Posthypoxic Period Research Articles

Posthypoxic disturbances in glutamatergic signal transduction in rat brain neurons: correction effect of preconditioning.

Severe 3-h hypobaric hypoxia was followed by impairment of Ca(2+)-mediated glutamatergic signal transduction in the posthypoxic period (no less than 72 h). This impairment manifested in changes in the calcium response to glutamate application in slices of rat brain cortex. Moderate hypoxic preconditioning prevented these disturbances developed over the first day after sever hypoxia.

Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells: H2O2 as a trigger and NF-kappaB as a signal transducer.

The haemorrhagic transformation in ischemic stroke involves disruption of the integrity of the microvascular beds, partially based on the action of matrix metalloproteinases (MMPs). The objective of the present study was to evaluate the contribution of microvascular endothelial cells from human brain (HBECs) to MMPs' expression and regulation under conditions relevant to brain ischemia. MMPs and their inhibitors were examined with zymography, Western-blotting, ELISA and MMP-activity assay in cultured HBECs. Four-hour hypoxia (pO(2)=60 mmHg) elevated the level of MMP-9 in the supernatant of the HBECs and this early response required collagen-matrix. Active oxygen species sustained the increased MMP-9 activity for at least 24 h. In the post-hypoxic period 20 micro mol/L H(2)O(2) caused a 6-fold increase in the specific activity of MMP-9 over the normoxic cells and a comparable effect was exerted by thrombin (50 nmol/L) and leukocyte elastase (10 nmol/L). The role of NF-kappaB, a redox-state sensitive transcription factor, was evaluated with immunofluorescence confocal microscopy and immunoblotting of nuclear and cytoplasmic extracts. The oxidative stress-dependent MMP-9 induction was accompanied by a significant increase in the NF-kappaB localized in the nuclei and these responses were blunted with a proteasome inhibitor (MG132). Consequently, according to our in vitro data HBECs are a source of MMP-9, which is under the control of triggers relevant to the ischemic/reperfused brain (reactive oxygen species, thrombus and inflammation related proteases) and this regulation is partially based on NF-kappaB activation. The reported regulation of endothelium-derived MMP-9 supports its potential involvement in the post-hypoxic disturbances of the cerebral microcirculation.

Mutant mice deficient in NOS-1 exhibit attenuated long-term facilitation and short-term potentiation in breathing.

The objective of the present study is to examine the potential role of nitric oxide (NO) in short-term potentiation (STP) and long-term facilitation (LTF) of breathing. Experiments were performed in wild-type (WT) and mutant mice deficient in nitric oxide synthase-1 (NOS-1), as well as in WT mice administered the NOS-1 inhibitor 7-nitroindazole (7-NI; 50 mg x kg(-1); I.P.). Respiratory responses following either single or recurrent episodes of hypoxia (7% O2, balance N2) were analysed in unanaesthetised animals by body plethysmography along with rate of O2 consumption (VO2)) and CO2 production (VCO2). After a single hypoxic challenge, respiration in WT mice remained elevated for 5 min, suggesting STP in ventilation. Following termination of three consecutive hypoxic challenges, respiration remained elevated during normoxia for as long as 30 min, indicating LTF in breathing under awake conditions. STP and LTF were significantly attenuated or absent in WT mice after 7-NI. A similar attenuation or absence of STP and LTF was also seen in NOS-1 mutant mice. Changes in VO2 and VCO2 were comparable among mice during the post-hypoxic period, suggesting that the absence of STP and LTF was not due to alterations in body metabolism. These results suggest endogenous NO is an important physiological modulator of ventilatory STP and LTF.

Mechanisms of changes in the erythroid hemopoietic stem during hypoxias of different severity.

Brain pathology (acute hypoxia and posthypoxic encephalopathy) is associated with less pronounced hyperplasia of the bone marrow erythroid stem (due to decreased count of proliferating committed precursors) and hemolytic anemia, while secretory activity of stromal cells of the hemopoiesis-inducing microenvironment is not impaired. Severe oxygen deficiency affects erythroid precursors and impairs production of functionally normal erythrocytes in the posthypoxic period.

Changes in the Activity of Cathepsin B in the Rat Brain Under Conditions of Hemic Hypoxia

We studied the effects of experimental hemic hypoxia of medium severity on the activity of lysosomal cathepsin B in different brain structures of the mother rats and their offspring. Hypoxia was evoked by daily i.p. injections of 60 μg/kg sodium nitrite into pregnant rat females (from the 5th to the 19th day of pregnancy). It resulted in noticeable changes in the activity of cathepsin B in the neocortex, cerebellum, midbrain, and pons Varolii of the “hypoxic” mother rats and their offspring. These changes differed in both the trend and the degree of expression depending on the duration of the post-hypoxic period.

