EFFECTS OF ALTERED OSMOLALITY ON RESPIRATION AND MORPHOLOGY OF MITOCHONDRIA FROM THE DEVELOPING BRAIN

Abstract— The effects of altered osmolality on respiration and fine structure were studied in isolated cerebral mitochondria from mature rats (60-100 days of age) and from rat pups in the first month of postnatal life (5, 10, 20 and 30 days). In the mature cerebral mitochondria, ADP-dependent respiration was inhibited in media of decreased osmolality. There was a transient inhibition of ADP-dependent respiration and a sustained increase in ADP-independent respiration in media of increased osmolality. In contrast, cerebral mitochondria from 5-day-old rats showed both inhibition of ADP-dependent respiration and increased ADP-independent respiration in hypo-osmolal media. In these mitochondria, inhibition of ADP-dependent respiration was stable and ADP-independent respiration was unchanged in media of increased osmolality. The transition to the mature respiratory response occurring with altered osmolality took place between 10 and 30 days of age. During this same age period, cerebral mitochondria showed an increasing resistance to matrix condensation in media of normal and increased osmolality.     

In vivo development of brain phosphocreatine in normal and creatine-treated rabbit pups

To study the effects of creatine (Cr) on brain energy metabolism and on hypoxia-induced seizures, 5- to 30-day-old rabbit pups were given subcutaneous Cr (3 g/kg) for 3 days before exposure to 4% O2 for 8 min. In saline-treated controls, hypoxic seizures were most frequent at 15 days (80% of pups) and 20 days (60%) of age. Seizures were prevented at 15 days and reduced 60% at 20 days in Cr-treated pups. In surface coil-localized brain 31P nuclear magnetic resonance spectra, with signal from both cerebral gray (GM) and white (WM) matter, the phosphocreatine (PCr)/nucleoside triphosphate (NTP) ratio doubled between 5 and 30 days of age in controls. In all Cr-injected pups, brain PCr/NTP increased to values seen in 30-day-old controls. When spectra were acquired in predominantly GM and WM slices in vivo, the PCr/NTP ratio was very low in GM at 5 days but reached adult levels by 15 days in controls. In WM, the ratio increased steadily from 5 to 30 days of age. In Cr-injected pups, PCr/NTP increased to mature levels in WM and in GM at all ages. In conclusion, hypoxic seizures occur midway in the time course of brain PCr/NTP increase in rabbit pups as previously described in rat pups. In both altricial pups, systemic Cr increases brain PCr/NTP ratio and prevents hypoxic seizures. These results suggest that mature levels of PCr and/or Cr in brain limit EEG activation either directly or indirectly by preventing hypoxic metabolic changes.     

In vitro effects of inorganic lead on isolated rat brain mitochondrial respiration

The effects of lead acetate on respiration in cerebral and cerebellar mitochondria from immature and adult rats were studied polarographically. With all substrates low lead concentrations produced an increase in respiration. Higher concentrations produced an inhibition of both this lead-induced respiration and ADP-dependent (State 3) respiration. Lead-induced respiration required inorganic phosphate and was inhibited by oligomycin, suggesting a coupling to oxidative phosphorylation. Inhibition of respiration was produced by much lower lead concentrations with NAD-linked citric acid cycle substrates than with succinate or -glycerophosphate. In partially disrupted mitochondria, NAD-linked substrate oxidation was inhibited at lead concentrations which did not affect NADH oxidation. Thus, in brain mitochondria the NAD-linked dehydrogenases, located in the matrix space, were more sensitive to inhibition by lead than were inner membrane enzymes. All in vitro lead effects on mitochondrial respiration were comparable in cerebral and cerebellar mitochondria isolated from both immature and adult rats.     

In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.     

The effect of low concentrations of sodium nitrites and nitrates on respiration and oxidative phosphorylation in rat liver mitochondria]

Nitrite incubation in mitochondria and nitrate intoxication of rats have been studied for their effect on aerobic energetic processes in the liver. Sodium nitrite in concentration of 2 mg/l causes an inhibition of ADP-stimulated respiration and provides uncoupling processes of oxidative phosphorylation and respiration in mitochondria, when adding succinate as a substrate. Low doses of nitrate in vivo promote oxygen economization in mitochondria. Intoxication of rats with nitrate in a dose of 50 mg/l for 30 days induces a decrease of the respiration rate after ADP-phosphorylation and an increase of the coefficient of oxidative phosphorylation efficiency (ADP/O). Intraperitoneal administration of adrenalin in a dose of 25 micrograms/100 g to rats after 30-day nitrate intoxication in a concentration of 10 mg/l induces no typical increase of ADP-stimulated respiration and rate of oxidative phosphorylation and succinate oxidation.     

