Hypoxia-ischemia induced neurological dysfunction and brain injury in the neonatal rat

Bilateral carotid artery occlusion (BCAO) followed by exposure to a hypoxic condition (8% oxygen for 10 or 15 min) was performed in postnatal day 4 SD rats. Brain injury and myelination changes were examined on postnatal day 21 (P21) and tests for neurobehavioral toxicity were performed from P3 to P21. BCAO followed by 10 or 15 min hypoxic insult resulted in mild and severe, respectively, brain injury, reduction in mature oligodendrocytes and tyrosine hydroxylase positive neurons and impaired myelination as indicated by decreased myelin basic protein immunostaining in the P21 rat brain. Hypoxia-ischemia also affected physical development (body weight gain and eye opening) and neurobehavioral performance, such as righting reflex, wire hanging maneuver, cliff avoidance, locomotor activity, gait analysis, responses in the elevated plus-maze and passive avoidance. BCAO followed by 15 min of hypoxia caused more severely impaired neurobehavioral performance as compared with BCAO followed by 10 min of hypoxia in the rat. The overall results demonstrate that hypoxia-ischemia-induced brain injury not only persists, but also is linked with neurobehavioral deficits in juvenile rats. The present data also indicate that the degree of brain injury and the deficits of neurobehavioral performance in the rat are dependent on the hypoxic-ischemic condition, i.e., the exposure time to hypoxia.     

Hypoxia-ischemia produces focal disruption of glutamate receptors in developing brain

We examined the impact of a perinatal hypoxic-ischemic insult on the distribution of glutamate receptors in developing brain. We used a well characterized rodent model for perinatal hypoxic-ischemic encephalopathy, unilateral carotid artery occlusion followed by exposure to 8% oxygen for 2.5 h in 7-day-old rat pups. This preparation results in focal neuronal damage in striatum, hippocampus, and cortex ipsilateral to ligation. Alterations in the regional distribution of glutamate binding in the first 24 h after the insult were assessed with quantitative in vitro [3H]glutamate autoradiography. In lesioned animals, we found progressive selective reductions in [3H]glutamate binding in forebrain ipsilateral to ligation in regions destined for neuronal damage. The earliest and most prominent unilateral reductions in binding were noted in the dentate gyrus of hippocampus (-45 +/- 9%, compared with contralateral hemisphere at 24 h). Acute reductions in specific glutamate binding appear to be a sensitive marker for hypoxic-ischemic neuronal damage in the immature brain. These observations suggest that neurons bearing glutamate receptors may be particularly susceptible to hypoxic-ischemic injury.     

Hypoxia-ischemia affects erythropoietin and erythropoietin receptor expression pattern in the neonatal rat brain

Erythropoietin (EPO), known for its role in erythroid differentiation, has been suggested to have non-hematopoietic functions in the brain, especially during development. In the present study, we investigated the expression of erythropoietin and erythropoietin receptor (EPOR) in the developing rat brain following hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia, and their brains were examined immediately, 24 h or 4 days after hypoxia-ischemia. RT-PCR and Western blot analysis revealed that hypoxia-ischemia only marginally affected EPO expression. Immunohistochemical study of brains 4 days after hypoxia showed that 60 min of hypoxia (resulting in cortical infarction and severe neuronal loss in other regions) led to the increased EPO immunoreactivity, especially in the boundaries of the damaged cerebral cortex, associated with astrocytosis. In contrast, EPOR was dramatically upregulated within 24 h after hypoxia-ischemia. These results suggest that there is a rapid response of EPOR to the hypoxic-ischemic stimulus, which seems to precede that of EPO, leading to the hypothesis that the EPO/EPOR system is implicated in the processes of neuroprotection from hypoxia-ischemia.     

Hypoxia-ischemia induces transforming growth factor beta 1 mRNA in the infant rat brain.

