Intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic brain injury estimated using the microdialysis-electron spin resonance method.

AIM: The intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic (HI) brain injury was estimated using the microdialysis-electron spin resonance method. MATERIAL AND METHODS: Seven-day-old Wistar rats were subjected to a modified Levine's procedure for producing HI brain injury. After HI insult, pups were returned and reared with their dams. Seven weeks after HI insult, their intracerebral antioxidant abilities were measured using the microdialysis-electron spin resonance method after the intraperitoneal injection of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl. Ascorbic acid, L-cysteine, and glutathione (GSH) were also determined. The rats without HI insult were used as a control. RESULTS: The decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in the non-ligated side of the cerebral hemisphere of the HI group was significantly larger than that of the control group. The amounts of ascorbic acid in the perfusate from the non-ligated side of the HI group were about four times larger than those of the control group. The amounts of L-cysteine and GSH of the HI group were about 10 times larger than those of the control group. CONCLUSIONS: The antioxidant ability in the non-ligated sides of the cerebral hemispheres of the mature rats 7 weeks after neonatal HI insult was higher than that of the control group. Higher amounts of ascorbic acid and GSH supported the higher antioxidant ability. The increase of the intracerebral antioxidant ability of the non-ligated side indicates the compensation of motor function for the lost side. The present results should offer important insights into the prognosis for hypoxic-ischemic encephalopathy.

Amniotic fluid insulin and C-peptide as predictive markers for fetal macrosomia, birth injuries, and delivery complications?

BACKGROUND: Gestational diabetes mellitus (GDM) occurs in 3-5% of all pregnancies. GDM increases both maternal and fetal risks, causes fetal macrosomia, and hence increases the rates of caesarean sections and delivery complications such as shoulder dystocia. An early predictive marker and consequent early treatment could be beneficial, so amniotic fluid insulin and C-peptide have been examined in several studies. Increased amniotic fluid insulin in early amniocentesis between the 14th and 20th gestational week predicted a later GDM. A potential direct association with fetal macrosomia remains to be determined. MATERIAL AND METHODS: This retrospective study investigated amniotic fluid insulin/C-peptide from amniocenteses between 14 and 20 weeks of gestation in correlation with fetal birth weight, type of delivery, and complications. To focus on effects of fetal hyperinsulinism apart from therapeutic confounders, we included patients who did not participate in GDM screening. Insulin and C-peptide were measured in 144 samples of frozen amniotic fluid. Birth weight, type of delivery, complications, and birth injuries were noted. RESULTS: Birth weights ranged from 760 g to 4410 g with a mean weight of 3424 g at an average of 40 weeks gestation. The mean amniotic fluid insulin was 4.36 U/ml and the mean C-peptide concentration was 0.076 ng/ml. There was no correlation between amniotic fluid insulin or C peptide and birth weight, type of delivery, complications, and birth injuries. CONCLUSIONS: Amniotic fluid insulin and C-peptide are unsuitable as predictive marker for fetal macrosomia, type of delivery, complications, or birth injuries.

The impact of simulated birth trauma and ovariectomy on the gene expression of detrusor muscarinic receptors in female rats.

INTRODUCTION AND HYPOTHESIS: This study aims to investigate the effects of simulated birth trauma and ovariectomy on detrusor muscarinic receptors (M2 and M3), urethral neuronal nitric oxide synthase (nNOS), and estrogen receptor beta (ER beta). METHODS: Forty primiparous rats were equally divided into five groups: group A--delivery, group B--delivery plus ovariectomy, group C--delivery plus balloon dilatation for 2 h, group D--delivery plus balloon dilatation for 4 h, and group E--delivery plus balloon dilatation for 2 h plus ovariectomy. The gene expression of M2, M3, nNOS, and ER beta were assessed by reverse transcription polymerase chain reaction. RESULTS: Significant decreases in mRNA expression of M2 receptors and nNOS (P < 0.05), and a significant increase in M3 mRNA expression (P < 0.05) were observed in groups D and E when compared with group A. CONCLUSIONS: Ovariectomy following birth trauma may synergistically impact the function of urinary tract, this being possibly related to the modification of the gene expression of muscarinic receptors.

