Search PubMed⌕ Search

Biomedical subjects

G G Haddad

Publications and source records attributed to G G Haddad.

At least 37 records · Page 2Linked to original sources
PubMed2000

delta-, but not mu- and kappa-, opioid receptor activation protects neocortical neurons from glutamate-induced excitotoxic injury.

Recent observations from our laboratory have led us to hypothesize that delta-opioid receptors may play a role in neuronal protection against hypoxic/ischemic or glutamate excitotocity. To test our hypothesis in this work, we used two independent methods, i.e., "same field quantification" of morphologic criteria and a biochemical assay of lactate dehydrogenase (LDH) release (an index of cellular injury). We used neuronal cultures from rat neocortex and studied whether (1) glutamate induces neuronal injury as a function of age and (2) activation of opioid receptors (delta, mu and kappa subtypes) protects neurons from glutamate-induced injury. Our results show that glutamate induced neuronal injury and cell death and this was dependent on glutamate concentration, exposure period and days in culture. At 4 days, glutamate (up to 10 mM, 4 h-exposure) did not cause apparent injury. After 8-10 days in culture, neurons exposed to a much lower dose of glutamate (100 microM, 4 h) showed substantial neuronal injury as assessed by morphologic criteria (>65%, n=23, P<0.01) and LDH release (n=16, P<0. 001). Activation of delta-opioid receptors with 10 microM DADLE reduced glutamate-induced injury by almost half as assessed by the same criteria (morphologic criteria, n=21, P<0.01; LDH release, n=16, P<0.01). Naltrindole (10 microM), a delta-opioid receptor antagonist, completely blocked the DADLE protective effect. Administration of mu- and kappa-opioid receptor agonists (DAMGO and U50488H respectively, 5-10 microM) did not induce appreciable neuroprotection. Also, mu- or kappa-opioid receptor antagonists had no appreciable effect on the glutamate-induced injury. This study demonstrates that activation of neuronal delta-opioid receptors, but not mu- and kappa-opioid receptors, protect neocortical neurons from glutamate excitotoxicity.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2000

Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.

We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed2000

Increased neuronal excitability after long-term O(2) deprivation is mediated mainly by sodium channels.

We have previously observed that prolonged O(2) deprivation alters membrane protein expression and membrane properties in the central nervous system. In this work, we studied the effect of prolonged O(2) deprivation on the electrical activity of rat cortical and hippocampal neurons during postnatal development and its relationship to Na(+) channels. Rats were raised in low O(2) environment (inspired O(2) concentration = 9.5+/-0.5%) for 3-4 weeks, starting at an early age (2-3 days old). Using electrophysiologic recordings in brain slices, RNA analysis (northern and slot blots) and saxitoxin (a specific ligand for Na(+) channels) binding autoradiography, we addressed two questions: (1) does long-term O(2) deprivation alter neuronal excitability in the neocortical and hippocampal neurons during postnatal development? and (2) if so, what are the main mechanisms responsible for the change in excitability in the exposed brain? Our results show that (i) baseline membrane properties of cortical and hippocampal CA1 neurons from rats chronically exposed to hypoxia were not substantially different from those of naive neurons; (ii) acute stress (e.g., hypoxia) elicited a markedly exaggerated response in the exposed neurons as compared to naive ones; (iii) chronic hypoxia tended to increase Na(+) channel mRNA and saxitoxin binding density in the cortex and hippocampus as compared to control ones; and (iv) the enhanced neuronal response to acute hypoxia in the exposed cortical and CA1 neurons was considerably attenuated by applying tetrodotoxin, a voltage-sensitive Na(+) channel blocker, in a dose-dependent manner. We conclude that prolonged O(2) deprivation can lead to major electrophysiological disturbances, especially when exposed neurons are stressed acutely, which renders the chronically exposed neurons more vulnerable to subsequent micro-environmental stress. We suggest that this Na(+) channel-related over-excitability is likely to constitute a molecular mechanism for some neurological sequelae, such as epilepsy, resulting from perinatal hypoxic encephalopathy.

Animals↗
PubMed2000

Mechanisms underlying hypoxia-induced neuronal apoptosis.

