Hypoxia preferentially destroys GABAergic neurons in developing rat neocortex explants in culture

The hypothesis that hypoxic ischemia before or during the human birth process preferentially destroys GABAergic nerve cells, particularly in the neocortex, was tested in a tissue culture model system. To that end, rat neocortex explants dissected from 6-day-old rat pups and cultured to a developmental stage approximately comparable to that of the newborn human neocortex were exposed to hypoxia for different periods. Quantitative light microscopic and immunocytochemical evaluation of the cultures demonstrated that GABAergic neurons were indeed the first neurons to die during hypoxia.     

Demonstration of long-range GABAergic connections distributed throughout the mouse neocortex

Gamma-aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67-green fluorescent protein (GFP) knock-in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP-positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long-range projections by tracing GFP-labeled GABAergic neurons with axon branches traveled rostro-caudally and medio-laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network.     

Permanent increase of the GAD67/synaptophysin ratio in rat cerebral cortex nerve endings as a result of hypoxic ischemic encephalopathy sustained in early postnatal life: a confocal laser scanning microscopic study

The aim of this study was to investigate whether perinatal hypoxia-ischemia preferentially destroys GABAergic nerve endings in rat cerebral cortex tissue which, in its turn, could then account for the reported higher risk of developing epilepsy later in life. To that end rat pups, with an age of 12–13 days postnatally, were unilaterally to hypoxic-ischemic conditions. After a survival period of to 2 to 6 months, the animals were sacrificed by perfusion fixation and their brains were used for cutting transversal vibratome and frozen sections. These sections were double-stained with primary antibodies against one of the two GABA synthesizing enzymes, glutamic acid decarboxylase with a mol. wt. of 66,600 (GAD₆₇) and one of the intrinsic membrane proteins synaptic vesicles, synaptophysin, followed by fluorophore-conjugated second antibodies. By using the confocal laser scanning microscope, we determined the ratio between the amount of GAD₆₇/synaptophysin immunofluorescence in nerve endings per unit volume of tissue in the hypoxia-damaged neocortex. It turned out that this ratio, contrary to expectations, was significantly higher in the hypoxia-damaged cortical areas than in matched areas on the contralateral side. It appeared, moreover, that this effect was directly proportional to the severity of the incurred damage. The conclusion was drawn that these observations do not support the hypothesis that perinatal hypoxia-ischemia ultimately leads to a preferential loss of GABAergic nerve endings in the damaged neocortex and, as such, to a shortage of inhibition.     

Cholinergic systems of the rat brain and neuronal reorganization under conditions of acute hypoxia

We studied the role of cholinergic systems of the rat brain in the mechanisms of acute response of the neocortex and hippocampus to acute hypobaric hypoxia. The activity of choline acetyltransferase and Na/K-ATPase and the protein content were measured in subfractions of synaptic membranes and the synaptoplasm of “light” and “heavy” synaptosomes. The same biochemical indices were measured in fractions of light and heavy synaptosomes from the nucleus of the solitary tract of the medulla oblongata. In the neocortex, we analyzed the ultrastructure of the synaptic pool. We found active involvement of cholinergic systems in the response to acute hypoxia in all brain structures studied. The synapses of cholinergic projection neurons from the basal nuclei of the frontal cortex (light fractions of synaptosomes), neocortical interneurons (heavy fractions of synaptosomes), and, presumably, GABAergic interneurons of the hippocampus (heavy fraction) were the most sensitive to hypoxia. At the limit of resistance to hypoxia, increased metabolic activity predominated in the synapses of rats that had poorly resistance to hypoxia, while in highly resistant rats this activity was diminished. This difference is related to the different duration of their exposure to critical altitude. We found a reduction in the number of synapses and staging in the development of the functional response of synapses to acute hypoxia. We propose a mechanism for the structural and functional reorganization of synaptic links during acute hypoxia, which is general for both poorly and highly resistant rats. We also hypothesize that a reduction of synapses is one of early triggers that switches neurons to levels of interaction that are adequate for hypoxia.     