Proinflammatory cytokine production by cultured neonatal rat microglia after exposure to blood products.

Periventricular germinal matrix hemorrhage is a devastating complication of preterm birth. Inflammation appears to play a role in brain damage after premature birth and hypoxia. The effects of rat blood plasma and serum on cytokine expression by cultured rat microglial cells were investigated. We analyzed mRNA expression levels of tumor necrosis factor (TNF)-alpha, interleukin-6 and protease activated receptor-1 and -4 by quantitative RT-PCR. Protein expression for TNFalpha was done using immunocytochemistry and ELISPOT assays. Plasma and serum had dose dependent toxic effects on microglia as measured by lactate dehydrogenase release assay and activated caspase-3 immunocytochemistry. High concentrations of plasma enhanced TNFalpha mRNA expression and protein production, while high concentrations of serum enhanced IL-6 mRNA expression. This study suggests that soluble components of blood might be differentially responsible for up regulating production of the cytokines TNFalpha and IL-6 by microglia from immature rodent brain.

Antioxidant status alters levels of Fas-associated death domain-like IL-1B-converting enzyme inhibitory protein following neonatal hypoxia-ischemia.

Activation of Fas death receptor (Fas DR) signaling cascade is seen after neonatal hypoxia-ischemia (HI). Cell survival is favored when signaling through the death-inducing signaling complex and cleavage of caspase 8 to its active form is blocked by FLIP, a dominant negative of caspase 8. H2O2 quickly downregulates expression of FLIP. Neonatal mice overexpressing glutathione peroxidase (GPx) have less injury and less H2O2 accumulation compared with neonatal mice overexpressing superoxide dismutase (SOD) or wild-type (WT) littermates. Expression of both FLIP(L) and FLIP(S) is increased in GPx-oxerexpressing mice relative to WT mice at 24 h and relative to SOD-overexpressing mice at 2 and 24 h following neonatal HI (ANOVA, p < 0.05). There is an increase in Fas DR expression at 24 h in both WT and GPx-overexpressing mice and significant differences between WT and SOD-overexpressing mice (ANOVA, p < 0.01). There is no difference in FADD expression among the 3 groups 24 h after HI. At 24 h following HI, the ratio of FLIP to Fas DR expression supports a significant negative correlation with injury score (r2 = 0.99, slope = -4.01), and expression of both the active fragment of caspase 8 and caspase 8 activity is increased in SOD overexpressors compared to GPx overexpressors at 24 h after HI (ANOVA, p < 0.05). The overall degree of injury previously seen in these 3 strains correlates well with changes in expression of Fas DR signaling proteins favoring neuroprotection in the GPx-overexpressing mice, i.e. increased FLIP expression and decreased caspase 8 activity compared to SODtg mice. The mechanism by which antioxidant status alters FLIP levels following neonatal HI may be related to the ability to detoxify H2O2 produced following neonatal HI.

Proton MR spectroscopy in neonates with perinatal cerebral hypoxic-ischemic injury: metabolite peak-area ratios, relaxation times, and absolute concentrations.