In vivo models of cerebral hypoxia-ischemia have shown that neuronal death may occur via necrosis or apoptosis. Necrosis is, in general, a rapidly occurring form of cell death that has been attributed, in part, to alterations in ionic homeostasis. In contrast, apoptosis is a delayed form of cell death that occurs as the result of activation of a genetic program. In the past decade, we have learned considerably about the mechanisms underlying apoptotic neuronal death following cerebral hypoxia-ischemia. With this growth in knowledge, we are coming to the realization that apoptosis and necrosis, although morphologically distinct, are likely part of a continuum of cell death with similar operative mechanisms. For example, following hypoxia-ischemia, excitatory amino acid release and alterations in ionic homeostasis contribute to both necrotic and apoptotic neuronal death. However, apoptosis is distinguished from necrosis in that gene activation is the predominant mechanism regulating cell survival. Following hypoxic-ischemic episodes in the brain, genes that promote as well as inhibit apoptosis are activated. It is the balance in the expression of pro- and anti-apoptotic genes that likely determines the fate of neurons exposed to hypoxia. The balance in expression of pro- and anti-apoptotic genes may also account for the regional differences in vulnerability to hypoxic insults. In this review, we will examine the known mechanisms underlying apoptosis in neurons exposed to hypoxia and hypoxia-ischemia.

Animals↗
PubMed2000

Enhancing our understanding of the molecular responses to hypoxia in mammals using Drosophila melanogaster.

Drosophila melanogaster has been used as a genetic model, especially in the past decade, to examine normative biological processes and disease conditions very effectively. These span a wide range of major issues such as aging, cancer, embryogenesis, neural development, apoptosis, and alcohol intoxication. Here, we detail how the Drosophila melanogaster can be used as a genetic model to study the molecular and genetic underpinnings of the response to hypoxia. In our study of the basis of anoxia tolerance, one of the potent approaches that we use is a mutagenesis screen to identify loss-of-function mutants that are anoxia sensitive. The major advantage of this approach is that it is not biased for any particular gene or gene product. Although our screen is in progress, we already have evidence that this approach is useful.

Animals↗
PubMed2000

Effect of extracellular HCO(3)(-) on Na(+) channel characteristics in hippocampal CA1 neurons.

The effect of HCO(3)(-)/CO(2) on membrane properties of isolated hippocampal CA1 neurons was studied with the use of the whole cell configuration of the patch-clamp technique. Neurons were acutely dissociated from 21- to 30-day-old mice. In the current-clamp mode, HCO(3)(-)/CO(2) significantly hyperpolarized CA1 neurons by more than 10 mV and decreased their input resistance. In addition, the overall excitability of these neurons was lower in the presence of HCO(3)(-)/CO(2) than in HEPES. Spontaneous and evoked action potential firing frequency was lower in the presence of HCO(3)(-)/CO(2) than in its absence. In the voltage-clamp mode, both activation and steady-state inactivation of a fast Na(+) current were shifted in the hyperpolarized direction in such a way that the window currents were smaller in HCO(3)(-)/CO(2) than in HEPES. Recovery from inactivation and deactivation from the open state of the fast Na(+) current was slower in HCO(3)(-)/CO(2) than in HEPES. We conclude that HCO(3)(-)/CO(2) decreases the intrinsic excitability of CA1 neurons by altering not only the passive properties of the neuronal membranes but also by changing several characteristics of the fast Na(+) current, including activation and inactivation kinetics as well as the recovery from inactivation and deactivation.

Action Potentials↗
PubMed1999

Isolation and characterization of the hypoxia-inducible factor 1beta in Drosophila melanogaster.

The hypoxia-inducible factor 1 (HIF-1), a heterodimer composed of alpha and beta subunits, plays an important role in the cellular response to O(2) deprivation. In this paper, Drosophila HIF-1beta (dHIF-1beta) homolog is cloned and characterized. Further, Northern analyses showed that dHIF-1alpha and dHIF-1beta expressed their highest level at an embryonic stage. From the pupal stage on, their expression was sharply reduced and maintained at a steady level. Anoxia treatment up-regulated the expression of the both alpha and beta subunits. Over-expression of dHIF-1alpha in transgenic embryos resulted in embryonic lethality, while over-expression of dHIF-1beta significantly prolonged fly recovery time from a 5-min anoxic stupor. The cloning and characterization dHIF-1beta reported in this paper provide a framework for further genetic dissection of the HIF-1 complex in its role in the cellular or tissue response to O(2) deprivation.

Amino Acid Sequence↗
PubMed1999

Sodium homeostasis in rat hippocampal slices during oxygen and glucose deprivation: role of voltage-sensitive sodium channels.

