Study of the hereditary differences in the cyclic nucleotide content of the blood serum in mice after exposure to stress and administration of phenazepam

Blood plasma content of cAMP and cGMP in C57BL/6, BALB/c mice and their reciprocal F1-hybrids has been studied at rest, upon exposure to stress induced in an open field technique and phenazepam injection at a dose of 0.05 and 0.1 mg/kg. Interstrain differences in baseline content and changes of nucleotide concentration in conditions of stress have been revealed. F1-hybrids inherit initial correlation between nucleotide content and the type of cAMP changes of C57BL/6 mice and cGMP changes of BALB/c mice. Phenazepam injection to C57BL/6 and BALB/c mice was shown to produce specific shifts in blood plasma cyclic nucleotide content.     

Oxidative changes in brain pyridine nucleotides and neuroprotection using nicotinamide

Pyridine nucleotides are critical during oxidative stress due to their roles in reductive reactions and energetics. The aim of the present study was to examine pyridine nucleotide changes in six brain regions of mice after an intracerebroventricular injection of the oxidative stress inducing agent, t-butyl hydroperoxide (t-BuOOH). A secondary aim was to investigate the correlation between NAD+ levels and DNA fragmentation. Here, we demonstrate that t-BuOOH induced a rapid oxidation of NADPH and a slow depletion of NAD+ in most brain regions. A slight increase in NADH also occurred in five brain regions. NAD+ depletion was associated with increased DNA fragmentation. This suggests the initiation of a death cascade involving poly(ADP-ribose) polymerase (PARP), NAD+, ATP depletion and consequent cell death in brain tissue. PARP activity was accelerated in some brain regions after 20 min of oxidative stress. To counteract oxidative stress induced toxicity, NAD+ levels were increased in the brain using an intraperitoneal injection of nicotinamide. A surplus of brain NAD+ prevented DNA fragmentation in some brain regions. Nicotinamide administration also resulted in higher brain NADH, NADP+ and NADPH levels in some regions. Their synthesis was further upregulated during oxidative stress. Nicotinamide as a precursor for NAD+ may provide a useful therapeutic strategy in the treatment of neurodegeneration.     

Chronic Stress- and Sex-Specific Neuromorphological and Functional Changes in Limbic Structures

Chronic stress produces sex-specific neuromorphological changes in a variety of brain regions, which likely contribute to the gender differences observed in stress-related illnesses and cognitive ability. Here, we review the literature investigating the relationship between chronic stress and sex differences on brain plasticity and function, with an emphasis on morphological changes in dendritic arborization and spines in the hippocampus, prefrontal cortex, and amygdala. These brain structures are highly interconnected and sensitive to stress and gonadal hormones, and influence a variety of cognitive abilities. Although much less work has been published using female subjects than with male subjects, the findings suggest that the relationship between brain morphology and function is very different between the sexes. After reviewing the literature, we present a model showing how chronic stress influences the morphology of these brain regions and changes the dynamic of how these limbic structures interact with each other to produce altered behavioral outcomes in spatial ability, behavioral flexibility/executive function, and emotional arousal.     

Mitochondrial activity in different regions of the brain at the onset of streptozotocin-induced diabetes in rats

Diabetes affects a variety of tissues including the central nervous system; moreover, some evidence indicates that memory and learning processes are disrupted. Also, oxidative stress triggers alterations in different tissues including the brain. Recent studies indicate mitochondria dysfunction is a pivotal factor for neuron damage. Therefore, we studied mitochondrial activity in three brain regions at early type I—diabetes induction. Isolated mitochondria from normal hippocampus, cortex and cerebellum revealed different rates of oxygen consumption, but similar respiratory controls. Oxygen consumption in basal state 4 significantly increased in the mitochondria from all three brain regions from diabetic rats. No relevant differences were observed in the activity of respiratory complexes, but hippocampal mitochondrial membrane potential was reduced. However, ATP content, mitochondrial cytochrome c, and protein levels of β-tubulin III, synaptophysin, and glutamine synthase were similar in brain regions from normal and diabetic rats. In addition, no differences in total glutathione levels were observed between normal and diabetic rat brain regions. Our results indicated that different regions of the brain have specific metabolic responses. The changes in mitochondrial activity we observed at early diabetes induction did not appear to cause metabolic alterations, but they might appear at later stages. Longer-term streptozotocin treatment studies must be done to elucidate the impact of hyperglycemia in brain metabolism and the function of specific brain regions.     