Antioxidant defense and lipid peroxidation in rat tissue after hypobaric hypoxia

Posthypoxic period is characterized by increased formation of lipid peroxidation products. Changes in the antioxidant enzyme systems include decreased catalase and glutathione reductase activities and elevated content of reduced glutathione.

Expression of matrix-degrading enzymes in pulmonary vascular remodeling in the rat.

Exposure of rats to hypoxia causes pulmonary arterial remodeling, which is partly reversible after return to air. We hypothesized that degradation of excess collagen in remodeled pulmonary arteries in the posthypoxic period is mediated by endogenous matrix metalloproteinases (MMPs). Total proteolytic, collagenolytic, and gelatinolytic activities, levels of stromelysin-1 and tissue inhibitor of metalloprotease-1 (TIMP-1), and immunolocalization of stromelysin-1 in main pulmonary arteries were determined after exposure of rats to 10% O2 for 10 days followed by normoxia. We observed transient increases in total proteolytic, collagenolytic, and gelatinolytic activities and expression of approximately 72-, 68-, and 60-kDa gelatinases by zymography within 3 days of cessation of hypoxic exposure. The level of TIMP-1 increased as the stromelysin-1 level increased. Immunoreactive stromelysin-1 was localized predominantly in the luminal region of normal and hypertensive pulmonary arteries. These results are consistent with the notion that endogenous MMPs may mediate the breakdown of excess collagen in remodeled pulmonary arteries during the early posthypoxic period.

Effects of hypoxia and post-hypoxic recovery on chick retinal pigment epithelium potentials and light-evoked responses in vitro

Purpose. To determine the cellular mechanisms involved in the hypoxia-induced alteration of the retinal pigment epithelium (RPE) potentials and the light-evoked responses of the RPE in chicks. In addition, to determine the mechanisms involved in the recovery of the RPE during the post-hypoxic period.Methods. In vitro preparations of chick retina-RPE-choroid were studied by potassium-selective microelectrodes placed in the subretinal space. In addition, single-barrel microelectrodes were used to obtain intracellular recordings from the RPE cells. The perfusate was bubbled continuously with 95% oxygen and 5% carbon dioxide for the control condition and replaced by 95% nitrogen and 5% carbon dioxide to induce hypoxia.Results. Hypoxia induced a significant reduction of the trans-tissue potential which was found to result from the depolarization of the apical membrane of the RPE. This depolarization was induced by an increase of subretinal [K+]o. The c-wave was also markedly decreased or abolished during hypoxia. There were two phases of post-hypoxic recovery: an initial small increase in the trans-tissue potential resulting from a basal membrane depolarization followed by an apical membrane hyperpolarization. The trans-tissue potential and the c-wave also were supernormal in two phases during this post-hypoxic period. The c-wave amplitude was temporarily elevated (263.7 ± 77.4% of pre-hypoxic control) because of the enhanced trans-epithelial c-wave and without a light-evoked decrease in subretinal [K+]o.Conclusions. The trans-tissue potential and the c-wave were markedly decreased during hypoxia. During the post-hypoxic period, both potential recovered with transient supernormalities in two phases. The results suggested that the hypoxic changes resulted directly from changes of the RPE membranes and indirectly from a change in the subretinal [K+]o but were not mediated by the light-evoked decrease in subretinal [K+]o. Curr. Eye Res. 17: 384–391, 1998.

Metabolism and acid-base status during hypoxic ventilatory depression.

The ventilatory response to acute systemic hypoxia has been thought to be determined by the balance between hypoxic stimulation via peripheral chemoreceptors and hypoxic inhibition of the respiratory neurons. In moderate-severe hypoxia, the latter predominates the former resulting in ventilatory "depression" (HVD). However, ventilation relative to metabolic rate (V.O2) during HVD is "not depressed" but remains increased because of associated reduction in O2 uptake (V.O2). The experiment presented here was conducted to elucidate the changes in CO2 output (V.CO2) and acid-base status during hypoxia and their role in ventilatory regulation. Ventilation, metabolic rate (V.O2, V.CO2), acid-base status and blood lactate concentration were measured during and after inhalation of hypoxic gases in halothane-anesthetized and spontaneously breathing rats. The HVD occurred at FIO2 0.08 with increased blood lactate concentration, increased venous PCO2 and a large drop in venous pH without significant changes in arterial pH and PCO2. Furthermore, the amount of reduction in V.CO2 during HVD was much smaller than that of V.O2 and the V.CO2/V.O2 ratio increased. These findings suggest that CO2 output becomes relatively higher than O2 consumption in moderate-severe hypoxia. The possible origin of CO2 accumulation in the venous blood, such as the buffering of lactic acid by bicarbonate, and its role in ventilatory stimulation are discussed. Since there was no large increase in V.E and metabolic rate in the post-hypoxic period, "O2 debt" during HVD was small.