The breakdown in brain ionic homeostasis during energy deprivation (anoxic depolarization [AD]) is intimately linked to neuronal injury. We studied the role of one particular route of Na+ influx, voltage-sensitive Na+ channels, in the AD induced by O2 and/or glucose deprivation. We recorded extracellular Na+ concentration ([Na+]e) and direct current potential (DCP) in the CA1 stratum pyramidale of hippocampal slices using Na+-selective microelectrodes. Tetrodotoxin (0.1-1 microM) delayed the occurrence of AD and reduced the peak change in both [Na+]e and DCP during AD. However the tetrodotoxin effects were overcome by a concomitant reduction in extracellular glucose during anoxia. We conclude that: (1) the activation of voltage-gated Na+ channels is involved in the triggering of AD; (2) there may be a critical level of energy depletion when AD occurs and different mechanisms may underlie AD during hypoxia, compared to O2 and glucose deprivation.

Animals↗
PubMed1999

Drosophila neurons respond differently to hypoxia and cyanide than rat neurons.

Compared with mammalian species, Drosophila melanogaster exhibits marked tolerance to hypoxia or anoxia. However, the underlying cellular mechanisms of tolerance are still largely unknown. In order to assess the electrophysiologic response to O(2) lack in Drosophila neurons and compare them to those in mammals, we used neurons from embryonic cultures of both Drosophila and rat. We studied the effects of hypoxia on membrane potential V(m), input resistance R(m), rheobase, and action potential characteristics before, during and after 3 to 5 min of hypoxia (measured PO(2)<20 Torr). In Drosophila neurons, on the one hand, V(m) reversibly hyperpolarized with hypoxia by an average of about 20 mV and input resistance decreased by 71% from control. In most cells studied, action potential (AP) amplitude decreased, its duration increased, and its threshold shifted in a hyperpolarized direction before AP generation was attenuated. On the other hand, V(m) in rat cortical neurons reversibly depolarized by an average of 10 mV with hypoxia. Input resistance was reversibly reduced by 58% and, in most cells studied, AP amplitude also decreased and its duration increased. In contrast to the effects of hypoxia on V(m), CN caused a depolarization by 22 mV and a slight increase in R(m) in Drosophila. In the rat, CN was similar to hypoxia in its effect on R(m). We conclude that (1) rat and Drosophila neurons decrease their excitability in hypoxia by activating different mechanisms; (2) the most likely explanation for the hyperpolarization and the decrease in R(m) in Drosophila neurons is the activation of a K(+) conductance; this activation, by itself, cannot explain the results in rat neurons and (3) hypoxia and cyanide have similar effects in rat neurons but are divergent in their effects in Drosophila neurons.

Animals↗
PubMed1999

bcl-2 prolongs neuronal survival during hypoxia-induced apoptosis.

In vivo models of hypoxic-ischemic brain injury have shown altered expression of a number of genes that are important in regulating neuronal survival. However, it is not clear as to whether hypoxia alone can alter the expression of genes regulating neuronal survival. We hypothesized that (1) hypoxia alone alters the expression of bcl-2 in neurons, (2) the severity and duration of hypoxia influence bcl-2 expression, and (3) the alteration of bcl-2 expression has an important role in regulating neuronal survival during hypoxia. Embryonic rat neocortical neurons cultured for 7-10 days were exposed to 0.1, 1, or 3% oxygen for various durations and were removed for analyses at 24-h intervals. Under all hypoxic conditions, neurons exhibited morphologic changes, as assessed by electron microscopy and Annexin V staining, consistent with apoptosis. Immunoblot and immunofluorescence analyses revealed an increase in neuronal bcl-2 protein during hypoxic exposure. Quantitative immunofluorescence analyses of bcl-2 immunostained neurons indicated that expression of bcl-2 was altered by the duration and severity of hypoxia. Attenuation of bcl-2 expression by antisense oligonucleotides decreased the proportion of surviving neurons by approximately 50% after 48 h of exposure to 0.1% oxygen. We conclude that observed increase in bcl-2, in part, plays an important role in neuronal survival during exposure to hypoxia.

Animals↗
PubMed1999

Gene regulation by O2 deprivation: an anoxia-regulated novel gene in Drosophila melanogaster.