Free radical induced increase in protein carbonyl is attenuated by low dose of adenosine in hippocampus and mid brain: implication in neurodegenerative disorders

There is strong evidence that oxidative stress participates in the etiology of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In the previous studies we have already shown that a combination of alpha-tocopherol and ascorbic acid protect neurons against tert-butyl hydroperoxide (t-BuOOH) induced neurotoxicity in different brain regions including hippocampus and mid brain. In this work, we examined the neuroprotective effect of low dose of adenosine against protein oxidation (protein carbonyls) in parallel with the level of reduced glutathione (GSH) in hippocampus and mid brain regions of mouse brain. The t-BuOOH was injected intraperitoneally in three concentrations (50, 100, 150 mg/kg b.w.) for 10 days. Results showed dose dependent increase in protein carbonyl (PC) in hippocampus and mid brain region. This increase was accompanied by a significant (p < 0.05) decline in GSH content in both brain regions of t-BuOOH treated mice. Adenosine (1 mg/kg b.w.) protected both hippocampus and mid brain neurons against protein oxidation as evidenced by reduction in protein carbonyl content. The GSH content was significantly (p < 0.05) increased after the treatment of adenosine in both brain regions. These data show that prior treatment with low dose of adenosine attenuates the oxidative protein damage with parallel increase in the GSH level in hippocampus and mid brain of t-BuOOH induced mice.     

Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency.     

Evidence for strong selective constraint acting on the nucleotide composition of 16S ribosomal RNA genes

Previous studies have shown that the guanine plus cytosine (G+C) content of ribosomal RNAs (rRNAs) is highly correlated with bacterial growth temperatures. This correlation is strongest in the double-stranded stem regions of the rRNA, a fact that can be explained by selection for increased structural stability at high growth temperatures. In this study, we examined the single-stranded regions of 16S rRNAs. We reasoned that, since these regions of the molecule are subject to less structural constraint than the stem regions, their nucleotide content might simply reflect the overall nucleotide content of the genome. Contrary to this expectation, however, we found that all of the single-stranded regions are characterized by very high adenine (A) and relatively low cytosine (C) contents. Moreover, the nucleotide content of these single-stranded regions is surprisingly constant between species, despite dramatic differences in optimal growth temperatures, and despite large differences in the overall genomic G+C content. This provides compelling evidence for strong stabilizing selection acting on 16S rRNA single-stranded regions. We found that selection favors purines (A+G), and especially adenine (A), in the single-stranded regions of these rRNAs.     

Genetic variability for regional brain gangliosides in five strains of young mice

The quantitative and qualitative distributions of gangliosides were determined in the cerebrum, cerebellum, and brain stem of five inbred strains (C57BL/6J, DBA/2J, LG/J, C3H/HeJ, BALB/cJ) of mice at 21 days of age. Genetic differences were found among the strains for wet weight, absolute amount of gangliosides per region, and concentration of ganglioside (expressed on both a wet and a dry weight basis) in all three regions of the brain. The water content of the various brain regions showed the least amount of genetic variability. Coefficients of genetic determination were used to estimate the magnitude of genetic influence on these traits in each brain region. Significant differences were also found among the five strains for the distribution of certain gangliosides. The DBA strain, which is susceptible to audiogenic seizure at this age, had the highest level of the myelin-enriched ganglioside G_M1 in all brain regions. Most of the genetic variation that influences the content and distribution of gangliosides among neurologically normal mice can be considered polygenic. Several possible sources of this genetic variation that may contribute to the differences observed among the strains are discussed.This work was supported by USPHS Grant NS 11853 and by a grant from the Swebilius Fund. T. N. S. is the recipient of a USPHS postdoctoral fellowship (1F32NS0443).     

Early nutritional stress impairs development of a song-control brain region in both male and female juvenile song sparrows (Melospiza melodia) at the onset of song learning

Birdsong is a sexually selected trait and is often viewed as an indicator of male quality. The developmental stress hypothesis proposes a model by which song could be an indicator; the time during early development, when birds learn complex songs and/or local variants of song, is of rapid development and nutritional stress. Birds that cope best with this stress may better learn to produce the most effective songs. The developmental stress hypothesis predicts that early food restriction should impair development of song-control brain regions at the onset of song learning. We examined the effect of food restriction on song-control brain regions in fledgling (both sexes, 23-26 days old) song sparrows (Melospiza melodia). Food restriction selectively reduced HVC volume in both sexes. In addition, sex differences were evident in all three song-control regions. This study lends further support to a growing body of literature documenting a variety of behavioural, physiological and neural detriments in several songbird species resulting from early developmental stress.     