BACKGROUND: Results from cerebral proton (1)H-MR spectroscopy studies of neonates with perinatal hypoxic-ischemic injury have generally been presented as metabolite peak-area ratios, which are T1- and T2-weighted, rather than absolute metabolite concentrations. We hypothesized that compared with (1)H-MR spectroscopy peak-area ratios, calculation of absolute metabolite concentrations and relaxation times measured within the first 4 days after birth (1) would improve prognostic accuracy and (2) enhance the understanding of underlying neurochemical changes in neonates with neonatal encephalopathy. METHODS: Seventeen term infants with neonatal encephalopathy and 10 healthy controls were studied at 2.4T at 1 (1-3) and 2 (2-4) (median [interquartile range]) days after birth, respectively. Infants with neonatal encephalopathy were classified into 2 outcome groups (normal/mild and severe/fatal), according to neurodevelopmental assessments at 1 year. The MR spectroscopy peak-area ratios, relaxation times, absolute concentrations, and concentration ratios of lactate (Lac), creatine plus phosphocreatine (Cr), N-acetylaspartate (NAA), and choline-containing compounds (Cho) from a voxel centered on the thalami were analyzed according to outcome group. RESULTS: Comparing the severe/fatal group with the controls (significance assumed with P < 0.05), we found that Lac/NAA, Lac/Cho, and Lac/Cr peak-area ratios increased and NAA/Cr and NAA/Cho decreased; Lac, NAA, and Cr T2s were increased; [Lac] was increased and [Cho], [Cr], and [NAA] decreased; and among the concentration ratios, only [Lac]/[NAA] was increased. Comparison of the normal/mild group with controls revealed no differences in peak-area ratios, relaxation times, or concentration ratios but decreased [NAA], [Cho], and [Cr] were observed in the infants with normal/mild outcome. Comparison of the normal/mild and severe/fatal groups showed increased Lac/NAA and Lac/Cho and decreased NAA/Cr and NAA/Cho peak-area ratios, reduced [NAA], and increased Lac T2 in the infants with the worse outcome. CONCLUSIONS: Metabolite concentrations, in particular [NAA], enhance the prognostic accuracy of cerebral (1)H-MR spectroscopy-[NAA] was the only measurable to discriminate among all (control, normal/mild, and severe/fatal outcome) groups. However, peak-area ratios are more useful prognostic indicators than concentration ratios because they depend on metabolite concentrations and T2s, both of which are pathologically modulated. Concentration ratios depend only on the concentrations of the constituent metabolites. Increased Cr T2 may provide an indirect marker of impaired cellular energetics, and similarly, NAA T2 may constitute an index of exclusively neuronal energy status. Our recommendation is to collect data that enable calculation of brain metabolite concentrations. However, if time constraints make this impossible, metabolite peak-area ratios provide the next best method of assigning early prognosis in neonatal encephalopathy.

Birth insults involving hypoxia produce long-term increases in hippocampal [125I]insulin-like growth factor-I and -II receptor binding in the rat.

Insulin-like growth factors-I and -II and insulin are structurally related mitogenic growth factors with multiple actions in the developing nervous system and adult CNS. Previous studies have demonstrated acute induction of insulin-like growth factors and their receptors, over a time course of several days, in response to hypoxic/ischemic insult to developing or adult brain. The current study tested whether birth insults involving hypoxia may produce long term changes in brain insulin-like growth factor or insulin receptor levels, lasting into adulthood. For this, rats were born vaginally (controls), by cesarean section, or by cesarean section with 15 min of added global anoxia (cesarean section+anoxia), and brain [125I]insulin-like growth factor-I, [125I]insulin-like growth factor-II and [125I]insulin receptor binding sites were assessed autoradiographically at adulthood. [125I]Insulin-like growth factor-I receptor binding sites were increased in all hippocampal subfields (CA1-CA3, dentate gyrus) in rats born either by cesarean section or by cesarean section+anoxia, compared with vaginal birth. [125I]Insulin-like growth factor-II binding was increased in all hippocampal subfields only in rats born by cesarean section+anoxia compared with either vaginal birth or cesarean section groups. [125I]Insulin-like growth factor-I and [125I]insulin-like growth factor-II binding in frontal cortex, striatum and cerebellum were unaffected by birth group, except for increased [125I]insulin-like growth factor-I binding in the cerebellar molecular layer of cesarean-sectioned animals. Birth group had no significant effect on [125I]insulin binding in any brain region. Affinity cross-linking experiments performed with hippocampal membranes from the three birth groups showed that i) [125I]insulin-like growth factor-I and [125I]insulin-like growth factor-II recognized bands of molecular weights characteristic of insulin-like growth factor-I and insulin-like growth factor-II receptors, respectively, and ii) [125I]insulin-like growth factor-I and [125I]insulin-like growth factor-II were displaced more potently by their respective unlabeled ligands than by related molecules. It is concluded that birth insults involving hypoxia can induce lasting increases in insulin-like growth factor-I and -II receptors in the CNS. There is specificity with respect to the subtype of insulin-like growth factor receptor affected by the particular birth insult and the brain region affected. It is suggested that enduring increases in levels of insulin-like growth factor receptors consequent to hypoxic birth insult may help to maintain hippocampal function at adulthood, and could modulate responsiveness to insulin-like growth factor administration.