Organisms, across the animal kingdom, vary in their tolerance or susceptibility to cell injury from O2 deprivation. In this study we have taken advantage of the genetically well studied fruit fly to dissect basic mechanisms underlying their ability to tolerate lack of O2. Using differential display and molecular techniques, we cloned and characterized a novel gene, named fau, which is up-regulated considerably following anoxia in Drosophila melanogaster. Northern blot analysis revealed that the transcript of this gene is approximately 0.9 kb in length with an open reading frame encoding a small hydrophilic protein ( approximately 14.4 kDa). This protein has no homology to previously described gene products but has many potential phosphorylation sites. In situ hybridization showed that this gene is located in region 7C-D on the Drosophila X-chromosome and its transcript concentrated in the lamina and cortical neurons of the Drosophila central nervous system (CNS). Transgenic flies showed that over-expression of fau significantly reduced the recovery time of the flies from anoxia. We conclude that (1) this study provided a framework on which the mechanisms underlying anoxia tolerance can be dissected in the fruit fly and (2) fau gene plays an important role in the regulation of tissue responsiveness to O2 deprivation.

Amino Acid Sequence↗
PubMed1999

Effect of prolonged O2 deprivation on Na+ channels: differential regulation in adult versus fetal rat brain.

Neuronal Na+ channels are functionally inhibited in the adult in response to acute O2 deprivation. Since prolonged hypoxia may not only affect channel function, but also its expression, we hypothesized that long-term hypoxia alters Na+ channel density. This alteration may depend on age, because we have found major differences in neuronal responses to hypoxia between the immature and adult. In the present work, we used northern blots, slot blots, saxitoxin binding and autoradiography to ask whether: (i) prolonged hypoxia alters Na+ channel messenger RNA and protein levels in the brain; (ii) there is a difference between the adult and prenatal brains regarding Na+ channel expression with hypoxic exposure; and (iii) regional differences in Na+ channel expression occur in hypoxia-exposed brains. Our results show the following. (1) Na+ channel messenger RNA and saxitoxin binding density decreased after prolonged hypoxia in adult brain homogenates; this is in sharp contrast to the changes observed in fetal brains, which tended to increase Na+ channel messenger RNA and protein after hypoxia. (2) Changes in saxitoxin binding density are related to alterations in the number of saxitoxin binding sites and not to binding affinity, since there was no major change in Kd values between the hypoxia and naive groups. (3) The hypoxia-induced Na+ channel expression was heterogeneous, with major differences between rostral regions (e.g., the cortex) and caudal regions (e.g., the medulla and pons). We speculate that down-regulation of Na+ channels during long-term hypoxia in mature brains is an adaptive cellular response, aimed at minimizing the mismatch between energy supply and demand, since maintenance of Na+ gradients is a major energy-requiring process. However, the prenatal brain does not depend on this adaptive mechanism in response to hypoxic stress.

Aging↗
PubMed1999

Use of genetic models in respiratory neurobiology and sleep.

In this review, two issues are highlighted: 1) the difficulties that can form major hurdles in trying to understand a disease or a fundamental biologic process at the genetic and molecular level and 2) the potential opportunities that genetic models such as the Drosophila or c-elegans can provide in answering clinically or biologically relevant questions. This review also lists in some detail the areas in which these models have been helpful and successes have been scored. For such models to be used, however, requires the "dissection" of a biologic or a disease process into a tractable phenotype that can be assayed in a genetic model and have relevant and interpretable conclusions. The hope is that questions pertaining to sleep, arousal, respiratory neurobiology, and their disorders can be formulated in such a way to be addressed in models that can lend themselves to very exciting discoveries.

Animals↗
PubMed1999

Intracellular pH regulation of CA1 neurons in Na(+)/H(+) isoform 1 mutant mice.

To understand the role of Na(+)/H(+) exchanger 1 (NHE1) in intracellular pH (pH(i)) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pH(i), using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO(2)/HCO(3)(-), steady-state pH(i) was higher in WT neurons than in mutant neurons. Using the NH(4)Cl prepulse technique, we also show that H(+) flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na(+) from the extracellular solution or by using 100 microM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO(2)/HCO(3)(-), the difference in H(+) flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 microM/s between them at pH 6.6. H(+) flux in CO(2)/HCO(3)(-) was responsive to diisothiocyanato-stilbene-2, 2'-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pH(i) and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO(3)(-), indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO(3)(-)-dependent and -independent transporters in neurons.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

O(2) deprivation inhibits Ca(2+)-activated K(+) channels via cytosolic factors in mice neocortical neurons.

