Characterisation of the ventilatory response to hypoxia in a model of transgenic anemic mice

Both polycythemia and the increase in hypoxic ventilatory response (HVR) are considered as important factors of acclimatization to hypoxia. The objective of this study was to characterise the ventilation pattern at different inspired oxygen fraction in a model of chronic anemic mice. These mice have a targeted disruption in the 5' untranslated region of the Epo gene that reduces Epo expression such that the homozygous animal is severely anemic. Ventilation in normoxia in Epo-TAg(h) mice was significantly greater than in wild type, and the difference was mainly due to a higher tidal volume. HVR was higher in Epo-TAg(h) mice at every FIO2 suggesting a higher chemosensitivity. Resting oxygen consumption was maintained in anemic mice. Maximal oxygen consumption was 30% lower while hemoglobin was 60% lower in anemic mice compared to wild type. This small decrease in maximal oxygen consumption is probably due a greater cardiac output and/or a better tissue oxygen extraction and would allow these anemic mice to acclimatize to hypoxia in spite of low oxygen carrying capacity. In conclusion, Epo-TAg(h) anemic mice showed increased ventilation and hypoxic ventilatory response. However, whether these adaptations will contribute to acclimatization in chronic hypoxia remains to be determined.     

Dopaminergic mechanisms of neural plasticity in respiratory control: transgenic approaches

Data supporting the hypothesis that dopamine-2 receptors (D(2)-R) contribute to time-dependent changes in the hypoxic ventilatory response (HVR) during acclimatization to hypoxia are briefly reviewed. Previous experiments with transgenic animals (D(2)-R 'knockout' mice) support this hypothesis (J. Appl. Physiol. 89 (2000) 1142). However, those experiments could not determine (1) if D(2)-R in the carotid body, the CNS, or both were involved, or (2) if D(2)-R were necessary during the acclimatization to hypoxia versus some time prior to chronic hypoxia, e.g. during a critical period of development. Additional experiments on C57BL/6J mice support the idea that D(2)-R are critical during the period of exposure to hypoxia for normal ventilatory acclimatization. D(2)-R in carotid body chemoreceptors predominate under control conditions to inhibit normoxic ventilation, but excitatory effects of D(2)-R, presumably in the CNS, predominate after acclimatization to hypoxia. The inhibitory effects of D(2)-R in the carotid body are reset to operate primarily under hypoxic conditions in acclimatized rats, thereby optimizing O(2)-sensitivity.     

Control of breathing in newborn mice lacking the beta‐2 nAChR subunit

Aim: To study the ventilatory and arousal/defence responses to hypoxia in newborn mutant mice lacking the β2 subunit of the nicotinic acetylcholine receptors. Methods: Breathing variables were measured non‐invasively in mutant (n =31) and wild‐type age‐matched mice (n = 57) at 2 and 8 days of age using flow barometric whole‐body plethysmography. The arousal/defence response to hypoxia was determined using behavioural criteria. Results: On day 2, mutant pups had significantly greater baseline ventilation (16%) than wild‐type pups (P < 0.02). Mutant pups had a decreased hypoxic ventilatory declines. Arousal latency was significantly shorter in mutant than in wild‐type pups (133 ± 40 vs. 146 ± 20 s, respectively, P < 0.026). However, the duration of movement elicited by hypoxia was shorter in mutant than in wild‐type pups (14.7 ± 5.9 vs. 23.0 ± 10.7 s, respectively, P < 0.0005). Most differences disappeared on P8, suggesting a high degree of functional plasticity. Conclusion: The blunted hypoxic ventilatory decline and the shorter arousal latency on day 2 suggested that disruption of the β2 nicotinic acetylcholine receptors impaired inhibitory processes affecting both the ventilatory and the arousal response to hypoxia during postnatal development.     