Transforming growth factor beta 1 (TGF beta 1) mRNA expression was examined after hypoxia-ischemia in rat brains using in situ hybridization. Twenty-one-day-old Wistar rats had unilateral ligation of the right carotid artery followed by either 15 or 90 min inhalational hypoxia. Fifteen min of hypoxia resulted in moderate damage with selective neuronal loss in cortical layer 3 and in the hippocampus of the ligated hemisphere. Seventy-two hours after hypoxia TGF beta 1 expression was markedly increased above control levels in those sites. Levels were normal after 120 h. Ninety min of hypoxia led to an infarction of the lateral cerebral cortex and hippocampus of the ligated hemisphere. One hour after hypoxia TGF beta 1 mRNA was expressed in the hippocampus of the damaged side. Seventy-two and 120 h after hypoxia, expressing cells were found throughout the cerebral cortex, piriform cortex, striatum, thalamus and hippocampus of the infarcted side. These data show that TGF beta 1 mRNA expression is induced after a hypoxic-ischemic insult in the brain. TGF beta 1 may be involved in post-asphyxial repair mechanisms.     

Ibotenate-induced neuronal degeneration in immature rat brain

Stereotaxic microinjections of the excitotoxin, ibotenic acid, were made into the striatum, hippocampus or cerebellum of the immature (7-day-old) rat. Two days later, pups were decapitated and the brains processed for light microscopic examination or neurochemical analyses. 10 micrograms ibotenate caused a complete loss of nerve cell bodies throughout the striatum and hippocampus while intracerebellar injections produced no detectable damage. In the striatum, catecholamine histofluorescence was abolished and dopamine uptake severely reduced, indicating also a loss of afferent nerve terminals. Co-injection of 10 micrograms ibotenate with equimolar amounts of the selective amino acid antagonist, (-)-2-amino-7-phosphonoheptanoic acid, resulted in the protection of both striatal cell bodies and dopaminergic nerve terminals. The neurotoxic properties of ibotenate described here are in marked contrast to those of kainic acid, a related excitotoxin. Differences in the ontogenetic pattern of receptors which mediate neurodegenerative events may account for the pharmacological and regional selectivity and the partial lack of axon-sparing properties of ibotenic acid lesions in the immature brain.     

L-glutamate increases internal free calcium levels in synaptoneurosomes from immature rat brain via quisqualate receptors

Internal free calcium concentrations ([Ca2+]i) have been monitored in synaptoneurosomes from 8-d-old rat whole brain previously loaded with the calcium-sensitive fluorescent probe Fura 2. Under basal conditions, [Ca2+]i was around 200 nM, this concentration increasing only slowly during storage of the synaptoneurosomes at room temperature (40% increase 2 hr after loading). Opening of sodium channels with veratridine- (10 microM) or KCl- (30 mM) induced depolarization caused rapid increases in synaptoneurosomal [Ca2+]i. [Ca2+]i was also markedly increased by addition of the Ca2+ ionophore A23187 (10-100 nM). The effect of veratridine, but not of KCl was prevented by previous addition of TTX (1 microM). KCl-induced [Ca2+]i increase was dependent on external Ca2+ and was partially blocked by the dihydropyridine derivative PN 200-110 (IC50 0.15 microM, maximal inhibition 55% at 3 microM). L-Glutamate elicited a concentration-dependent fast increase in synaptoneurosomal [Ca2+]i in the 8-d-old (but not in the adult) rat brain (EC50 = 2 microM). The effect of glutamate was stereospecific, the EC50 of the D-isomer being 47 times higher than that of L-isomer. The magnitude of the L-glutamate response differed in several brain regions, being highest in the cerebral cortex and lowest in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia-ischemia induces heat shock protein-like (HSP72) immunoreactivity in neonatal rat brain

The expression of heat shock protein immunoreactivity in rat brain was evaluated in a model of neonatal hypoxia-ischemia. One-week-old rats were subjected to left carotid artery coagulation and exposure to 8% O2/92% N2 for 2 h (moderate injury) or 3.5 h (severe injury). Animals were sacrificed 1, 12 and 24 h after the hypoxic insult. Cells immunoreactive for the 72 kDa heat shock protein (HSP72) were observed in ipsilateral cortex as early as 1 h after the termination of the hypoxia. After 12 h, neurons in the ipsilateral hippocampus and cortex stained intensely for HSP72 immunoreactivity in the moderately injured group. In the severely injured brains, bilateral staining was observed in neurons and vessels of the hippocampus and cortex. Therefore, cells containing HSP72 immunoreactivity may serve as an early marker for neuronal injury from hypoxia-ischemia in the neonatal rat brain and more importantly may illustrate previously unrecognized areas of central nervous system vulnerability.     