纯氧或空气环境复苏对新生大鼠缺氧性大脑皮质神经元超微结构的影响

目的应用缺氧动物模型比较纯氧环境(pure oxygen environment,POE,100%氧)与空气环境(roomair environment,RAE,21%氧)复苏对新生大鼠缺氧性大脑皮质神经元超微结构的影响。方法7日龄20只SD(Sprague Dawley)乳鼠建立缺氧2.5 h模型后,分为纯氧环境组(POE)和空气环境组(RAE),每组再根据复氧后时间点分为2个亚组,即24 h组和72 h组,每亚组5只。按时间点取各组乳鼠大脑半球右侧额顶皮质行电镜样品制备,透射电镜观察。结果复氧各组均可见神经元、神经毡和细胞间隙水肿。RAE 24 h组神经元核膜结构不清,细胞器减少,线粒体肿胀、空泡化、嵴断裂;粗面内质网扩张,常呈空泡状,核糖体减少;高尔基复合体囊泡扩张;溶酶体较多。RAE 72 h组细胞器改变同前,但类似凋亡的细胞核较多,坏死细胞亦较其他组多见。POE 24 h组病变较RAE 24 h组轻。POE 72 h组细胞内线粒体及粗面内质网较丰富,病变亦比RAE 72 h组轻。结论POE复苏较RAE复苏可更能缓解缺氧致神经元超微结构的损伤、减少细胞凋亡及减轻脑水肿。提示,纯氧环境对缺氧复苏后大脑皮质神经元有一定的保护作用,并表明本动物模型适合缺氧新生大鼠大脑皮质神经元研究。     

Prenatal epoxiconazole exposure effects on rat postnatal development

Although some studies have pointed out to embryo/fetal toxicity, knowledge about the potential toxicity of the fungicide epoxiconazole is still limited. Once the results of these previous studies have raised some concern, this study studied the effects of epoxiconazole maternal exposure on the physical endpoints in the development of rat pups. To accomplish that, the effects of epoxiconazole (50.0, 100.0, and 150.0 mg/kg) were examined when rats were exposed at two different developmental stages: during the first 6 days of pregnancy or in the organogenesis period (6-15 days). After parturition, pups were tested for growth and maturational milestones. Maternal exposure to the fungicide, independently of phase, resulted in significantly early mean time to vaginal opening and delayed time to testes descent in pups. Weight gain rate in pups and their mothers was not affected for the tested exposure period. The findings of this study emphasize that epoxiconazole maternal exposure may lead to alterations in developmental patterns in nursing pups, consistent with the known influence of epoxiconazole on steroid hormone synthesis.     

Ascorbic Acid Ameliorates Gestational Lead Exposure-Induced Developmental Alteration in GAD67 and c-Kit Expression in the Rat Cerebellar Cortex

In the present study, we investigated the effects of ascorbic acid on lead-exposed developing cerebellum. Female rats were divided into the following three groups: control (distilled water), lead (0.2% lead acetate), and lead plus ascorbic acid (100 mg/kg/day, 10% solution). To evaluate the effect of lead exposure and ascorbic acid treatment accurately on the cerebellar development for the gestational period, we halted further treatment with lead and ascorbic acid in the dams after delivery of the pups. Although the ascorbic acid slightly decreased the lead level in pups, lead level was still high in the group treated with lead plus ascorbic acid group compared with the control group. The blood lead levels indicated that the ascorbic acid could facilitate both the excretion and transfer of lead from a dam to its pups via milk. At postnatal day 21, lead exposure significantly reduced the number of Purkinje cells in the cerebellar cortex of pups. Additionally, lead treatment induced degenerative changes such as reduction of glutamic acid decarboxylase (GAD67) and c-kit expressions are observed in the developing cerebellar cortex. In the cerebellum of the pups from the lead plus ascorbic acid group, reduction of the number of Purkinje cells, GAD67 expression, and c-kit immunopositivity were remarkably restored compared with the lead group. Our present results suggested that ascorbic acid treatment to lead-exposed dam exerted protective effects on the developing cerebellum against lead-induced neurotoxicity.     