Cardioventilatory effects of acclimatization to aquatic hypoxia in channel catfish

The mechanisms responsible for altering cardioventilatory control in vertebrates in response to chronic hypoxia are not well understood but appear to be mediated through the oxygen-sensitive chemoreceptor pathway. Little is known about the effects of chronic hypoxia on cardioventilatory control in vertebrates other than mammals. The purpose of this study was to determine how cardioventilatory control and the pattern of response is altered in channel catfish (Ictalurus punctatus) by 1 week of moderate hypoxia. Fish were acclimatized for 7 days in either normoxia (P(O(2)) approximately 150 Torr) or hypoxia (P(O(2)) approximately 75 Torr). After acclimatization, cardioventilatory, blood-gas and acid/base variables were measured during normoxia (P(O(2)) 148+/-1 Torr) then at two levels of acute (5 min) hypoxia, (P(O(2)) 72.6+/-1 and 50.4+/-0.4 Torr). Ventilation was significantly greater in hypoxic acclimatized fish as was the ventilatory sensitivity to hypoxia (Delta ventilation/Delta P(O(2))). The increase in ventilation and hypoxic sensitivity was due to increases in opercular pressure amplitude, gill ventilation frequency did not change. Heart rate was greater in hypoxic acclimatized fish but decreased in both acclimatization groups in response to acute hypoxia. Heart rate sensitivity to hypoxia (Delta heart rate/Delta P(O(2))) was not affected by hypoxic acclimatization. The ventilatory effects of hypoxic acclimatization can be explained by increased sensitivity to oxygen but the effects on heart rate cannot.     

Effect of Hypoxic Preconditioning on Free Radical Processes in Tissues of Rats with Different Resistance to Hypoxia

We studied the effect of single hypoxic preconditioning exposure (hypobaric hypoxia, 5000 m, 60 min) on free radical processes, glutathione system, and antioxidant defense enzymes in tissues of rats with different resistance to acute hypoxia. The intensity of free radical processes was shown to increase or decrease on day 1 after hypoxic preconditioning. These changes were tissue-specific and opposite in animals with genetically determined differences in the resistance to hypoxia. Hypoxic preconditioning contributes to the immediate resistance. The effect was more pronounced in low resistant animals, who did not exhibit signs of oxidative stress in tissues during the early posthypoxic period. By contrast, hypoxic preconditioning was followed by activation of free radical processes in tissues of highly resistant animals. These rats were characterized by low ability for the development of immediate resistance. Activation of free radical processes in the early period of adaptation (first hours after hypoxic preconditioning) does not play a role in the induction of immediate adaptive mechanisms for hypoxia.     

LOCALIZATION OF ERYTHROPOIETIN GENE EXPRESSION IN PROXIMAL RENAL TUBULAR CELLS DETECTED BY DIGOXIGENIN-LABELLED OLIGONUCLEOTIDE PROBES

Erythropoietin (EPO) is the main humoral stimulus of erythropoiesis. In adult mammals, the kidney releases EPO in response to hypoxic stress. Conflicting data have suggested either renal tubular or peritubular cell origins of EPO synthesis in vivo. In situ hybridization studies were performed to define further the kidney cell type(s) capable of increasing EPO gene expression during hypoxic stimulation. EPO gene expression was stimulated in mice exposed to acute hypobaric hypoxia. Kidneys from hypoxic and control normoxic mice were obtained. Six digoxigenin-labelled oligonucleotide probes complementary to EPO exon sequences were utilized for in situ hybridization for EPO messenger RNA. Positive hybridization signals were identified in some proximal tubular cells, confined to the inner third of the renal cortex of hypoxic mouse kidney.     

Amelioration of hypoxia-induced striatal 5-HT(2A) receptor, 5-HT transporter and HIF1 alterations by glucose, oxygen and epinephrine in neonatal rats

