Circadian pattern of ventilation during prolonged hypoxia in conscious rats

In mammals, metabolic rate is well known to present a circadian oscillation. Because metabolism is a major determinant of the magnitude of the hypoxic ventilatory response, we hypothesized that the level of this response would follow a circadian pattern. To this end, we measured pulmonary ventilation (VE), oxygen consumption (V(O(2))), body temperature (Tb) and activity simultaneously and continuously in conscious adult rats by non-invasive methods. Measurements were made in a 12:12-h light (L):dark (D) cycle for 3 days in air, and then for 4 days in 10.5% normobaric hypoxia (HX). In normoxia, all variables oscillated, with the highest values in D. In HX, the circadian Tb and V(O(2)) oscillations were blunted, due to a decrease in their D values. The hypoxic VE response (% increase in VE from the corresponding air value) was greater in L than in D. This L-D difference was proportionate to that of the V(O(2)) response such that the hyperventilatory response (% increase in VE/V(O(2))) was similar throughout the day. The VE/V(O(2)) response was also similar between L and D when it was compared for the same level of activity; in this case, however, there was no L-D difference in the VE or V(O(2)) response. We conclude that the circadian oscillation in the hypoxic VE response was related to the time-of-day changes in the effect of HX on V(O(2)), and that, in the rat, the presence of a circadian clock does not compromise the hyperventilatory response to HX, as this response presents no time-of-day variation.     

Sleep and respiration of rats during hypoxia.

1. The effects of hypoxia on slow-wave sleep (SWS) and of SWS on respiratory responses to hypoxia were investigated on rats provided with chronically implanted cortical electrodes. 2. During the daytime (5-7 hr periods) the proportion of time spent in SWS was 45% (S.E. +/- 1.0%) when the rats breathed air. Exposure to 10% O2 (equivalent to 18,000 ft.) reduced this proportion to 27% (S.E. +/- 2.5%). During hypoxia the intensity of e.e.g. activity in SWS (mean, rectified slow-wave voltage) rarely equalled the normal values characteristic of the same rats in fully developed SWS breathing air. The normal pattern of 5-15 min episodes of SWS was changed by hypoxia to a series of brief (2-3 min) incompletely developed episodes. 3. Addition of CO2 to inspired gas failed to prevent the reduction of SWS during hypoxia. CO2 in normal O2 did not alter sleep significantly. The effects of hypoxia on sleep therefore depend upon changes in O2 pressure rather than upon changes in CO2. 4. The effect of SWS on respiration of rats breathing air was to decrease frequency and minute volume by 10-20%. In hypoxia, however, the frequency increased markedly when the animals entered SWS ; minute volume was not significantly changed. It follows that stimulation of breathing by hypoxia is greater during SWS than during wakefulness. 5. The anomalous increase of respiratory frequency when hypoxic rats entered SWS was abolished by addition of CO2 to the hypoxic gas mixture. 6. Steady-state gaseous metabolism (Vo2 Vco2) was decreased 18 +/- 3% during hypoxia and was increased 31 +/- 4% during exposure to 5% CO2. The implications of these changes for interpretation of respiratory responses to O2 and CO2 are discussed.     

Aggravated hypoxia during breath-holds after prolonged exercise

Hyperventilation prior to breath-hold diving increases the risk of syncope as a result of hypoxia. Recently, a number of cases of near-drownings in which the swimmers did not hyperventilate before breath-hold diving have come to our attention. These individuals had engaged in prolonged exercise prior to breath-hold diving and it is known that such exercise enhances lipid metabolism relative to carbohydrate metabolism, resulting in a lower production of CO₂ per amount of O₂ consumed. Therefore, our hypothesis was that an exercise-induced increase in lipid metabolism and the associated reduction in the amount of CO₂ produced would cause the urge to breathe to develop at a lower P O₂, thereby increasing the risk of syncope due to hypoxia. Eight experienced breath-hold divers performed 5 or 6 breath-holds at rest in the supine position and then 5 or 6 additional breath-holds during intermittent light ergometer exercise with simultaneous apnoea (dynamic apnoea, DA) on two different days: control (C) and post prolonged sub-maximal exercise (PPE), when the breath-holds were performed 30 min after 2 h of sub-maximal exercise. After C and before the prolonged submaximal exercise subjects were put on a carbohydrate-free diet for 18 h to start the depletion of glycogen. The respiratory exchange ratio ( RER) and end-tidal P CO₂, P O₂, and SaO₂ values were determined and the data were presented as means (SD). The RER prior to breath-holding under control conditions was 0.83 (0.09), whereas the corresponding value after exercise was 0.70 (0.05) ( P <0.01). When the three apnoeas of the longest duration for each subject were analysed, the average duration of the dynamic apnoeas was 96 (14) s under control conditions and 96 (17) s following exercise. Both P O₂ and P CO₂ were higher during the control dynamic apnoeas than after PPE [PO₂ 6.9 (1.0) kPa vs 6.2 (1.2) kPa, P <0.01; P CO₂ 7.8 (0.5) kPa vs 6.7 (0.4) kPa, P <0.001; ANOVA testing]. A similar pattern was observed after breath-holding under resting conditions, i.e., a lower end-tidal P O₂ and P CO₂ after exercise (PPE) compared to control conditions. Our findings demonstrate that under the conditions of a relatively low RER following prolonged exercise, breath-holding is terminated at a lower P O₂ and a lower P CO₂ than under normal conditions. This suggests that elevated lipid metabolism may constitute a risk factor in connection with breath-holding during swimming and diving.     