Effect of thyroidectomy on phosphorylative oxidation and RNA synthesis in various regions of the brain in the adult monkey

The subcellular effects of thyroidectomy in selected brain regions of Cynomolgus monkey were analyzed. 20 days after operation the respiratory rates, the activities of succinate cytochrome c reductase, glycerol-3-phosphate dehydrogenase and of oligomycin-sensitive ATPase were decreased in mitochondria isolated from all brain structures. The highest reduction (30%) was found in cerebral cortex and hippocampus. Cerebellar and striatal activities were reduced by about 20%. A smaller decrease (15%) was observed in thalamus. The effects of thyroidectomy on in vitro RNA synthesis were followed in cerebral cortex, cerebellum and thalamus. In the three analyzed regions, the activities of nucleolar and nucleoplasmic RNA polymerases dropped by 40%. Replacement therapy with T4 (2.5 micrograms/kg/day) or T3 (1 microgram/kg/day) administered immediately after thyroidectomy for 20 days, maintained mitochondrial and nuclear activities at normal level.     

Lactate, 3-hydroxybutyrate,and glucose as substrates for the early postnatal rat brain

The dependence of cerebral energy metabolism upon glucose, 3-hydroxybutyrate, and lactate as fuel sources during the postnatal period was investigated. The brain of 6 day old suckling pups used very little glucose, but by the 15th postnatal day glucose was the major catabolite. Hydroxybutyrate was not a major brain fuel at either 6 or 15 days of age. Its utilization accounted for only 19% of the brain's total energy needs at 15 days of age, even though blood ketone concentrations are near maximal at this time. Seventy percent of the cerebral metabolic requirements were met by lactate in animals aged 6 days. The major role played by lactate as a substrate for brain metabolism in young pups was not a result of abnormally elevated blood lactate concentrations. The slow catabolism of glucose in young brain can not be explained by low rates of influx or inadequate enzymatic capacity.     

Evidence for decreased GDP binding to brown-adipose-tissue mitochondria of obese Zucker (fa/fa) rats in the very first days of life.

GDP binding to brown-adipose-tissue mitochondria of obese Zucker-rat (fa/fa) pups aged 2-14 days was significantly less than in lean control rats. Scatchard analysis in 10-day-old pups suggests that there was a large decrease in GDP-binding sites. However, a significant increase in fat content in brown adipose tissue of 2-day-old pre-obese pups raised the question of the sequential order and causal relationship between these two derangements.     

Reduction in Ultrasonic Vocalizations in Pups Born to Rapid Eye Movement Sleep Restricted Mothers in Rat Model

The effects of rapid eye movement sleep restriction (REMSR) in rats during late pregnancy were studied on the ultrasonic vocalizations (USVs) made by the pups. USVs are distress calls inaudible to human ears. Rapid eye movement (REM) sleep was restricted in one group of pregnant rats for 22 hours, starting from gestational day 14 to 20, using standard single platform method. The USVs of male pups were recorded after a brief isolation from their mother for two minutes on alternate post-natal days, from day one till weaning. The USVs were recorded using microphones and were analysed qualitatively and quantitatively using SASPro software. Control pups produced maximum vocalization on post-natal days 9 to 11. In comparison, the pups born to REMSR mothers showed not only a reduction in vocalization but also a delay in peak call making days. The experimental group showed variations in the types and characteristics of call types, and alteration in temporal profile. The blunting of distress call making response in these pups indicates that maternal sleep plays a role in regulating the neural development involved in vocalizations and possibly in shaping the emotional behaviour in neonates. It is suggested that the reduced ultrasonic vocalizations can be utilized as a reliable early marker for affective state in rat pups. Such impaired vocalization responses could provide an important lead in understanding mother-child bonding for an optimal cognitive development during post-partum life. This is the first report showing a potential link between maternal REM sleep deprivation and the vocalization in neonates and infants.     

Effects of continuous gastric infusion of food dyes on developing rat pups

The consequences of chronic percutaneous gastric infusion of food dyes on the activity levels and shock avoidance performance of normal developing rats and those treated with 6-hydroxydo p-amine (6-OHDA) were investigated. All pups were artificially reared from four days of age and a liquid diet containing either food dyes (D) or no colorings (N) was infused into both 6-OHDA (T) and sham-treated pups (V). Pups receiving food dyes were significantly more active than comparable pups not receiving food dyes at all ages tested, an effect observed in both 6-OHDA and shamtreated pups. Pups treated with food dyes exhibited an impaired avoidance performance in a shuttle box at 28 days of age. VD and TN pups displayed an over 100% impairment in avoidance performance in comparison with VN pups. These results suggest that food dyes and/or their metabolites may exert deleterious effects on the behavioral repertoire of the developing rat pup.     

