Cerebral Cortex Of Immature Rats Research Articles

Kinetic study of benzyl [1-14C]acetate as a potential probe for astrocytic energy metabolism in the rat brain: Comparison with benzyl [2-14C]acetate

Brain uptake of [14C]acetate has been reported to be a useful marker of astrocytic energy metabolism. In addition to uptake values, the rate of radiolabeled acetate washout from the brain appears to reflect CO2 exhaustion and oxygen consumption in astrocytes. We measured the time–radioactivity curves of benzyl [1-14C]acetate ([1-14C]BA), a lipophilic probe of [1-14C]acetate, and compared it with that of benzyl [2-14C]acetate ([2-14C]BA) in rat brains. The highest brain uptake was observed immediately after injecting either [1-14C]BA or [2-14C]BA, and both subsequently disappeared from the brain in a single-exponential manner. Estimated [1-14C]BA washout rates in the cerebral cortex and cerebellum were higher than those of [2-14C]BA. These results suggested that [1-14C]BA could be a useful probe for estimating the astrocytic oxidative metabolism. The [1-14C]BA washout rate in the cerebral cortex of immature rats was lower than that of mature rats. An autoradiographic study showed that the washout rates of [1-14C]BA from the rat brains of a lithium–pilocarpine-induced status epilepticus model were not significantly different from the values in control rat brains except for the medial septal nucleus. These results implied that the enhancement of amino acid turnover rate rather than astrocytic oxidative metabolism was increased in status epilepticus.

Antioxidant enzymes in cerebral cortex of immature rats following experimentally-induced seizures: upregulation of mitochondrial MnSOD (SOD2)

We have recently demonstrated the evidence of oxidative stress in brain of immature rats during seizures induced by dl-homocysteic acid (dl-HCA). The aim of the present study was to investigate the antioxidant defense mechanisms under these conditions. Seizures were induced in immature 12-day-old rats by bilateral icv infusion of dl-HCA (600nmol/side), and the activities of the main antioxidant enzymes were examined in supernatants of the cerebral cortex during the acute phase of seizures and at several periods of survival, up to 5 weeks, following these seizures. In control animals individual antioxidant enzymes revealed different changes during the studied postnatal period (PD 12 till PD 47). Total superoxide dismutase (SOD), CuZn SOD (SOD1), Mn SOD (SOD2) and glutathione peroxidase (GPX) activities were increasing while, catalase activity decreased and the activity of glutathione reductase (GR) remained unchanged. In HCA-treated animals, the activity of total SOD, SOD1 and particularly SOD2 significantly increased at 20h and 6 days of survival. Importantly, upregulation of SOD2 was also confirmed in mitochondria at the protein level by immunoblotting. The activities of other antioxidant enzymes including catalase and GPX did not significantly differ upon HCA treatment from the appropriate controls at any of the studied time intervals. The pronounced and selective upregulation of SOD2 points to enhanced ROS levels in the mitochondrial matrix. This may be associated with inhibition of respiratory chain complex I that we have demonstrated in our previous studies. The present findings suggest that oxidative stress occurring in the brain of immature rats during and following the seizures induced by dl-HCA is apparently due to both the increased free radical production and the limited antioxidant defense.

Effect of Ghrelin on nuclear factor-κB and tumor necrosis factor-α in the cerebral cortex of immature rats with pilocarpine-induced epilepsy

Objective To explore the changes of gene and protein expressions of nuclear factor-κB(NF-κB) and tumor necrosis factor-α (TNF-α) in immature rats with pilocarpine-induced epilepsy treated with ghrelin. Methods The pllocarpine-induced epilepsy model in immatured rats were built, then the rats were divided into three groups: Ghrelin-treated group, saline-treated group and model group, meanwhile the normal control group was set. The NF-κB and TNF-α levels of gene and protein in the cerebral cortex of immature rats were detected. Results The expression levels of gene and protein of NF-κB and TNF-α were increased in model group,but decreased in the normal control group;NF-κB and TNF-α levels in Ghrelin treated group were obviously lower than those of saline-treated group and model group(P < 0. 05). Conclusion The protective mechanism of Ghrelin for nerve cell is cutting down the expressions of NF-κB and TNF-α in the cerebral cortex of immature rats with epilepsy and lessening inflammatory reaction in neurocytes. Key words: Epilepsy; Cerebral cortex; Pilocarpine; Ghrelin; rats

Sustained deficiency of mitochondrial complex I activity during long periods of survival after seizures induced in immature rats by homocysteic acid