Effect of hypoxia on amniotic fluid erythropoietin levels in fetal rats.

Erythropoietin (EPO) levels in amniotic fluid and serum were measured in hypoxic (fraction of inspired oxygen, FiO2, 0.09) and posthypoxic (following a 24-hour period of hypoxia, FiO2 0.09) fetal rats on day 21 of gestation. Each of the study groups comprised 12-20 fetuses. Each of the control groups consisted of 21 or 22 fetuses. Fetal serum EPO levels at 3, 6, 9, 12, and 24 h of hypoxia were significantly higher than the control level. Amniotic fluid EPO levels at 9, 12, and 24 h of hypoxia were also significantly increased compared to the control level. Fetal serum EPO levels returned to baseline during the 12- to 48-hour period after hypoxia. During the 0- to 48-hour posthypoxic period, amniotic fluid EPO levels were significantly higher than the control level. These data demonstrate that rates of appearance and turnover of amniotic fluid EPO are different from those of fetal serum EPO.

Poor in vivo reproducibility of gastric intramucosal pH determined by saline-filled balloon tonometry

Purpose: The reproducibility of gastric intramucosal pH (pHi) determinations by saline-filled balloon tonometry has not been assessed adequately. We examined the agreement of pHi obtained using pairs of tonometry catheters under various conditions in a canine model of global hypoxia. Methods: Two gastric balloon tonometry catheters were inserted into each of 16 anesthetized dogs. Cimetidine was administered to six of the animals (group 1). Hypoxia was induced for 30 minutes by reducing the F lO 2 to 0.08. pHi was determined under basal conditions, at completion of hypoxia, and during the posthypoxic period. Results: The mean bias of the paired pHi determinations was 0.03 for group 1 and 0.00 for group 2 ( P = NS). The corresponding 95% limits of agreement spanned 0.24 and 0.28 pH units, respectively. Agreement of pHi determinations between tonometry catheters was not significantly affected by varying of the equilibration period, by administration of cimetidine, or by the presence of posthypoxic conditions. Conclusions: Clinically important disagreement was observed in simultaneous pHi determinations obtained using separate gastric balloon tonometry catheters exposed to identical in vivo conditions. Poor reproducibility of individual pHi determinations may limit the accuracy of pHi obtained by gastric balloon tonometry in the clinical setting.

Effect of hypoxia and reoxygenation on regional activity of nitric oxide synthase in brain of newborn piglets

The activity of nitric oxide synthase (NOS) was measured in homogenates from cortex, striatum, hippocampus, cerebellum, pons, thalamus and midbrain of the brain of newborn piglets and the effects of hypoxia and posthypoxic period on this activity was evaluated. The control activities were 19.7, 31.5, 26.8, 16.7, 33.6, 19.3 and 39.4 pmol/mg protein per min, respectively. A 1 h period of hypoxia (an Fi0 2 of 7%) resulted in statistically significant decreases in the activity of NOS in every region of the brain except for the cortex, where the activity was not significantly altered compared to control. By 2 h of reoxygenation following such a hypoxic episode, the NOS activities increased to above control levels in all regions of the brain, but this increase was statistically significant compared to control only in thalamus. Since hypoxia induced the greatest decrease in NOS activity in the cerebellum, the kinetic constants of the enzyme were measured in homogenates from this region of brain. The decreased activity following the hypoxic episode was associated with an approximately four-fold increase in the apparent affinity ( K M ) for arginine with no significant change in the maximal rate of reaction ( V ax). The decrease in NOS activity subsequent to a hypoxic episode may contribute to the disturbances in cellular metabolism in the immature brain induced by episodes of hypoxia-reoxygenation.

Dynamics of the impulse activity of neurons of the neocortex of monkeys in a visual recognition task after brief oxygen deprivation.