Food restriction during pregnancy alters brain’s antioxidant network in dams and their offspring

Dietary restriction increases life span and protects distinct organisms against a series of diseases, among which, those related to oxidative stress, like neurodegenerative diseases. Interferences in the maternal environment are known to reprogram the offspring metabolism response, impacting in the risk of chronic diseases development in adulthood. We aimed to assess the effects of 40% food restriction on reactive species levels, enzymatic and non-enzymatic antioxidant defenses, and oxidative damage parameters in the cerebellum and total cerebral cortex of pregnant rats and their offspring. Dams and pups showed oxidative modulation caused by food restriction in both structures. Dichlorofluorescein oxidation, reflecting reactive species levels, was reduced in the cerebellum of dams and offspring, while the cerebral cortex was not affected. Decreased mitochondrial superoxide levels were found in the cerebellum and cerebral cortex of pups, while nitric oxide was increased in the cortex. We also measured the activities of important antioxidant enzymes responsible by reactive oxygen species elimination. Superoxide dismutase activity was increased in the cerebellum of dams and in both structures of pups, while it was decreased in dams’ cerebral cortex. Both brain structures were affected concerning to catalase, glutathione peroxidase, and glutaredoxin activities, which were reduced in pups and dams. Non-enzymatic defenses were decreased in pups, while dams showed an adaptive pattern in the cerebellum and no alteration in the cerebral cortex. Even though the results suggest increased oxidative status, lipids and proteins were not oxidatively affected. Our data suggest that intrauterine food restriction may disrupt oxidative status, impairing the antioxidant network.     

Germination,Viability and Clearance of Stachybotrys chartarum in the Lungs of Infant Rats

Observing that the conidia of Stachybotrys chartarum can germinate in the lung of infant rats, it became important to ascertain whether an infection can ensue. Viable conidia of S. chartarum were instilled into the lungs of 4 and 14 day-old rat pups. Germination was observed frequently in the lungs of 4 day-old but rarely in the 14 day-old pups. In the 4 day-old pups, pulmonary inflammation with hemorrhagic exudates was observed and resulted in about 15% mortality rate compared to 0% for the controls instilled with phosphate buffered saline. Acute neutrophilic inflammation and intense interstitial pneumonia with poorly formed granulomas observed three days following exposure were associated with fungal hyphae and conidia. The surviving experimental pups showed significantly slower weight gain for seven days. Dilution plating and quantitative PCR analysis were used to follow total fungal load in the rat pups lung homogenates. In the 4 day-old rat pups viable fungi decreased rapidly and were less than 1% by day seven. Similarly, fungal DNA decreased exponentially and was only 0.03% by fourteen days after exposure. However, 14 day-old rat pups showed neither the lethal effects of exposures to viable conidia of S. chartarum nor the slower weight gain, and the fungal load decreased even more rapidly. We conclude that S. chartarum conidia can initially germinate and form hyphae but even in the immature rat pups do not establish an effective infection, although a very limited persistence cannot be excluded.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effect of mammillary body lesions in adult rats on the survival and development of their young

The lesion of the mammillary bodies in the adult female did not alter the oestrous cycle, sexual behavior, parturition, or the number and weight of pups at birth. Neither did it alter maternal behavior (nesting behavior, cleaning of the pups, placentophagia, nursing position, retrieving, etc.). Nevertheless, more than 30% of the pups born from the mothers with total lesions of the mammillary bodies died during the first 10 days after birth. The weight of the surviving pups was greatly impaired and the pups showed severe undernourishment. The effects of the lesion of the mammillary bodies on the survival and weight of the pups were dependent on the mass of tissue destroyed in that structure.     