Acute hydrocephalus upregulates monoamine oxidase mRNA in neonatal rat brain

Metabolic derangement of the dopamine system in hydrocephalic brain has been observed, but the change of monoamine oxidase (MAO), the major enzyme to metabolize dopamine, is not known. The metabolic changes of dopamine and MAO mRNA in the striatum were examined in acute hydrocephalic rats whose ventricular size and intracranial pressure were controlled to a similar degree. The tissue levels of dopamine and its metabolites as well as MAO-A and MAO-B mRNA elevated significantly in hydrocephalus. Cerebrospinal fluid (CSF) diversion reversed these changes and induced an initial decline, followed by an elevation of these substances in extracellular fluid. In summary, the metabolism of dopamine system in the striatum was up-regulated in acute hydrocephalus and CSF diversion reversed this metabolic derangement.     

Androgen receptors in the perinatal rat brain

Androgen receptors were identified and characterized in the cytosol and nuclear fractions of the perinatal female rat brain. Cytosol receptors have sedimentation coefficients in the range of 8 to 10S and bind to DNA cellulose columns. These receptors are detected in low concentrations in hypothalamus-preoptic area-amygdala-septum (HPAS) during the last few days of fetal life, less than 1/10th of levels found in adult HPAS. They undergo the most dramatic increase in concentration between postnatal days 7 and 15. Their appearance thus coincides temporally with the appearance of estrogen and progestin receptors, but the time course of their development is delayed, by comparison. Cell nuclear androgen receptors were also identified by gel exclusion chromatography and protamine sulfate precipitation after salt extraction of neonatal brain cell nuclei following in vivo labeling with both a synthetic and a natural androgen. Two well-known anti-andorgens, cyproterone acetate and flutamide, are shown to inhibit the in vivo androgen binding to these cell nuclear receptors. The results are discussed in relation to androgen action in the perinatal rat brain, and the information currently available suggests that androgens influence both gonadotropin secretion and aspects of brain sexual differentiation.     

Alpha-II-spectrin after controlled cortical impact in the immature rat brain

Proteolytic processing plays an important role in regulating a wide range of important cellular functions, including processing of cytoskeletal proteins. Loss of cytoskeletal proteins such as spectrin is an important characteristic in a variety of acute central nervous system injuries including ischemia, spinal cord injury and traumatic brain injury (TBI). The literature contains extensive information on the proteolytic degradation of alpha-II-spectrin after TBI in the adult brain. By contrast, there is limited knowledge on the characteristics and relevance of these important processes in the immature brain. The present experiments examine TBI-induced proteolytic processing of alpha-II-spectrin after TBI in the immature rat brain. Distinct proteolytic products resulting from the degradation of the cytoskeletal protein alpha-II-spectrin by calpain and caspase 3 were readily detectable in cortical brain parenchyma and cerebrospinal fluid after TBI in immature rats.     

Chronic fluoxetine treatment upregulates 5-HT uptake sites and 5-HT2 receptors in rat brain: An autoradiographic study

This study was undertaken to investigate the effect of chronic treatment with fluoxetine, a selective serotonin uptake inhibitor used widely in the treatment of depression, on the distribution and density of 5-HT uptake sites, 5-HT₂ receptors, and vesicular amine uptake sites in rat brain. Fluoxetine (10 mg/kg i. p.) was administered daily for 21 days. The density of 5-HT uptake sites labelled by [³H]paroxetine, 5-HT₂ receptors labelled by [³H]ketanserin in presence of tetrabenazine and vesicular amine uptake sites labelled by [³H]ketanserin in the presence of mianserin were measured by quantitative autoradiography in 22 areas of rat brain, using coronal tissue sections. Chronic administration of fluoxetine produced significant increases in the density of 5-HT uptake sites in layers of frontoparietal cortex (by 32–43%), of striate cortex (by 55%), in CA1 field of hippocampus (by 111%) and in superior colliculus (by 20%). Fluoxetine treatment also resulted in upregulation of 5-HT₂ receptors in layers of frontparietal cortex (31–38%) and in CA2-3 fields of hippocampus (by 39%). The density of tetrabenazine-sensitive vesicular amine uptake sites in the caudate-putamen was also significantly increased (by 66%). The observed alterations in 5-HT uptake site and 5-HT₂ receptor densities are likely a part of adaptive neuronal changes that occur after chronic administration of fluoxetine and may be related to the antidepressant effect of the drug. © 1993 Wiley-Liss, Inc.     