Our previous work demonstrated the marked decrease of mitochondrial complex I activity in the cerebral cortex of immature rats during the acute phase of seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side) and at short time following these seizures. The present study demonstrates that the marked decrease ( approximately 60%) of mitochondrial complex I activity persists during the long periods of survival, up to 5 weeks, following these seizures, i.e. periods corresponding to the development of spontaneous seizures (epileptogenesis) in this model of seizures. The decrease was selective for complex I and it was not associated with changes in the size of the assembled complex I or with changes in mitochondrial content of complex I. Inhibition of complex I was accompanied by a parallel, up to 5 weeks lasting significant increase (15-30%) of three independent mitochondrial markers of oxidative damage, 3-nitrotyrosine, 4-hydroxynonenal and protein carbonyls. This suggests that oxidative modification may be most likely responsible for the sustained deficiency of complex I activity although potential role of other factors cannot be excluded. Pronounced inhibition of complex I was not accompanied by impaired ATP production, apparently due to excess capacity of complex I documented by energy thresholds. The decrease of complex I activity was substantially reduced by treatment with selected free radical scavengers. It could also be attenuated by pretreatment with (S)-3,4-DCPG (an agonist for subtype 8 of group III metabotropic glutamate receptors) which had also a partial antiepileptogenic effect. It can be assumed that the persisting inhibition of complex I may lead to the enhanced production of reactive oxygen and/or nitrogen species, contributing not only to neuronal injury demonstrated in this model of seizures but also to epileptogenesis.

Mitochondrial complex I inhibition in cerebral cortex of immature rats following homocysteic acid-induced seizures

The major finding of the present study concerns the marked decrease of respiratory chain complex I activity in the cerebral cortex of immature rats following seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side). This decrease was already evident during the acute phase of seizures (60–90 min after infusion) and persisted for at least 20 h after the seizures. It was selective for complex I since activities of complex II and IV and citrate synthase remained unaffected. Inhibition of complex I activity was not associated with changes in complex I content. Based on enhanced lipoperoxidation and decreased aconitase activity, it can be postulated that oxidative modification is most likely responsible for the observed inhibition. Mitochondrial respiration, as well as cortical ATP levels remained in the control range, apparently due to excess capacity of the complex I documented by energy thresholds. On the other hand, the enhanced production of reactive oxygen species by inhibited complex I was observed in mitochondria from HCA-treated animals. The decrease of complex I activity was substantially attenuated when animals were treated with substances providing an anticonvulsant effect and also with selected free radical scavengers. We can assume that inhibition of complex I may elicit enhanced formation of reactive oxygen species and contribute thus to neuronal injury demonstrated in this model.

Evidence that intracellular Ca 2+ mediates the effect of α-ketoisocaproic acid on the phosphorylating system of cytoskeletal proteins from cerebral cortex of immature rats

In this study we investigated the involvement of Ca 2+ on the effects of α-ketoisocaproic acid (KIC), the main metabolite accumulating in maple syrup urine disease (MSUD), on the phosphorylating system associated with the intermediate filament (IF) proteins in slices from cerebral cortex of 9-day-old rats. We first observed that KIC significantly decreased the in vitro phosphorylation of IF proteins in brain slices. KIC-induced dephosphorylation was mediated especially by the protein phosphatase PP2B, a Ca 2+-dependent protein phosphatase, but also by PP2A. We also demonstrated the involvement of Ca 2+-dependent mechanisms in the KIC effects using the specific L-voltage-dependent Ca 2+ channels (L-VDCC) inhibitor nifedipine, the NMDA antagonist DL-AP5 and the intracellular Ca 2+ chelator BAPTA-AM. Blockage of Ca 2+ channels or chelating intracellular Ca 2+ completely prevented the effects of KIC on the phosphorylating system associated to IF proteins. In addition, we verified that KIC increased 45Ca 2+ uptake in brain slices after 3 and 30 min incubation. Taken together, our present data indicate that KIC increase intracellular Ca 2+ levels, probably promoting the activation of calcineurin. These results might be associated with the increased dephosphorylation of the IF proteins in slices of cerebral cortex of immature rats exposed to KIC at similar concentrations from those found in blood and tissues of patients with MSUD.

Guanine nucleotide- and muscarinic agonist-dependent phosphoinositide metabolism in synaptoneurosomes from cerebral cortex of immature rats.