钾离子通道在大鼠慢性低氧所致肺血管低反应中的作用

目的与方法：采用阻断剂及离体肺内动脉血管环方法,研究不同类型钾离子通道对慢性低压低氧的大鼠模型的低氧性肺血管收缩反应（HPV）的作用,旨在探讨钾离子通道在慢性低氧肺血管低反应机制中的作用。结果：①慢性低氧15 d、30 d可降低肺血管对急性低氧的收缩反应。②分别阻断正常对照组、慢性低氧组大鼠肺血管钙激活性钾通道（K_Ca）、ATP敏感的钾通道（K_ATP）,均使其HPV反应明显增强,其中慢性低氧组增强幅度显著高于正常对照组（P<0.01）。③阻断正常对照组、慢性低氧组大鼠肺血管延迟整流性钾通道（K_DR）对其HPV均无明显影响。结论：K_Ca、K_ATP在HPV反应起着重要的调节作用,慢性低氧可使此调节作用显著加强,这可能是导致肺血管低反应一个重要机制。     

Changes in hypoxia resistance in rats during daytime

The resistance to acute hypoxia in male Wistar rats was evaluated by the period of survival after exposure to high-altitude hypoxia (11.5 km above see level). The study was performed during daytime (13.00–21.00) in autumn. The fatal rat population was characterized by the log-normal distribution of survival periods. The rats with low and moderate resistance to hypoxia exhibited similar diurnal variations in it with gradual decrease by the end of daytime more pronounced in low-resistant rats. The rats with high resistance showed relatively constant resistance to hypoxia which decreased only at 21:00. All groups revealed a relatively stable resistance to hypoxia from 16:00 to 18:00. These variations in the resistance to hypoxia should be taken into consideration when planning experimental research. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 3, pp. 256–260, March 1999     

Beta-adrenergic agonist-mediated desensitization in senescent rats

The capacity of senescent rats to develop the catecholamine refractory state was investigated in CDF (F-344) rats of 3 and 24 months of age. Beta-adrenergic receptor number, receptor-agonist affinity, and adenylate cyclase activity in heart membranes were assessed, following the chronic in vivo administration of the beta-adrenergic agonist, metaproterenol. Drug treatment leads to marked myocardial hypertrophy, receptor down-regulation, and reduced isoproterenol-stimulated adenylate cyclase activity. The extent of catecholamine-refractoriness was not different in the older rats, indicating the catecholamine desensitization of myocardial beta-adrenergic responsiveness is not impaired in senescence. Receptor agonist affinity and the percent of receptors in the high-affinity state decrease with age. These parameters are further reduced by agonist treatment but to a lesser extent in the older animals. Thus, the effects of age and agonist desensitization are not additive and suggest that aged animals may already be partially desensitized.     

Drug induced hypothermia in adult and senescent rats

Temperature regulation was evaluated in senescent (34-40 month old) and adult (8-9 month old) female Iva:WIWU and Emd:Wi-AF/Han rats. Injection of 1.5 mg/kg BW apomorphine HCl or 1.0 mg/kg BW oxotremorine sesquifumarate produced comparable maximal hypothermic responses in adult and senescent rats. However, the latency to reach maximal hypothermia after oxotremorine (but not apomorphine) was longer in senescent than in adult rats of both strains.     

Influenza in senescent mice: impaired cytotoxic T-lymphocyte activity is correlated with prolonged infection.

Influenza and pneumonia are leading causes of death in the elderly. Cytotoxic T-lymphocyte activity is responsible for viral clearance after infection and declines with age. We hypothesized that following intranasal infection with influenza virus, aged mice would have decreased anti-influenza cytotoxic T-lymphocyte activity that would correlate with prolonged pulmonary viral shedding. To test this, young (1.5-4.0 month) and aged (22-25 month) BALB/c mice were infected intranasally with influenza A/Port Chalmers/1/73(H3N2). Mice were killed at 3-19 days following infection. Their splenic cytotoxic T-lymphocyte activity was measured by a secondary in vitro chromium release assay. Pulmonary viral titres were quantified by growth of titrated lung specimens in fertilized hens' eggs. Serum antibody titres were measured by an ELISA. Young mice responded in a relatively homogeneous fashion. They developed maximal cytotoxic T-lymphocyte activity of 60.9 +/- 2.0% by Days 11-13, and all except one cleared virus from the lung by Day 7. In contrast, old mice were heterogeneous. Their cytotoxic T-lymphocyte activity peaked at 46.9 +/- 5.0% and was delayed by 5-7 days. Forty-five per cent were still shedding virus at Days 7 and 8, and shedding persisted for at least 13 days in some mice. There was a strong correlation in both young and aged mice between the presence of virus in the lungs and decreased splenic cytotoxic T-lymphocyte activity (chi 2 = 30.2, P much less than 0.001). No significant difference was found between young and aged animals in serum IgG1 anti-H3 antibody titres. We conclude that following influenza infection in aged mice, impaired cytotoxic T-lymphocyte activity leads to prolonged duration of infection. These observations may lead to a better understanding of the excess morbidity and mortality in elderly persons that occur with influenza.     