O(2) deprivation induces membrane depolarization in mammalian central neurons. It is possible that this anoxia-induced depolarization is partly mediated by an inhibition of K(+) channels. We therefore performed experiments using patch-clamp techniques and dissociated neurons from mice neocortex. Three types of K(+) channels were observed in both cell-attached and inside-out configurations, but only one of them was sensitive to lack of O(2). This O(2)-sensitive K(+) channel was identified as a large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), as it exhibited a large conductance of 210 pS under symmetrical K(+) (140 mM) conditions, a strong voltage-dependence of activation, and a marked sensitivity to Ca(2+). A low-O(2) medium (PO(2) = 10-20 mmHg) markedly inhibited this BK(Ca) channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O(2) deprivation on BK(Ca) channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pH(i)), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BK(Ca) channel activity by reducing the sensitivity of this channel to Ca(2+). In contrast, the reducing agents glutathione and DTT increased single BK(Ca) channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BK(Ca) is modulated by O(2) deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca(2+) sensitivity resulting from cytosolic alternations such as in pH(i) and phosphorylation. Because of their large conductance and prevalence in the neocortex, BK(Ca) channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.

Adenosine Triphosphate↗
PubMed1999

Neuroprotective interactions in rats between paclitaxel and cisplatin.

Paclitaxel and cisplatin are associated with dose-limiting neurotoxicity that may result from their differing effects on microtubule stability in peripheral nerves. We hypothesized that such different actions of paclitaxel and cisplatin could be exploited to minimize their neurotoxicity by giving them in combination. Paclitaxel (9-18 micromol/kg/week or 7.7-15.4 mg/kg/week) and cisplatin (5-10 micromol/kg/week or 1.5-3 mg/kg/week) were given alone and in combination to female Wistar rats. Treatment was given once per week for a total of 7-10 weeks. Paclitaxel and cisplatin were given 24 h apart when they were given in combination. Changes in sensory nerve conduction velocity (SNCV) and dorsal root ganglia (DRG) morphology were measured. The nature of their interaction was analyzed using an isobologram. Their antitumor activity alone or in combination was also determined in C57B1/6 mice bearing colon 38 tumors. Reductions in SNCV occurred with paclitaxel alone (P = 0.009), cisplatin alone (P = 0.012), and cisplatin given 24 h before paclitaxel (P < 0.0001). In contrast, there was no significant change in SNCV with paclitaxel given 24 h before cisplatin (P = 0.11). An isobologram showed that the SNCV effects of the drug combinations were less than additive or antagonistic. Cisplatin-induced morphometric changes in DRG neurons were less marked when cisplatin was given with paclitaxel (P = 0.004). Concentrations of platinum in dorsal root ganglia, sural nerves, and sciatic nerves were not altered by giving paclitaxel before cisplatin. Tumor growth delays (TGD) were greater after treatment with paclitaxel (23.4 micromol/kg or 20 mg/kg) given 24 h before cisplatin (23.3 micromol/kg or 7 mg/kg) (TGD = 7.5 days) than after paclitaxel (23.4 micromol/kg or 20 mg/kg) (TGD = 2.0 days) or cisplatin (23.3 micromol/kg or 7 mg/kg) (TGD = 3.5 days) alone. Paclitaxel and cisplatin antagonized each other's neurotoxicity in Wistar rats. Combining cytotoxic agents with opposing effects on peripheral nerves has potential for minimizing neurotoxicity in patients.

Animals↗
PubMed1998

Hypoxia-induced apoptosis: effect of hypoxic severity and role of p53 in neuronal cell death.

In various animal models of cerebral hypoxia-ischemia, it is not clear whether neuronal apoptosis results from hypoxia alone or whether other factors mediate this process. We hypothesized that (1) hypoxia alone can induce neuronal apoptosis, (2) hypoxic severity alters the time course of neuronal apoptosis, (3) hypoxia increases neuronal p53, and this increase in p53 is critical for neuronal apoptosis. Embryonic neocortical neurons cultured for 7-10 days were placed in an incubator with levels set at 0.1%, 1%, and 3% O2 and were removed at 24-h intervals for study. Under all hypoxic conditions, observed changes in cellular morphology and DNA fragmentation, detected by the TUNEL method and gel electrophoresis, were consistent with apoptosis. These alterations were seen after a shorter period with increasing hypoxic severity. Immunoblot analysis revealed an increase in p53 protein in hypoxia-exposed neurons. Analysis of immunofluorescence-stained neurons revealed increases in p53 with increased duration and severity of hypoxia. Antisense oligonucleotides for p53 significantly increased the number of surviving neurons during hypoxic exposure. We conclude that hypoxia-induced neuronal apoptosis is, in part, a p53-dependent process whose time course is influenced by hypoxic severity and duration.

Animals↗