Guanine nucleotide-, neurotransmitter-, and fluoride-stimulated accumulation of [3H]inositol phosphates ([3H]InsPs) was measured in [3H]inositol-labeled synaptoneurosomes from cerebral cortex of immature (7-day-old) and adult rats, in order to clarify the role of GTP-binding proteins (G-proteins) in modulating phosphoinositide (PtdIns) metabolism during brain development. GTP(S) [Guanosine 5'-O-(3-thio)triphosphate] time- and concentration-dependently stimulated PtdIns hydrolysis. Its effect was potentiated by full (carbachol, metacholine) and partial (oxotremorine) cholinergic agonists through activation of muscarinic receptors. The presence of deoxycholate was required to demonstrate agonist potentiation of the guanine nucleotide effect. The response to GTP(S) was higher in adult than in immature rats, while the effect of cholinergic agonists was similar at the two ages examined. At both ages, histamine potentiated the effect of GTP(S), while norepinephrine was ineffective. At both ages, guanosine 5'-O-(2-thio)diphosphate [GDP(S)] and pertussis toxin significantly decreased GTP(S)-induced [3H]InsPs formation. The phorbol ester phorbol 12-myristate 13-acetate (PMA), on the other hand, did not inhibit the guanine nucleotide response in synaptoneurosomes from immature rats. NaF mimicked the action of GTP(S) in stimulating PtdIns hydrolysis. Its effect was not affected by carbachol and was highly synergistic with that of AlCl3, according to the concept that fluoroaluminate (AlF4-) is the active stimulatory species. No quantitative differences were found in the response to these salts between immature and adult animals. These results provide evidence that, in both the immature and adult rat brain, neuroreceptor activation is coupled to PtdIns hydrolysis through modulatory G-proteins.

Spontaneous hemorrhage in the cerebral cortex of immature rats

We report the presence of spontaneous hemorrhages in the brain of normal neonatal rats. The quantitative evaluation showed that the hemorrhaging was most frequent in 4- to 5-day-old rats. Nearly all the lesions were found in the cerebral cortex of the lateral part of the forebrain hemisphere, particularly in the middle cortical layers. Their average incidence amounted to 6.71 ± 2.95 per six 5-μm sections from 5-day-old rats. The lesions were nearly absent in the newborn and 7-day-old animals. We suggest that vascular wall immaturity and some extravascular factors, like developmental increase of blood pressure, may account for the perinatal hemorrhaging.

Transient increase in ligand binding to quisqualate and kainate sites in cerebral cortex of immature rats

The postnatal changes in the specific binding of [ 3H]kainate and [ 3H]AMPA (RS-α-amino-3-hydroxy-5-methyl-4-isoxasolopropionic acid, an agonist of quisqualate receptors) were studied in cerebral cortex of rats, aged 2–360 days. The binding of the two ligands was assayed in whole-tissue homogenates. Similar developmental time courses were found for kainate and AMPA binding, characterized by high perinatal levels, a further increase during the first few days after birth, an early maximum value around the age of one week, and a gradual decrease to adult values which were attained at an age of 3–4 weeks. As revealed by Scatchard analysis, the transient elevation of ligand binding was derived from an increased density of binding sites, which, in the case of AMPA, was accompanied also by an increase in binding affinity. The results indicate that, in the immature cerebral cortex, kainate and quisqualate receptors may play a role other than in synaptic transmission.

Identification of the ‘estrogen-induced protein’ in uterus and brain of untreated immature rats

The appearance of a specific estrogen-induced protein (IP) in the rat uterus [l] represents one of the earliest effects of estrogen on uterine macromolecular synthesis [2]. This effect can be reproducibly demonstrated following in vitro as well as in vivo estrogen treatment (3-S], making IP the most convenient marker protein available for studying the mechanism of action of estrogen in the rat uterus. IP has been routinely detected using a doubleisotope ratio method [6]. This method is convenient, but it cannot yield information on content or rate of synthesis of IP in non-treated organs, or in organs in which IP may be present but not susceptible to induction by estrogen. We have reported initial results, on the time course and age dependence of IP induction [7], using ~uoro~aphy of ~35S]met~on~e-labelled proteins separated by sodium dodecyl sulfate polyac~l~ide gel electrophoresis [S]. We describe here the characterization of IP by partial protease digestion, which enables the identification of IP as a major constitutive protein in uterus,pituitary, hypothalamus and cerebral cortex of immature rats. (A short report of some of these findings was presented [9] .)

The effect of phenylpyruvate on oxidative-phosphorylation in brain mitochondria.

Abstract— A mitochondrial fraction isolated from the cerebral cortex of immature rats was shown to possess the characteristics of intact, functional mitochondria. Phenylpyruvate, in low concentrations, had an inhibitory effect upon oxygen utilization by this system without uncoupling phosphorylation. The inhibition was specific for the keto acid and to pyruvate as substrate. It was concluded that phenylpyruvate inhibits the oxidation of pyruvate and the implications of this are discussed in relation to phenylpyruvic oligophrenia.