The preconditioning modified neuronal expression of apoptosis-related proteins of Bcl-2 superfamily following severe hypobaric hypoxia in rats

The patterns of expression of the Bcl-2, Bax, and Bcl-xL proteins were examined immunocytochemically in rat hippocampus and neocortex after severe hypobaric hypoxia (180 Torr for 3 h) and severe hypoxia preconditioned by intermittent mild hypoxia (360 Torr for 2 h daily, for 3 consecutive days, 24 h prior to severe hypoxia). As revealed by TUNEL assay, severe hypobaric hypoxia produced extensive apoptotic damage to the neurons of hippocampal CA1-CA4 and the neocortex but not the dentate gyrus granule cells. Remarkable posthypoxic up-regulation of Bax expression maximal at 24 h was detected in the CA1-CA4 areas of hippocampus and neocortex 3-72 h after severe hypoxia. The preconditioning to severe hypoxia protected neurons from the posthypoxic apoptotic transformations, the up-regulation of Bax expression, and resulted in persistent overexpression of Bcl-2 and Bcl-xL. We conclude that the protective action of hypoxic preconditioning is at least in part mediated by shifting of neuronal Bax/Bcl-2-Bcl-xL ratio to a favor of antiapoptotic proteins Bcl-2 and Bcl-xL.     

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.     

GABAergic neocortical neurons are resistant to NMDA receptor-mediated injury

N-methyl-D-aspartate (NMDA) receptors are thought to mediate much of the central neuronal loss produced by certain neurologic insults, including hypoxia-ischemia, hypoglycemia, and trauma. Therefore, the specific vulnerability of GABAergic inhibitory neurons to NMDA receptor-mediated toxicity might be an important determinant of the potential for epileptogenesis following these insults. We have examined the fate of GABAergic cortical neurons in mouse cell cultured neuronal population) were identified either by immunoreactivity with antisera to GABA or by autoradiography following high-affinity uptake of 3H-GABA. Cultures exposed for 5 min to 20 to 750 microM NMDA showed NMDA concentration-dependent, widespread neuronal loss. However, GABAergic neurons were relatively spared, and thus represented an enhanced fraction of neuronal survivors. These observations suggest that GABAergic cortical neurons may possess some intrinsic resistance to NMDA receptor-mediated neurotoxicity, a property which might convey an anticonvulsant "inhibitory safety factor" to neocortex against certain forms of injury.     

Multiple Mild Hypobaric Hypoxia Induces Expression of Thioredoxin-1 in the Hippocampus and Neocortex of Rats

It is known that mild hypobaric hypoxia preconditioning may prevent brain damage induced by ischemic stroke and other forms of hypoxia. Activation of the antioxidant systems is one of the key molecular mechanisms that protect cells from oxidative stress during and after severe hypoxia/ischemia. Specifically, the cytoplasmic protein antioxidant thioredoxin-1 efficiently protects neurons from oxidative stress and hypoxia-induced damage. It has been previously shown that preconditioning with triple mild hypobaric hypoxia treatment significantly increased the expression of some endogenous antioxidants, including thioredoxin- 1, in the rat brain after subsequent severe hypoxia. This increase correlated with prevention or at least substantial amelioration of behavioral disturbances and structural brain damage. However, triple preconditioning per se, that is, without or prior to beginning of the subsequent severe hypoxia, did not increase but even significantly decreased the expression of thioredoxin-1 and some other antioxidants. In the present study we estimated the effects of a six-time mild hypobaric hypoxia on the expression of thioredoxin-1 in various regions of the hippocampus and neocortex of rats. Using immunocytochemistry, we demonstrate that six-time treatment with mild hypoxia significantly increases the thioredoxin-1 expression in neurons of the hippocampus and neocortex of rats 24 h after the last hypoxia treatment.     

Preferential loss of GABAergic neurons in hypoxia-exposed neocortex slab cultures is attenuated by the NMDA receptor blocker D-2-amino-7-phosphonoheptanoate

The preferential loss of GABAergic neurons in neocortex slab cultures after exposure to hypoxia was reinvestigated and confirmed using an improved morphometric analysis based on stereological principles. This preferential cell loss could be attenuated by treating the hypoxic slab cultures with the N-methyl-D-aspartate (NMDA) receptor blocker D-2-amino-7-phosphonoheptanoate (D-APH).     