DNA damage, repair, and antioxidant systems in brain regions: a correlative study

8-Hydroxy-2'-deoxyguanosine (oxo(8)dG) has been used as a marker of free radical damage to DNA and has been shown to accumulate during aging. Oxidative stress affects some brain regions more than others as demonstrated by regional differences in steady state oxo(8)dG levels in mouse brain. In our study, we have shown that regions such as the midbrain, caudate putamen, and hippocampus show high levels of oxo(8)dG in total DNA, although regions such as the cerebellum, cortex, and pons and medulla have lower levels. These regional differences in basal levels of DNA damage inversely correlate with the regional capacity to remove oxo(8)dG from DNA. Additionally, the activities of antioxidant enzymes (Cu/Zn superoxide dismutase, mitochondrial superoxide dismutase, and glutathione peroxidase) and the levels of the endogenous antioxidant glutathione are not predictors of the degree of free radical induced damage to DNA in different brain regions. Although each brain region has significant differences in antioxidant defenses, the capacity to excise the oxidized base from DNA seems to be the major determinant of the steady state levels of oxo(8)dG in each brain region.     

Foot-shock Stress-Induced Regional Iron Accumulation and Altered Iron Homeostatic Mechanisms in Rat Brain

Like in other organs, iron in the brain plays an important role in various biological processes. Previous studies have shown that systemic iron homeostasis in mammalians was changed under specific stress conditions. The present study aimed to investigate effects of stress on brain iron homeostasis in rats using a foot-shock stress model. Young adult male Sprague-Dawley rats were randomly assigned to foot-shock stress group subjected to 30 min of cutaneous foot-shock (0.80 mA, 1 pulse/s, 300 ms duration) daily for 1 week or control group left undisturbed. Then, the rats were sacrificed and iron concentration in serum, liver, and some brain regions were measured using atomic absorption spectrophotometry. Expression of ferritin, Transferrin receptor (TfR), divalent metal transporter 1 (DMT1, with or without iron-responsive element), lactoferrin (Lf), and iron regulatory protein 1 (IRP1) in rat hippocampus were determined using western blot analysis. The results showed that stress induced decreased serum iron concentration, increased liver iron content, and elevated iron contents in specific brain regions including hippocampus, striatum, and frontal cortex. In the hippocampus, stress caused decreased expression of ferritin, increased expression of TfR and IRP1, and no change in expression of DMT1 or Lf. Results of this study demonstrated that foot-shock stress induced region specific iron accumulation and altered iron homeostatic mechanisms in the brain in addition to a changed systemic iron homeostasis characterized by decreased serum iron concentration and increased liver iron content. And, elevated IRP1 expression might be associated with the increased TfR and decreased ferritin expression, leading to subsequent iron accumulation and possible increased vulnerability to oxidative damage in hippocampus.     

Changes in the levels of neural cell specific proteins in the developing rat brain

The levels of S-100 protein (S-100) and neuron-specific enolase (NSE) in the developing rat brain were determined by a sensitive enzyme immunoassay and the results were compared with those obtained by other methods. Changes with development in the levels of S-100, NSE, and 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase), biochemical markers for astroglia, neurons and oligodendroglia respectively, were determined in various brain regions including the cerebral hemisphere (CH), brain stem (BS) and cerebellum (Ce). The peak increments of S-100, NSE, and CNPase activity were reached later than that of the brain weight in all of the regions. The ratios of S-100/NSE and CNPase/NSE rose during the 21 days after birth in the CH and BS; the S-100/NSE ratio in the CH began to decrease from the 21st day, whereas the CNPase/NSE ratio continued to rise even after the 30th day, suggesting different maturation periods of the different glial cells. In the Ce, the change of these ratios showed a pattern different from those in the other regions. In the CH of rats with experimental microencephaly induced by methylazoxymethanol (MAM), the ratios were almost normal, in spite of the reduction of the brain weight to about 50% of the control.Dedicated to Professor Yasuzo Tsukada.     

Increased level of immunoreactive opioid peptides in the brain and adrenals of rats as affected by adaptation to physical loading

The effect of long-term (7 weeks) swimming training on the content of beta-endorphin, met- and leu-enkephalins in various brain regions and adrenal glands has been studied in Wistar rats. It has been shown that the adaptation to exercise induced an increase in the content of opioid peptides in most of the brain regions and in adrenal glands. This increase in the level of opioid peptides seems to play an important role in the increase of the resistance to stress.     

