C) were injected with DF. Cerebral concentrations of malondialdehyde (MDA, lipid peroxidation marker) and the levels of GSH and vitamin E were determined post-mortem, (1) immediately after anoxia, (2) 3 days, (3) 7 days, and (4) 2 weeks after anoxia. There were no post-anoxic changes in MDA, GSH and vitamin E concentrations in newborn rats kept at body temperature of 33 °C. In contrast, perinatal anoxia at elevated body temperatures intensified oxidative stress and depleted the antioxidant pool in a temperature-dependent manner. Both the depletion of antioxidants and lipid peroxidation were prevented by post-anoxic DF injection. The data support the idea that hyperthermia may extend perinatal anoxia-induced brain lesions.

Glutathione deficiency attenuates endotoxic fever in rats

Abstract Purpose: Glutathione constitutes the first line of the cellular defence mechanism against oxidative stress, and according to published data it is required by a number of factors that are involved in fever mechanism. The aim of the present study was to investigate whether or not glutathione deficiency can modulate a course of the fever induced by endotoxin (LPS). Material and methods: Intraperitoneal injection of LPS from Escherichia coli was used to provoke fever in Wistar rats. The level of liver glutathione was decreased by administration of phorone (Pho). Deep body temperature (Tb) in free running rats was recorded using a biotelemetry system. The concentration of TNF-α was estimated. Next, the supplementation of TNF-α was done using recombinant rat TNF-α. Results: Animals with decreased glutathione level responded with diminished fever after LPS injection (average Tb in Pho/LPS-treated and oil/LPS-treated animals were 36.90 ° ± 0.10 °C and 37.80 ° ± 0.15 °C, respectively). This response was accompanied by a significant attenuation of LPS-induced increase in TNF-α concentration (in the Pho/LPS-treated group it was 10.68 pg/mL ± 2.24, vs. 113.35 pg/mL ± 13.93 in oil/LPS-treated rats). Supplementation with TNF-α partially restored fever. Conclusion: Based on these data, we conclude that glutathione deficiency modifies the LPS-induced fever, in a TNF-α related manner.

The influence of abscisic acid on the ethylene biosynthesis pathway in the functioning of the flower abscission zone in Lupinus luteus.

Flower abscission is a highly regulated developmental process activated in response to exogenous (e.g. changing environmental conditions) and endogenous stimuli (e.g. phytohormones). Ethylene (ET) and abscisic acid (ABA) are very effective stimulators of flower abortion in Lupinus luteus, which is a widely cultivated species in Poland, Australia and Mediterranean countries. In this paper, we show that artificial activation of abscission by flower removal caused an accumulation of ABA in the abscission zone (AZ). Moreover, the blocking of that phytohormones biosynthesis by NDGA (nordihydroguaiaretic acid) decreased the number of abscised flowers. However, the application of NBD - an inhibitor of ET action - reversed the stimulatory effect of ABA on flower abscission, indicating that ABA itself is not sufficient to turn on the organ separation. Our analysis revealed that exogenous ABA significantly accelerated the transcriptional activity of the ET biosynthesis genes ACC synthase (LlACS) and oxidase (LlACO), and moreover, strongly increased the level of 1-aminocyclopropane-1-carboxylic acid (ACC) - ET precursor, which was specifically localized within AZ cells. We cannot exclude the possibility that ABA mediates flower abscission processes by enhancing the ET biosynthesis rate. The findings of our study will contribute to the overall basic knowledge on the phytohormone-regulated generative organs abscission in L. luteus.

Deferoxamine improves antioxidative protection in the brain of neonatal rats: The role of anoxia and body temperature

After hypoxic-ischemic insult iron deposited in the brain catalyzes formation of reactive oxygen species. Newborn rats, showing reduced physiological body temperature and their hyperthermic counterparts injected with deferoxamine (DF), a chelator of iron, are protected both against iron-mediated neurotoxicity and against depletion of low-molecular antioxidants after perinatal asphyxia. Therefore, we decided to study the effects of DF on activity of antioxidant enzymes (superoxide dismutase-SOD, glutathione peroxidase-GPx and catalase-CAT) in the brain of rats exposed neonatally to a critical anoxia at body temperatures elevated to 39°C. Perinatal anoxia under hyperthermic conditions intensified oxidative stress and depleted the pool of antioxidant enzymes. Both the depletion of antioxidants and lipid peroxidation were prevented by post-anoxic DF injection. The present paper evidenced that deferoxamine may act by recovering of SOD, GPx and CAT activity to reduce anoxia-induced oxidative stress.

Thermal and motor behavior in experimental autoimmune encephalitis in Lewis rats

Abstract Thermoregulation in patients, who suffer from multiple sclerosis (MS) is impaired and may result in either increases or decreases in body temperature. Disturbances in body temperature correlate with acute relapses, and for this reason, it is an important issue in everyday life of those who suffer from MS. Although rat experimental autoimmune encephalitis (EAE) appeared useful for the examination of current therapies against MS, it has not been thoroughly investigated in terms of body temperature. The purpose of this study was to examine the effect of EAE induction on thermal and motor behavior in the rats. Subcutaneous injection of encephalitogenic emulsion into both pads of hind feet of the Lewis rats provoked symptoms of EAE. Body temperature (Tb) and motor activity of rats were measured using biotelemetry system. We report a significant increase in body temperature within 24 h prior to the EAE manifestation (12 h average of Tb for EAE induced animals was higher by 1.07 ± 0.06 °C during day-time and by 0.5 ± 0.05 °C during night time in comparison to the control rats). On the other hand, the onset of EAE symptoms was associated with gradual decrease of body temperature, and during the first night-time Tb was lower by 1.03 ± 0.08 °C in comparison to the control rats. The inhibition of the motor activity started from the night time, 2 days before EAE onset. On the basis of our data, we concluded that the pattern of body temperature changes after EAE induction may be considered as useful symptom (prodrom) to predict precisely the time of EAE onset. Furthermore, we suggest that EAE in rats may be a suitable model to study mechanism of body temperature alternations observed in MS patients.