Alterations in neurotransmitters and its receptors expression induce brain injury during neonatal hypoxic insult. Molecular processes regulating the serotonergic receptors play an important role in the control of respiration under hypoxic insult. The present study focused on the serotonergic regulation of neonatal hypoxia and its resuscitation methods. Receptor binding assays and gene expression studies were done to evaluate the changes in 5HT_2A receptors and its transporter in the corpus striatum of hypoxic neonatal rats and hypoxic rats resuscitated with glucose, oxygen and epinephrine. Total 5HT and 5HT_2A receptor number was increased in hypoxic neonates along with an up regulation of 5HT_2A receptor and 5HT transporter gene. The enhanced striatal 5HT_2A receptors modulate the ventilatory response to hypoxia. Immediate glucose resuscitation was found to ameliorate the receptor and transporter alterations. Hypoxia induced ATP depletion mediated reduction in blood glucose levels can be encountered by glucose administration and oxygenation helps in overcoming the anaerobic condition. The adverse effect of immediate oxygenation and epinephrine supplementation was also reported. This has immense clinical significance in establishing a proper resuscitation for the management of neonatal hypoxia.     

Developmental plasticity of the hypoxic ventilatory response after perinatal hyperoxia and hypoxia

Both genetic and environmental factors influence the normal development of the respiratory control system. This review examines the role perinatal O2 plays in the development of normoxic breathing and the hypoxic ventilatory response in mammals. Hyperoxia and hypoxia elicit plasticity in respiratory control that is unique to development and may persist weeks to years after return to normoxia. Specifically, both hyperoxia and hypoxia during early postnatal development attenuate the adult hypoxic ventilatory response, but the underlying mechanisms for this plasticity differ. Hyperoxia attenuates the hypoxic ventilatory response through potentially life-long changes in carotid body function. Neonatal hypoxia appears to have short-term effects on carotid body function, but persistent changes in the hypoxic ventilatory response may instead reflect changes in respiratory mechanics or related neural pathways. Overall, it appears that a relatively narrow range of environmental O2 is consistent with "normal" postnatal respiratory control development, predisposing animals to potentially maladaptive plasticity in the face of disease or atypical environmental conditions.     

Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem and carotid bodies

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non-erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epo's impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild-type (WT) animals developed a life-threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.     

Immediate and Delayed Effects of Hypobaric Hypoxia during Early Postnatal Period on the Functioning of Digestive Enzymes

We studied immediate and delayed effects of hypobaric hypoxic stress experienced by rat pups in the early postnatal period on the digestive system. It was shown that hypoxia leads to remarkable changes in the functioning of the small intestine, as well as liver and kidney, especially in adult animals.     

Soluble erythropoietin receptor is present in the mouse brain and is required for the ventilatory acclimatization to hypoxia

While erythropoietin (Epo) and its receptor (EpoR) have been widely investigated in brain, the expression and function of the soluble Epo receptor (sEpoR) remain unknown. Here we demonstrate that sEpoR, a negative regulator of Epo's binding to the EpoR, is present in the mouse brain and is down-regulated by 62% after exposure to normobaric chronic hypoxia (10% O₂ for 3 days). Furthermore, while normoxic minute ventilation increased by 58% in control mice following hypoxic acclimatization, sEpoR infusion in brain during the hypoxic challenge efficiently reduced brain Epo concentration and abolished the ventilatory acclimatization to hypoxia (VAH). These observations imply that hypoxic downregulation of sEpoR is required for adequate ventilatory acclimatization to hypoxia, thereby underlying the function of Epo as a key factor regulating oxygen delivery not only by its classical activity on red blood cell production, but also by regulating ventilation.     

慢性低压低氧暴露对小鼠海马CA1区神经元树突棘形态及细丝蛋白A表达的影响

目的:探讨低压低氧暴露对小鼠海马CA1区神经元树突棘形态及细丝蛋白A表达的影响。方法:6～8周龄C57BL/6雄性小鼠分为常氧暴露7 d组、常氧暴露14 d组、低压低氧暴露7 d组和低压低氧暴露14 d组。低压低氧暴露组置于低压舱模拟6 000 m海拔高原进行低压低氧暴露。Golgi染色法观察小鼠海马CA1区树突的分支数,以及基树突棘和顶树突棘长度和密度的变化; Western blot方法检测小鼠海马细丝蛋白A表达水平的变化;免疫组织荧光染色法检测小鼠海马CA1区细丝蛋白A的表达及分布变化。结果:与常氧暴露组相比,低压低氧暴露后,小鼠海马CA1区树突分支数的差异无统计学显著性,但基树突棘和顶树突棘的长度显著增加(P 0. 05),密度显著降低(P 0. 01)。低压低氧暴露后,小鼠海马细丝蛋白A表达水平低于常氧暴露组(P 0. 01或P0. 05)。免疫组织荧光染色显示细丝蛋白A在小鼠海马CA1区表达,低压低氧暴露后,海马CA1区细丝蛋白A表达水平降低(P 0. 05)。结论:慢性低压低氧暴露可影响小鼠海马CA1区细丝蛋白A表达,并导致海马CA1区神经元树突棘形态发生改变。     