Nitric oxide in the pathophysiology of hyperthermic brain injury. Influence of a new anti-oxidant compound H-290/51

Summary The possibility that nitric oxide (NO) is involved in the pathophysiology of brain injury caused by heat stress (HS) was examined using neuronal nitric oxide synthase (NOS) immunohistochemistry in a rat model. In addition, to find out a role of oxidative stress in NOS upregulation and cell injury, the effect of a new antioxidant compound H-290/51 (Astra Hässle, Mälndal, Sweden) was examined in this model. Subjection of conscious young rats to 4 h HS in a biological oxygen demand (BOD) incubator at 38°C resulted in a marked upregulation of NOS in many brain regions compared to control rats kept at room temperature (21 ± 1°C. This NOS immunoreactivity was found mainly in distorted neurons located in the edematous regions not normally showing NOS activity. Breakdown of the blood-brain barrier (BBB) permeability, increase in brain water content and marked neuronal, glial and myelin reaction were common findings in several brain regions exhibiting upregulation of NOS activity. Pretreatment with H-290/51 significantly attenuated the upregulation of NOS in rats subjected to HS. In these animals breakdown of the BBB permeability, edema and cell changes were considerably reduced. Our results suggest that hyperthermic brain injury is associated with a marked upregulation of NOS activity in the CNS and this upregulation of NOS and concomitant cell injury can be reduced by prior treatment with an antioxidant compound H 290/51. These observations indicate that oxidative stress seems to be an important endogenous signals for NOS upregulation and cell reaction in hyperthermic brain injury.     

Delayed Increase of Brain Noradrenaline After Acute Footshock Stress in Rats

Several lines of evidence strongly suggest that accumulation of noradrenaline (NA) in the brain may underlie the hyperarousal symptoms experienced in post-traumatic stress disorder. In animal experiments, however, the effect of stress on NA content appears complex; acute stress reduces the level, while chronic stress tends to increase it. To explain this discrepancy, it is necessary to observe the long-term effects of acute stress on NA metabolism in the brain. In this study, rats were exposed to intermittent intense footshock stress for 1 h, and the brain NA content was measured for 7 days after the stress stimulus. Hypothalamic NA content was immediately reduced and recovered within 24 h. However, a significant NA increase was observed 7 days after the footshock. In the cerebral cortex and hippocampus, an increase in NA content was observed 1 day after the stress and lasted for at least 7 days. The fact that the content of 3-methoxy-4-hydroxyphenylglycol, a major NA metabolite, only transiently increased in all these regions possibly reflects NA release. These results indicate that increase in the brain NA content can be induced by acute stress, though its emergence is delayed. Importantly, this suggests that both acute and chronic stress may lead to NA accumulation under the same mechanism.     

Regulation of brain water during acute hyperosmolality in ovine fetuses, lambs, and adults.

In adult rats, when plasma osmolality increases, water flows across the blood-brain barrier down its concentration gradient from brain to plasma, and brain volume deceases. The brain responds to this stress by gaining osmotically active solutes, which limit water loss. This phenomenon is termed brain volume (water) regulation. We tested the hypothesis that brain volume regulation is more effective in young lambs and adult sheep than in fetuses, premature lambs, and newborn lambs. Brain water responses to acute hyperosmolality were measured in the cerebral cortex, cerebellum, and medulla of fetuses at 60 and 90% of gestation, premature ventilated lambs at 90% of gestation, newborn lambs, young lambs at 20-30 days of age, and adult sheep. After exposure of the sheep to increases in systemic osmolality with mannitol plus NaCl, brain water content and electrolytes were quantified. The ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress. There were significant differences from an ideal osmometer in the cerebral cortex of fetuses at 90% of gestation, cerebral cortex, and cerebellum of newborn lambs, and cerebral cortex, cerebellum, and medulla of young lambs and adult sheep; however, there were no differences in the brain regions of fetuses at 60% of gestation and premature lambs, cerebellum and medulla of fetuses at 90% of gestation, and medulla of newborn lambs. We conclude that 1) brain water loss is maximal and brain volume regulation impaired in most brain regions of fetuses at 60 and 90% of gestation and premature lambs; 2) brain volume regulation develops first in the cerebral cortex of the fetuses at 90% of gestation and in the cerebral cortex and cerebellum of newborn lambs, and then it develops in the medulla of the lambs at 20-30 days of age; 3) brain water loss is limited and volume regulation present in the brain regions of young lambs and adult sheep; and 4) the ability of the brain to exhibit volume regulation develops in a region- and age-related fashion.     

Catecholamine Content Changes in Brain Regions of Spontaneously Hypertensive Rats Under Immobilization Stress

We compared the effect of immobilization stress on noradrenaline (NA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) content in two brain regions--diencephalon and pons-medulla oblongata--in young and adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). In SHR, NA content decreased with time after the onset of the stress, whereas levels of its metabolite MHPG increased. In WKY, NA and MHPG showed no change. The MHPG/NA ratio in both regions increased relative to the duration of the stress in SHR, whereas it remained almost constant in WKY. The rate of increase in the ratio was much higher in the diencephalon of adult (12-week-old) than of young (4-week-old) SHR. In SHR, NA turnover in the brain is readily affected by environmental stress, and these changes in the noradrenergic system may induce or sustain hypertension.