Effects of birth insult and stress at adulthood on excitatory amino acid receptors in adult rat brain.

Birth complications involving fetal hypoxia and stress at adulthood, which are risk factors for schizophrenia, can produce alterations in subcortical dopamine (DA) function in rat models. As adults, rats born either by cesarean section (C-section) or by C-section with added global anoxia show increased stress-induced DA release from nucleus accumbens and increased amphetamine-induced locomotion, compared to vaginally born controls. Moreover, stress at adulthood interacts with these birth insults to modulate DA receptor and transporter levels. Glutamatergic transmission at the level of the nucleus accumbens, prefrontal cortex, and hippocampus are known to modulate subcortical DA activity. Thus, altered excitatory amino acid (EAA) function might contribute to the dopaminergic changes observed in rats after birth insult and/or stress at adulthood. To test this possibility, rats born vaginally, by C-section, or by C-section with 15 min of anoxia, were either repeatedly stressed (15 min of tail pinch daily for 5 days) at adulthood or received no stress, and levels of EAA receptor binding were measured by ligand autoradiography in limbic brain regions. As adults, rats born by C-section showed increases in AMPA receptor binding in nucleus accumbens shell, NMDA receptor binding in cingulate cortex, and kainate receptor binding in the hippocampal CA1 region. Anoxic rats showed increases in CA1 kainate receptor and anterior olfactory NMDA receptor binding. Stress at adulthood increased AMPA receptor binding in several regions of prefrontal cortex and reduced NMDA receptor binding in infralimbic cortex and dentate gyrus, across all birth groups. Two instances of interactions between birth insult and stress at adulthood were observed. Stress reduced cingulate cortex NMDA receptor binding and increased olfactory tubercle kainate receptor binding only in C-sectioned animals, but not in controls. The possibility that the observed EAA receptor changes contribute to dopaminergic dysfunction in these animal models is discussed, in light of known glutamate-DA interactions.

Differential vulnerability of male versus female rats to long-term effects of birth insult on brain catecholamine levels.

There are gender differences in the prevalence and severity of several human behavioral disorders, in which both obstetric complications and dysregulation of brain monoamine systems have been implicated. In animal studies, males are more susceptible than are females to lasting behavioral deficits following various perinatal insults. The current study compared monoamine levels in brain regions from adult male and female rats that had been born under various conditions-vaginal birth (control), Caesarean section (C-section), or C-section with 15 min of added anoxia (Anoxia). At adulthood, male rats born by C-section had increased dopamine (DA) levels in the nucleus accumbens and dorsal striatum, and decreased amygdalar norepinephrine (NE), compared to vaginally born males. C-sectioned female rats had increased NE levels in the thalamus in comparison to vaginally born females. The only monoamine change observed in the Anoxia groups was a decrease in nucleus accumbens NE in females. Thus addition of 15 min of anoxia to the C-section procedure reversed several of the monoamine changes produced by C-section alone. Birth group had no effect on serotonin in several brain regions in either sex. Male, but not female, rats born by C-section had decreased plasma epinephrine levels at birth and slightly increased brain lactate at 5 h after birth. Pups of both sexes in the Anoxia groups had high levels of plasma catecholamines at birth. The possible functional significance of the lasting, region-specific changes in brain DA and NE due to birth insult and possible roles of hormones at birth in producing these monoamine changes in the two sexes are discussed.