Microglial response to N-methyl-D-aspartate-mediated excitotoxicity in the immature rat brain

The intracerebral injection of N-methyl-D -aspartate (NMDA) has been proposed as a model for hypoxic-ischemic insult in the immature brain. In this light, the aim of this study was to describe the time course of the microglial reaction in the areas undergoing primary degeneration at the site of intracortical NMDA injection as well as in areas undergoing secondary anterograde and/or retrograde degeneration. Fifty nanomoles of NMDA were injected in the sensorimotor cortex of 6-day-old rats. After survival times ranging from 10 hours to 28 days, cryostat sections were stained for routine histology and for the demonstration of microglial cells by means of tomato lectin histochemistry. The areas affected by primary degeneration caused by the intracortical injection of NMDA were the neocortex, the hippocampus, and the rostral thalamus. Secondary degeneration (retrograde and anterograde) was observed in the ventrobasal complex of the thalamus. The cortical lesion also caused Wallerian degeneration of the cortical descending efferents as observed in the basilar pons. Microglial reactivity in all these areas was present at 10 hours postinjection and was restricted to the areas undergoing neuronal or axonal degeneration. Reactive microglial cells were stained intensely and showed a round or pseudopodic morphology. At 3 days, an apparent increase in the number of tomato lectin-positive cells was observed in the areas undergoing neuronal death. By 7 days after the injection, the lesion became nonprogressive, and by 14 and 28 days, microglial cells showed moderate lectin binding and a more ramified morphology. © 1996 Wiley-Liss, Inc.     

Nuclear magnetic resonance study of the immature rat brain

Using rat brains, the maturational change on the water contents and on the proton NMR signals were studied in this laboratory. Water contents, the proton longitudinal relaxation time (T1) and the transverse relaxation time (T2) were measured on the samples from gray and white matters, separately. A constant decrease of these parameter values was observed in relation to the development of the brain. Water content of 1 week-old rat was 88.393 +/- 1.19% in the gray matter (G) and 88.63 +/- 1.96% in the white matter (W). These values decreased gradually and reached adult rat level when the animals became 5 weeks old: 81.47 +/- 1.67% in G and 79.45 +/- 2.39% in W. No marked change on the brain water content was observed thereafter, T1 values of immature (one week-old) rat brain were 1.755 +/- 0.088 sec in G, and 1.832 +/- 0.154 sec in W, while T1 values of the adult rat brain were 1.327 +/- 0.043 sec in G, and 1.297 +/- 0.039 sec in W, respectively. A marked difference was observed on the values of T1 between the immature and the adult rats. T2 value of the 1 week-old rat brain was 101.05 +/- 10.20 msec in G, and 102.75 +/- 8.10 msec in W, respectively. T2 of the mature rat brain was 56.90 +/- 2.61 msec in G, and 54.52 +/- 2.35 msec in W, respectively. A statistically significant correlation was proved between the water content and the proton relaxation time (either T1 and T2).(ABSTRACT TRUNCATED AT 250 WORDS)     

NF-kappaB and IkappaBalpha expression following traumatic brain injury to the immature rat brain