A model of brief oxygen supply disruption was created in monkeys trained to a delayed visual spatial choice. A substantial reorganization of the impulse activity of neurons of the visual and prefrontal cerebral cortex, accompanied by disturbances in behavioral reactions, was observed during brief hypoxia (2.5 min); the motor reaction was not restored for several hours in the monkeys during a delayed visual spatial choice. The reorganization of the activity consisted in the appearance of successive phases of hyperactivation and inhibition. The frequency of the impulse activity in the phase of hyperactivation was higher in prefrontal cortex neurons. Successive phases of inhibition and hyperactivation were also identified in the posthypoxic period of restoration in the structure of the activity. The duration of the posthypoxic inhibition and the ratio of the frequencies of posthypoxic hyperactivation to the baseline frequency in the reactions of prefrontal cortex neurons was substantially greater than in the neurons of the visual cortex.

Lipophilic antioxidant U-18 and superoxide dismutase prevent cultured hippocampal neurons from destruction during hypoxia and in the posthypoxic period

The lipophilic antioxidant U-18 from the class of hindered phenols prevents the destruction of cultured hippocampal neurons during hypoxia and also in the posthypoxic reoxygenation period, apparently by being stably incorporated into their phospholipid membranes and by safeguarding these from free-radical damage in the course of reoxygenation. On the other hand, the protection afforded to the cultured hippocampal neurons by superoxide dismutase is probably due to its ability to interfere with the posthypoxic neuron-degrading processes mediated through hyperproduction of superoxide radicals in the neuronal cytoplasm.

Effect of hypoxia and reoxygenation on the activity of transglutaminase in brain of newborn piglets

The transglutaminase activity in five regions of the brain of newborn piglets was measured and the effects of hypoxia and posthypoxic period on this activity evaluated. Enzyme activity was measured in homogenates from cortex, hippocampus, striatum, thalamus and midbrain. The control activities were 7.2, 6.2, 6.0, 5.7 and 4.6 pmol/mg protein/min, respectively. The activities at the end of an 18 min period of hypoxia induced by an F iO 2 of 9% were not significantly different from control activities. By 3 h after the hypoxic episode, however, the transglutaminase activities were significantly above control levels in all five regions of the brain. Measurements of the kinetic constants of tranglutaminase indicated that increases in enzyme activity were associated with an increase in Vmax with no significant change in the apparent affinity of the enzyme for the substrate, putrescine. The increased activity of transglutaminase during the posthypoxic period, with no changes immediately after hypoxia, suggest that the increases could be due to increased enzyme synthesis rather then activation of existing enzyme. The rise in transglutaminase activity subsequent to a hypoxic episode may contribute significantly to the long-term disturbances in cellular metabolism in the immature brain induced by hypoxic episodes.

Respiratory responses to progressive ambient hypoxia in the sturgeon, Acipenser baeri

Changes in respiratory and acid-base variables were studied in siberian sturgeon, Acipenser baeri, during progressive deep hypoxia followed by recovery under normoxic conditions. During hypoxia, both ventilatory frequency and amplitude increased and this sturgeon was able to maintain standard oxygen consumption down to a low critical level of ambient P O 2 (P W O 2 <40 mmHG). During the posthypoxic period an O 2 debt was repaid by an elevated oxygen consumption (nearly double control value at 1 h), indicating that a shift to anaerobic metabolism had occurred during exposure to severe hypoxia. Gradually increasing ambient hypoxia initially induced a respiratory alkalosis. Below the critical P W O 2 level and during normoxic recovery, a sudden flush of lactate into the blood was associated with a typical metabolic acidosis which was almost totally compensated 3.5 h after return to normoxia. Thus, as for most other fish, respiratory responses of the sturgeon to progressive hypoxia reveal a typical O 2 regulatory behavior.

Influence of airway resistance on hypoxia-induced periodic breathing.

We studied the effects of changing upper airway pressure on the variability of the dynamic response of ventilation to a hypoxic disturbance in 11 spontaneously breathing dogs. Supralaryngeal pressure, instantaneous inspiratory flow, end-expiratory lung volume, and the inspiratory and expiratory O2 and CO2 concentrations were continuously recorded at baseline and after a 1.5-min hypoxic stimulus (abrupt normoxic recovery). Arterial blood gases were obtained at baseline, at the end of the hypoxic period, and after 1 min of recovery. Airway resistances were modified during the recovery by changing the composition of the inspired gas (all with an inspiratory O2 fraction of 20.9%) among four different trials: two trials were realized with air (density 1.12 g/l), and the other two were with He or SF6 (respective density 0.42 and 4.20) in random order. There was no difference between baseline minute ventilation, arterial blood gases, and supralaryngeal resistance values preceding the trials. The hypoxemic and hypocapnic levels and the hypoxia-induced hyperventilation reached during the hypoxic tests were identical for the different hypoxic stimuli. The supralaryngeal resistance measured at peak flow was dramatically influenced by the composition of the inspired gas: 8.8 +/- 1.8 and 6.9 +/- 1.7 (SE) cmH2O.l-1.s with air, 7.2 +/- 2.2 with He, 21.9 +/- 5.5 with SF6 (P less than 0.05). Ventilatory fluctuations were consistently seen during the posthypoxic period. They were characterized by a strength index value (M) (Waggener et al. J. Appl. Physiol. 56: 576-581, 1984).(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia induced metabolism dysfunction of rat astrocytes in primary cell cultures