Substrate mobilization during prolonged exercise in 6-hydroxydopamine treated rats

Summary Exercise-induced lipolysis and glycogenolysis were studied in rats that had undergone chemical sympathectomy, surgical adrenomedullectomy, or a combination of these treatments. Sympathectomy was accomplished by injection of 6-hydroxydopamine. The exercise test administered immediately prior to sacrifice consisted of running 90 min (30 min at 22.5, 28.5, and 32.0 m/min, respectively, without interruption). Control animals were sacrificed at rest. Lipolysis, as measured by the rise in plasma and adipose tissue free fatty acid levels, was significantly (P<0.01) elevated over control values in all exercised groups. However, progressive (P<0.05) depression in lipolysis occurred as adrenergic control was successively eliminated. No treatment completely inhibited lipolysis or reduced work capacity. None of the treatments inhibited liver or muscle glycogenolysis in the exercised animals. The results demonstrate that prolonged exercise induces significant lipolysis and glycogenolysis in the rat after treatments designed to eliminate the sympathetic control over these processes. These data point to factors other than the adrenergic system as playing a role in substrate mobilization during endurance exercise.     

Phosphate excretion during 24 h of hypoxia in conscious rats

The objective of this study was to investigate renal phosphate excretion during 24 h of hypoxia in conscious rats fed by total parenteral nutrition. Wistar rats weighing 190 g were exposed to hypoxia (inspired oxygen fraction = 0.10) or normoxia (inspired oxygen fraction = 0.21) for 24 h in a normobaric chamber. Renal clearance and hormonal studies were performed. The results showed a greater fractional excretion of phosphate (5.37 pL 0.07%, P < 0.05) and hypophosphataemia (7.40 pL 0.12 mg dL^-1, P < 0.01) in hypoxic rats (n = 10) than in normoxic rats (n = 13; 3.50 pL 0.37% and 8.02 pL 0.16 mg dL^-1, respectively). In addition, during hypoxia there was a significant decrease in the excretion of urinary adenosine 3′,5′-cyclic monophosphate per glomerular filtrate (2.97 pL 1.27 nmol dL^-1, P < 0.005), a parameter of the renal action of parathyroid hormone, and a stable level of serum parathyroid hormone (10.2 pL 2.6 ng mL^-1) (cf. normoxia: 8.57 pL 0.70 nmol dL^-1 and 8.0 pL 1.7 ng mL^-1, respectively). However, creatinine clearance and the renal adenosine triphosphate level, both of which affect adenosine 3′,5′-cyclic monophosphate excretion, were not different between the two groups. These data suggest that exposure of conscious rats to 24 h of hypoxia causes renal hyporesponsiveness to physiological levels of parathyroid hormone, which is manifested as a decrease in adenosine 3′,5′-cyclic monophosphate excretion. Phosphaturia is not a direct net effect of hypoxia and secondary hypocapnia on renal phosphate transport, which is known to be regulated by parathyroid hormone through adenosine 3′,5′-cyclic monophosphate.     

Enhanced apoptosis in prolonged cultures of senescent porcine pulmonary artery endothelial cells

Senescent or aged endothelial cells in culture remain metabolically active after cessation of division, and are generally believed to eventually die. However, mechanisms underlying the terminal aging of cultured cells, i.e. from senescence to death, are poorly understood. Here, we report that culturing of replicative senescent endothelial cells for a prolonged period of time without passaging leads to enhanced programmed cell death or apoptosis. Senescent (passage 45) and young (passage 3) porcine pulmonary artery endothelial cells (PAEC) were cultured for 0-42 days post confluence. The cells attached to culture dishes and floating in medium were collected at 0, 7, 14, 21, 28, 35 and 42 days post confluence and were assessed for markers of apoptosis. Morphology studies showed that ratios between senescent and young cells attached to dishes declined to 45% after 42 days postconfluence. Apoptotic cells in prolonged cultures of senescent PAEC increased from 5 to 35% as determined by protein mass, DNA breakage, and caspase-3 activation. Steady state levels of Bcl-2, an anti-apoptotic protein, in senescent prolonged cultures decreased to less than 20% for all time points compared with young cells. Relative levels of Bad, a pro-apoptotic protein, in senescent cells were elevated from 60 to 130% during prolonged culturing. These results indicate that terminal cellular aging enhances apoptosis and the levels of Bcl-2/Bad may be associated with the apoptotic process in porcine lung endothelial cells.     

Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.