Alterations in Tau Phosphorylation in Rat and Human Neocortical Brain Slices Following Hypoxia and Glucose Deprivation

Tau is a microtubule-associated protein which is regulated by phosphorylation. Highly phosphorylated tau does not bind microtubules and is the main component of the paired helical filaments seen in Alzheimer's and related neurodegenerative diseases. Recent reports suggested that patterns of tau phosphorylation changed following ischemia and/or reperfusionin vivo.We used anin vitromodel employing rat and human neocortical slices to investigate changes in tau phosphorylation which accompany oxygen and glucose deprivation. Western blotting with polyclonal and phosphorylation-sensitive Tau-1 monoclonal antisera was used to monitor changes in tau which accompanied conditions of oxygen and glucose deprivation and reestablishment of these nutrients.In vitrohypoglycemia/hypoxia caused tau to undergo significant dephosphorylation in both rat and human neocortical slices after 30 and 60 min of deprivation. This dephosphorylation was confirmed using immunoprecipitation experiments after radiolabeling tau and other proteins with³²Pi. Okadaic acid, a phosphatase inhibitor, was able to prevent tau dephosphorylation in both control and ischemic slices. Lubeluzole, a benzothiazole derivative within vivoneuroprotective activity, did not significantly alter patterns of tau phosphorylation. Restoration of oxygen and glucose following varied periods ofin vitrohypoxia/hypoglycemia (15–60 min) led to an apparent recovery in phosphorylated tau. These data suggest that tau undergoes a rapid, but reversible dephosphorylation following brief periods ofin vitrohypoxia/hypoglycemia in brain slices and that changes in tau phosphorylation help determine the extent of recovery following oxygen and glucose deprivation.     

Cellular factors controlling neuronal vulnerability in the brain: a lesson from the striatum

In many acute and chronic neurologic disorders, both deficits in energy metabolism and defects in glutamate-mediated excitatory synaptic transmission have been proposed as important pathogenic factors. Brain cells, however, express variable vulnerability to these insults, as indicated by the fact that certain brain areas and even different cell types in the same area are preferentially spared until the very late stages of various diseases. This can be clearly seen in the striatum, where GABAergic projection cells but not cholinergic interneurons are precociously damaged in the course of both acute metabolic insults (such as hypoxia, hypoglycemia, and ischemia) and chronic neurodegenerative disorders (such as Huntington's disease). A well-mapped pattern of cell loss, in fact, is a common finding in the striatum of patients suffering from these pathologic conditions. Physiologic and molecular studies have been directed in recent years to the identification of the cellular mechanisms underlying the cell-type specific vulnerability of striatal cells. These studied recognized that, in striatal spiny and aspiny cells, specific membrane ion channels, glutamate receptor subtypes and subunits, and intracellular enzymatic activities are involved in the cascade of events responsible for opposite responses and vulnerabilities to oxygen or glucose deprivation and to glutamate receptor-mediated toxicity. Specific molecules able to target these cellular factors might be employed as therapeutic agents during acute and chronic neurologic disorders affecting this brain area.     

Amelioration of hypoxic and hypoglycemic damage to cerebral endothelial cells

Induction of the 70 kDa heat shock protein (HSP70) by hypoxia and/or hypoglycemia and the effects of prior heat shock on injury owing to hypoxia and/or hypoglycemia were studied in rat cerebral endothelial cells. Hypoxia and/or hypoglycemia treatment resulted in increased expression of HSP70 only when such treatment was sufficient to cause detectable injury and when the initial treatment was followed by exposure of the cells to 24 h of normoxia and normoglycemia. Heat shock induced 24 h prior to treatment with 48 h of hypoxia slightly reduced endothelial cell damage as measured by fraction of lactate dehydrogenase release (10% decrease in injury). There was a more dramatic effect of prior heat shock on the moderate damage produced by 12 h of combined hypoxia and hypoglycemia (45% decrease), whereas the severe damage produced by 24 h of hypoxia and hypoglycemia was decreased by prior heat shock by only 16%. These results indicate that the hypoxia and hypoglycemia occurring in conjunction with ischemia are more likely to result in heat shock protein expression when there is injury to the tissue. Furthermore, heat shock protects cerebral endothelial cells from hypoxia and hypoglycemia either by slowing the initial development of injury or by delaying the onset of injury.     