NF-kappaB is one of the most important modulators of stress and inflammatory gene expression in the nervous system. In the adult brain, NF-kappaB upregulation has been demonstrated in neurons and glial cells in response to experimental injury and neuropathological disorders, where it has been related to both neurodegenerative and neuroprotective activities. Accordingly, the aim of this study was to evaluate the cellular and temporal patterns of NF-kappaB activation and the expression of its endogenous inhibitor IkappaBalpha following traumatic brain injury (TBI) during the early postnatal weeks, when the brain presents elevated levels of plasticity and neuroprotection. Our results showed that cortical trauma to the 9-day-old rat brain induced a very fast upregulation of NF-kappaB, which was maximal within the first 24 hours after injury. NF-kappaB was mainly observed in neuronal cells of the degenerating cortex as well as in astrocytes located in the corpus callosum adjacent to the injury, where a pulse-like pattern of microglial NF-kappaB activation was also found. In addition, astrocytes of the corpus callosum, and microglial cells to a lower extent, also showed de novo expression of IkappaBalpha within the time of NF-kappaB activation. This study suggests an important role of NF-kappaB activation in the early mechanisms of neuronal death or survival, as well as in the development of the glial and inflammatory responses following traumatic injury to the immature rat brain.     

Transient hypoxia/hypoglycemia upregulates endothelin B receptors in cultured rat astrocytes

Endothelins are potent vasoactive peptides that bind to their specific receptors, playing an important role in the CNS under physiological and pathophysiological conditions. Astrocytes, which have been shown to express these receptors, also have a considerable role to play under physiological and pathophysiological conditions, particularly those involved in delayed neuronal death. We carried out in vitro receptor autoradiographic binding experiments using specific ligands for endothelin receptors on cultured rat astrocytes. On astrocytes, the specific binding sites for (125)I-PD151242 (a selective endothelin A receptor antagonist) and (125)I-IRL 1620 (a selective endothelin B receptor agonist) were detected. We also characterized the qualitative and quantitative changes of endothelin receptors 24 h after subjecting cultured rat astrocytes to a transient 4-h hypoxia/hypoglycemia insult, used as a model of delayed neuronal death. After transient hypoxia/hypoglycemia, the number of endothelin B receptors increased significantly on cultured astrocytes, but this did not occur among the endothelin A receptors. These findings suggest that the astrocytic effects associated with endothelin in delayed neuronal death include gliosis or the repair process or both, manifested primarily by an increase in the number of endothelin B receptors. This rise does not require interaction with other types of CNS cells. Endothelin A receptors might have a role taking their number into consideration on rat astrocytes under both physiological and pathophysiological conditions.     

Progestin receptors in the developing rat brain and pituitary

Repeated application of electrical stimulation to the amygdala at a frequency of 3 Hz resulted in the development of afterdischarge and behavioral seizures. The rate of low-frequency kindling was faster than that of kindling with conventional 60 Hz stimulation, but the form of the seizures kindled with the two frequencies of stimulation was identical. Low-frequency kindling was obtained only when pulses of sufficient duration and intensity were administered.     

MK-801 upregulates the expression of d-amino acid oxidase mRNA in rat brain

We have evaluated the effect of the systemic administration of MK-801 (0.4 mg/kg) on the gene expression of D-amino acid oxidase (DAO) in several brain areas of the rat. The levels of DAO mRNA in all the brain areas significantly increased and peaked at 4 h after the administration. The present results suggest that there is a link between the expression of DAO mRNA and the N-methyl-D-aspartate (NMDA) receptor activity.     

Perinatal exposure to environmental tobacco smoke upregulates nicotinic cholinergic receptors in monkey brain

In humans, perinatal exposure to environmental tobacco smoke (ETS) is associated with neurobehavioral deficits. In the current study, we exposed Rhesus monkeys to ETS in late gestation and in the early neonatal period, and examined changes in neurotransmitter receptors in the brainstem and caudal portion of the cerebral cortex. Nicotinic acetylcholine receptors were markedly upregulated and the effect was selective in that there were no changes in m(2)-muscarinic acetylcholine receptors or in beta-adrenergic receptors. Nicotinic receptor upregulation is indicative of chronic cell stimulation by nicotine, and is a hallmark of nicotine-induced neuroteratogenesis. These results indicate that perinatal ETS exposes the fetus and neonate to quantities of nicotine that are sufficient to alter brain development.