Birth insult interacts with stress at adulthood to alter dopaminergic function in animal models: possible implications for schizophrenia and other disorders.

Altered subcortical dopaminergic activity is thought to be involved in the pathophysiology of several disorders including schizophrenia, substance abuse and attention deficit hyperactivity disorder. Epidemiological studies have implicated perinatal insults, particularly obstetric complications involving fetal or neonatal hypoxia, as etiological risk factors for schizophrenia. This suggests the possibility that perinatal hypoxia might have lasting effects on dopaminergic function. In animal models, dopaminergic systems appears to be particularly vulnerable to a wide range of perinatal insults, resulting in persistent alterations in function of mesolimbic and mesostriatal pathways. This review summarizes recent work characterizing long-term changes in dopaminergic function and biochemistry in models of Caesarean section (C-section) birth and of C-section birth with added global anoxia in the rat and guinea pig. C-section birth and C-section with anoxia appear to be two distinct hypoxic birth insults, with somewhat differing patterns of lasting effects on dopamine systems. In addition, birth insult alters the manner in which dopaminergic function is regulated by stress at adulthood. The possible relevance of these finding to effects of human birth procedures is discussed.

Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: current concepts.

Multiple, biochemical cascades contribute to the pathogenesis of neonatal hypoxic-ischemic brain injury. This article summarizes experimental evidence that supports the role of excitatory amino acids, calcium, free radicals, nitric oxide, proinflammatory cytokines, and bioactive lipids. Specific vulnerabilities that distinguish the response of the immature brain from that of the mature brain are highlighted. These include increased susceptibility to excitotoxicity and free radical injury, greater tendency to apoptotic death, and heightened vulnerability of developing oligodendrocytes. Available supportive evidence from human studies is also included. Implications for clinical neuroprotective strategies are discussed.

Role of the new growth hormone-releasing secretagogues in the diagnosis of some hypothalamopituitary pathologies.

Growth hormone (GH)-releasing hormone (GHRH) and somatostatin have a dominant role in regulating GH secretion. However, results of studies using the new class of GH secretogogues, particularly GHRP-6, indicate that there may also be other, as yet undefined, hypothalamic mechanisms involved. Studies in adults with hypothalamopituitary disconnection (functional pituitary stalk transection), show GHRP-6-mediated GH release to be completely blocked, indicating a main action at the hypothalamic rather than the pituitary level. The synergistic effect of GHRH plus GHRP-6 administration on GH release seen in normal adults (and virtually unaffected by age, obesity, or sex) is also absent in these patients, providing further support for this conclusion. Studies of the effects of GHRP-6 in children with GH deficiency due to perinatal pituitary stalk transection have produced similar findings. It is suggested that the combined GHRH plus GHRH-6 test should be a promising tool for diagnosing GH deficiency states in both children and adults, and may identify a subgroup of patients with GH deficiency caused by interruption of the hypothalamopituitary connection.

[Vitamin supply of premature infants with intracranial birth injuries fed in different ways]. / Obespechennost' vitaminami nedonoshennykh detei s vnutricherepnoi rodovoi travmoi, nakhodivshikhsia na raznykh vidakh vskarmlivaniia.

A total of 136 premature infants with intracranial birth trauma were studied during the first 2 months of life for vitamin B, C, A and carotinoid supply with regard to their body mass at birth and type of feeding. Premature children with different patterns of birth trauma showed low supply with thiamine, riboflavin, niacin and vitamin A in the presence of breast feeding. Combined feeding raised the supply of the children under observation with thiamine and riboflavin. The data obtained attest to the necessity of further study in this direction with the purpose of developing pathogenetically validated schemes for vitamin therapy of premature children.