Resveratrol inhibits interleukin-6 production in cortical mixed glial cells under hypoxia/hypoglycemia followed by reoxygenation

Reactive oxygen intermediates (ROIs) are important mediators of a variety of pathological processes, including inflammation and ischemia/reperfusion injury. Cytokines and chemokines are detected at mRNA level in human and animal ischemic brains. This suggests that hypoxia/reoxygenation may induce cytokine production through generation of ROIs. In this study, we investigated the cytokine induction and inhibition by antioxidants in rat cortical mixed glial cells exposed to in vitro ischemia-like insults (hypoxia plus glucose deprivation). The results showed that interleukin-6 (IL-6) mRNA and protein, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), were induced during hypoxia/hypoglycemia followed by reoxygenation in the mixed glial cells. The accumulation of IL-6 mRNA was induced as early as 15 min after hypoxia/hypoglycemia and its level was further increased after subsequent reoxygenation. Among the antioxidants studied, only resveratrol suppressed IL-6 gene expression and protein secretion in mixed glial cultures under hypoxia/hypoglycemia followed by reoxygenation. These findings suggest that resveratrol might be useful in treating ischemic-induced inflammatory processes in stroke.     

The Effects of Severe Hypoxia and Hypoxic Postconditioning on the Glutathione-Dependent Antioxidant System of the Rat Brain

In this study we investigated the effects of severe hypobaric hypoxia (SH) and severe hypobaric hypoxia accompanied by postconditioning using mild hypobaric hypoxia (PostC) on the glutathione-dependent antioxidant system in the rat hippocampus and neocortex. SH (3 h, 180 mmHg, 5% O₂) led to oxidative stress that was associated with a decrease in the total glutathione level, as well as in antioxidant capacity. PostC (2 h, 360 mmHg, 10% O₂) led to incomplete recovery of the total glutathione level and up-regulated glutathione peroxidase activity. In the neocortex, SH did not lead to the development of posthypoxic pathology. A small decrease in total glutathione, glutathione peroxidase activity, and antioxidant capacity on the 1st day after SH was corrected by the 2nd day. In contrast, glutathione reductase activity decreased by the 4th day after exposure to SH. PostC led to a consistent decrease in the total glutathione level but normalized glutathione reductase activity. We found that the studied brain structures develop a specific response to SH. In the hippocampus, SH led to oxidative stress, whereas the neocortex was not affected by exposure to SH. Partial differences between brain areas are based on better antioxidant defense of the neocortex in comparison with the hippocampus. PostC corrects posthypoxic pathology in the hippocampus with involvement of the glutathione- dependent antioxidant system. In the neocortex, PostC did not lead to a significant biochemical response.     

Phorbol ester alters the electrophysiological responses to hypoxia and ischemic-like conditions in the rat hippocampal slice

The effect of incubation with the protein kinase C activator, 4β-phorbol 12,13-dibutyrate (β-PDBu) on the electrophysiological responses to hypoxia and combined hypoxia and hypoglycemia was investigated in the rat hippocampal slice. Preincubation with β-PDBu prevents adenosine-mediated inhibition of synaptic transmission under normoxic, normoglycemic conditions. β-PDBu preincubation also reduces the adenosine-mediated hypoxia-induced depression of synaptic transmission revealing a substantial adenosine-independent hypoxia-induced depression of synaptic transmission. During combined hypoxia and hypoglycemia, slices preincubated in β-PDBu display a significant shortening of the time to anoxic depolarization, an effect of β-PDBu that is not mimicked by application of the adenosine antagonist cyclopentyltheophylline (8-CPT). It is concluded that the state of PKC activation may influence the electrophysiological responses to hypoxia and ischemia.     

Rat cultured astrocytes release guanine-based purines in basal conditions and after hypoxia/hypoglycemia

Brain ischemia stimulates release from astrocytes of adenine-based purines, particularly adenosine, which is neuroprotective. Guanosine, which has trophic properties that may aid recovery following neurological damage, is present in high local concentrations for several days after focal cerebral ischemia. We investigated whether guanine-based purines, like their adenine-based counterparts, were released from astrocytes and whether their release increased following hypoxia/hypoglycemia. HPLC analysis of culture medium of rat astrocytes showed spontaneous release of endogenous guanine-based purines at a higher rate than their adenine-based counterparts. The concentration of guanosine (≈120 nM) and adenosine (≈43 nM) in the culture medium remained constant, whereas concentrations of adenine and guanine nucleotides, particularly GMP, and their metabolites increased with time. Exposure of the cultures to hypoxia/hypoglycemia for 30 min increased the extracellular concentration of adenine-based purines by 2.5-fold and of guanine-based purines by 3.5-fold. Following hypoxia/hypoglycemia extracellular adenine nucleotide levels increased further. Adenosine concentration increased, but not proportionally to nucleotide levels. Accumulation of adenosine metabolites indicated it was rapidly metabolized. Conversely, the concentrations of extracellular guanine-based nucleotides remained elevated and the concentration of guanosine continued to increase. These data indicate that astrocytes are a major source of guanine-based purines, the release of which is markedly increased following hypoxia/hypoglycemia, permitting them to exert neurotrophic effects. GLIA 25:93–98, 1999. © 1999 Wiley-Liss, Inc.     