Lead-induced alterations of apoptosis and neurotrophic factor mRNA in the developing rat cortex, hippocampus, and cerebellum

Previous reports have recently shown the prototypic neurotoxicant, lead, to induce apoptosis in the brains of developing organisms. In the current study, timed-pregnant rats were exposed to lead acetate (0.2% in the drinking water) 24 h following birth at postnatal day 1 (PND 1). Dams and pups were continuously exposed to lead through the drinking water of the dam until PND 20. Postnatal exposure in the pups resulted in altered mRNA levels of the following apoptotic and neurotrophic factors: caspase 2 and 3, bax, bcl-x, brain-derived neurotrophic factor (BDNF). Ribonuclease protection assays were conducted to measure the factors simultaneously at the following postnatal time points: 9, 12, 15, 20, 25, days. Our results suggest a brain region- and time-specific response following lead acetate exposure. The region most vulnerable to alterations occurs in the hippocampus with alterations beginning at PND 12, in which caspase 3, bcl-x, BDNF increase with lead exposure. Significant treatment effects were not observed for both the cortex and cerebellum.     

纯氧与空气环境复苏对低氧新生大鼠大脑皮质神经元凋亡的影响

目的应用无创性低氧动物模型,比较纯氧环境(pure oxygen environment,POE,100%氧)与空气环境(room air environment,RAE,21%氧)复苏对低氧新生大鼠大脑皮质神经元凋亡的影响。方法 30只SpragueDawley(SD)7日龄乳鼠在建立低氧2.5 h模型后,分别于纯氧环境与空气环境中进行复苏。实验分为正常对照组(无预缺氧和复氧实验,n=10)、纯氧环境组(POE,n=10)和空气环境组(RAE,n=10),依复苏后时间点每组又分为24 h组和72 h组,每亚组5只。按时间点取各组乳鼠大脑皮质行尼氏体染色、原位末端标记(TUNEL)技术、图像分析和透射电镜观察神经元凋亡。结果与正常对照组相比,低氧乳鼠复氧后各组大脑皮质内锥体层均可见散在的、单个的、成群的或2～4个排列成串的凋亡锥体神经元,细胞凋亡现象RAE 72 h组最明显,POE 72 h组凋亡细胞个数和面密度都较RAE 72 h组少(P0.01)。电镜下RAE 72 h组类似凋亡细胞核也较其他各组多见。结论在本动物模型POE复苏较RAE复苏早期可更能减少细胞凋亡,缓解低氧致神经元的损伤,表明纯氧早期对新生儿低氧性脑损伤有一定的保护作用。     

Calcium disodium edetate enhances type A monoamine oxidase activity in monkey brain

The effects of metal chelators on monoamine oxidase (MAO) isozymes, MAO-A and MAO-B, in monkey brain mitochondria were investigated in vitro. MAO-A activity increased to about 40% with 0.1 μM calcium disodium edetate (CaNa₂EDTA) using serotonin as a substrate, and this activation was proportional to the concentration of CaNa₂EDTA. On the other hand, MAO-A activities were decreased gradually with an increasing concentration of o-phenanthroline and diethyldithiocarbamic acid, but these metal chelators had no effect on MAO-B activity in monkey brain. The activation of MAO-A activity by CaNa₂EDTA was reversible. CaNa₂EDTA did not activate both MAO-A and MAO-B activities in rat brain mitochondria. Zn and Fe ions were found in the mitochondria of monkey brain. Zn ions potently inhibited MAO-A activity, but Fe ions did not inhibit either MAO-A or MAO-B activity in monkey brain mitochondria. These results indicate that the activating action of CaNa₂EDTA on MAO-A was the result of the chelating of Zn ions contained in mitochondria by CaNa₂EDTA. These results also indicate the possibility that Zn ions may regulate physiologically the level of serotonin and norepinephrine content in brain by inhibiting a MAO-A activity.     

Accumulation of lead in root cells of Pisum sativum

The ever-increasing environmental pollution necessitates organisms to develop specific defense systems in order to survive and function effectively. Lead is taken up by plants mainly through roots and over 96% are accumulated there.Pea plants were cultivated hydroponically for 4 days with 0.1, 0.5 and 1 mM Pb(NO₃)₂. Uptake of lead ions from nutrient solution and accumulation in root stems and leaves during 96-h cultivation was estimated. The root tip cells were observed with transmission electron microscope to analyse their ultrastructure and lead localization. Pb was accumulated in the cell wall, cell membrane, vacuoles, mitochondria and peroxisomes. The fractions of mitochondria and peroxisomes were isolated from pea roots purified by means Percoll gradient, and were observed by means of electron microscope with the attachment for X-ray microanalysis. Visible deposits containing Pb were observed in both cell organelles.