In order to study the astroglial contribution to hypoxic injury on brain tissue metabolism, modifications of glutamine synthetase (GS) lactate dehydrogenase (LDH) enolase and malate dehydrogenase activity produced by reduced oxygen supply have been determined in primary cultures of astrocytes prepared from newborn rat cerebral cortex. Enzymatic activities were measured immediately after the hypoxic treatment (9 h) and during post injury recovery. GS level is significantly decreased in response to low oxygen pressure and increased above control value during the post hypoxic recovery period. The magnitude of GS reduction by hypoxia depends on the age of the cells in culture. Lactate dehydrogenase and enolase levels were significantly enhanced during the two periods considered. No modification of the MDH level was observed. The synthesis of LDH isoenzymes containing mainly M subunits is specifically induced by hypoxia. Our results suggest that astroglial cells may represent a particularly sensitive target toward hypoxia injury in brain tissue. Low oxygen pressure available may modify some fundamental metabolical functions of these cells such as glutamate turnover and lactic acid accumulation.

Cerebral energy metabolism and intracellular pH during severe hypoxia and recovery: A study using 1H, 31P, and 1H [13C] nuclear magnetic resonance spectroscopy in the guinea pig cerebral cortex in vitro

1H and 31P nuclear magnetic resonance spectroscopy was used to study intracellular pH (pHi), high-energy phosphates, lactate, and amino acids in cortical brain slices superfused in Krebs-Henseleit bicarbonate buffer during and after severe hypoxia at 0, 10, and 50 mM glucose. An extensive drop in phosphocreatine (PCr) and a rapid build-up of intracellular lactate and H+ were the first signs of hypoxia. Adenosine triphosphate (ATP) was significantly more resistant to hypoxia provided that glucose was present. In the preparations that had been exposed to hypoxia in the presence of glucose, PCr became detectable within 2 min of reoxygenation, and both PCr and ATP concentrations were restored to 72-80% of normoxic levels within 30 min. Lactate was washed out, and pHi returned to normal within 4-8 min. Using 1-[13C]glucose as a tracer, we demonstrated that the rate of lactate production in the immediate posthypoxic period was at the prehypoxic level, indicating that the elevated lactate during this period was due solely to that produced during hypoxia. During reoxygenation of the preparations that were denied glucose during hypoxia, only 30% of total creatine + PCr and 18% of PCr were restored, and ATP was not detectable. The lactate concentration rose twofold in this period, and pHi became significantly more alkaline than before the hypoxic insult. Thus acute metabolic damage was considerably greater if glucose was absent during the insult, suggesting that either anaerobic ATP production or low pH may exert some protective effect against acute cell damage.

Mechanistic Distinctions between Excitotoxic and Acidotic Hippocampal Damage in an in vitro Model of Ischemia

Excitotoxicity is believed to underlie the selective loss of vulnerable neurons after transient ischemia, while lactic acidosis seems to be the principal feature and probable cause of tissue infarcts. Primary hippocampal cultures containing both neurons and astrocytes derived from fetal rats were used to examine the relative contributions of and interactions between excitotoxic and acidotic cell injury. Hypoxia-induced damage was energy dependent and involved the N-methyl-D-aspartate (NMDA) receptor. Glucose above 1 mM could completely protect against hypoxia-induced injury in a pH range of 7.4-6.5, while the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (500 microM) during the posthypoxic period provided only partial protection in the absence of glucose. Astrocyte cultures were undamaged by ischemic-like treatment in this pH range, suggesting that hypoxia-induced cell death in mixed cultures was restricted to neurons. Lowering the extracellular pH to 7.0 and 6.5 caused no neuronal damage in normoxic controls, but in each case provided significant protection against hypoxic neuronal injury. In contrast, a second type of neurotoxicity was observed after a 6-h exposure to pH 6.0, while exposure to pH 5.5 was required to kill astrocytes. This acidotic damage appeared to be energy independent and did not involve the NMDA receptor. These results suggest that excitotoxic neuron death has an energetic component and that acidosis may produce both protective and damaging effects in the hippocampus during ischemic insults.