Central cholinergic systems in the mechanisms of hypoxic preconditioning: Diverse pathways of synaptic reorganization in vivo

We studied the effects of single moderate hypobaric hypoxia (HBH, 10% O2, 60 min) on the central cholinergic systems. For this purpose, we determined the activity of the marker of cholinergic neurons choline acetyltransferase (ChAT) and the protein content in subfractions of synaptic membranes and synaptoplasm, which were isolated from the “light” and “heavy” synaptosomes of the pons varolii and medulla oblondata (“pontomedulla”), neocortex, and hippocampus. Experiments were performed with intact rats and rats with low and high resistance to hypoxia that first underwent severe hypobaric hypoxia (4% O₂). We found that HBH influenced the synaptic pool of the pontomedulla in all groups of rats, the neocortex in groups of intact and highly resistant rats, and the hippocampus in neither group. HBH affected cytosolic and membrane-bound ChAT and proteins; the changes depended on the group of rats. We reviewed the role of cytosolic and membrane-bound ChAT in the regulation of exchange and secretion of acetylcholine in vitro. We show that in vivo acute adaptation (1) in the intact brain via transformation of cholinergic synaptic pool, which is associated with the formation and activation of contacts of the hypoxia-resistant morphological type; (2) occurs via inhibition of cholinergic activity in different populations of synapses and reduction of non-cholinergic, presumably GABAergic, populations in the brains of poorly and highly resistant rats. Our study demonstrates (1) numerous varieties of plastic possibilities of the brain and (2) one of natural mechanisms of hypoxic preconditioning of the intact brain, viz., the formation and activation of morphological hypoxia-resistant cholinergic synapses in the pontomedulla and neocortex.     

Loss of calbindin-immunoreactivity in CA1 hippocampal stratum radiatum and stratum lacunosum-moleculare interneurons in the aged rat

Alterations in hippocampal circuitry may underly age-related learning and memory impairment. We showed in a previous study that the GABA_B-mediated slow inhibitory postsynaptic potential (IPSP) induced in CA1 pyramidal neurons by electrical stimulation of stratum radiatum, is depressed in the hippocampus of the aged rat. This could be due to alterations in GABAergic interneuron functions. We report in this study that the number of hippocampal calbindin-immunoreactive (CaBP-IR) GABAergic interneurons is decreased in the aged rat. The mean number of CaBP-IR interneurons per slice decreases by 50% in the aged rat. The most severe loss was observed in the stratum radiatum of CA1 (78%), with a less consistent loss of immunoreactivity in CA3 (35%). In contrast, the mean number of interneurons containing parvalbumin (PV), was not significantly decreased in the aged rat. Our results show a loss of CaBP immunoreactivity in a population of GABAergic interneurons, which might be related to an altered function of these interneurons and consequently of GABAergic synaptic transmission in the aged rat. In contrast, PV immunoreactivity in interneurons located close to the pyramidal layer does not decrease in the hippocampus of the aged rat.     

Epileptiform activity preferentially arises outside tumor invasion zone in glioma xenotransplants

Seizures occur commonly with brain tumors. The underlying mechanisms are not understood. We analyzed network and cellular excitability changes in tumor-invaded and sham neocortical tissue in vitro using a rat glioblastoma model. Rat C6 glioma cells were transplanted into rat neocortex, yielding diffusely invading gliomas resembling human glioblastomas. We hypothesized that network excitability would increase in regions neighboring the tumor, and that initiation of epileptic discharges might be correlated to a higher density of intrinsically bursting neurones. Voltage-sensitive dye imaging revealed epileptic activity to be initiated in paratumoral zones (1-2 mm from main tumor mass), in contrast to control tissue, where epileptic foci appeared randomly throughout the neocortex. Neuronal firing patterns revealed significantly more intrinsically bursting neurones within these initiation zones than in regions directly adjacent to the tumor or in control tissue. We conclude that gliomas are associated with a higher density of intrinsically bursting neurones, and that these may preferentially initiate epileptiform events.