Ibuprofen blocks time-dependent increases in hypoxic ventilation in rats

Recently, inflammatory processes have been shown to increase O(2)-sensitivity of the carotid body during chronic sustained hypoxia [Liu, X., He, L., Stensaas, L., Dinger, B., Fidone, S., 2009. Adaptation to chronic hypoxia involves immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. Am. J. Physiol. Lung Cell Mol. Physiol. 296, L158-L166]. We hypothesized that blocking inflammation with ibuprofen would reduce ventilatory acclimatization to hypoxia by blocking such increases in carotid body O(2) sensitivity. We tested this in conscious rats treated with ibuprofen (4mg/kg IP daily) or saline during acclimatization to hypoxia ( [Formula: see text] for 7 days). Ibuprofen blocked the increase in hypoxic ventilation observed in chronically hypoxic rats treated with saline; ibuprofen had no effects on ventilation in normoxic control rats. Ibuprofen blocked increases in inflammatory cytokines (IL-1β, IL-6) in the brainstem with chronic hypoxia. The data supports our hypothesis and further analysis indicates that ibuprofen also blocks inflammatory processes in the central nervous system contributing to ventilatory acclimatization to hypoxia. Possible mechanisms linking inflammatory and hypoxic signaling are reviewed.     

Control of breathing in newborn mice lacking the beta-2 nAChR subunit

AIM: To study the ventilatory and arousal/defence responses to hypoxia in newborn mutant mice lacking the beta2 subunit of the nicotinic acetylcholine receptors. METHODS: Breathing variables were measured non-invasively in mutant (n = 31) and wild-type age-matched mice (n = 57) at 2 and 8 days of age using flow barometric whole-body plethysmography. The arousal/defence response to hypoxia was determined using behavioural criteria. RESULTS: On day 2, mutant pups had significantly greater baseline ventilation (16%) than wild-type pups (P < 0.02). Mutant pups had a decreased hypoxic ventilatory declines. Arousal latency was significantly shorter in mutant than in wild-type pups (133 +/- 40 vs. 146 +/- 20 s, respectively, P < 0.026). However, the duration of movement elicited by hypoxia was shorter in mutant than in wild-type pups (14.7 +/- 5.9 vs. 23.0 +/- 10.7 s, respectively, P < 0.0005). Most differences disappeared on P8, suggesting a high degree of functional plasticity. CONCLUSION: The blunted hypoxic ventilatory decline and the shorter arousal latency on day 2 suggested that disruption of the beta2 nicotinic acetylcholine receptors impaired inhibitory processes affecting both the ventilatory and the arousal response to hypoxia during postnatal development.     

Ventilatory response to acute hypoxia in transgenic mice over-expressing erythropoietin: effect of acclimation to 3-week hypobaric hypoxia

We used transgenic mice constitutively over-expressing erythropoietin ("tg6" mice) and wild-type (wt) mice to investigate whether the high hematocrit (hct), consequence of Epo over-expression affected: (1) the normoxic ventilation (V (E)) and the acute hypoxic ventilatory response (HVR) and decline (HVD), (2) the increase in ventilation observed after chronic exposure to hypobaric hypoxia (430mmHg for 21 days), (3) the respiratory "blunting", and (4) the erythrocythemic response induced by chronic hypoxia exposure. V (E) was found to be similar in tg6 and wt mice in normoxia (FIO2=0.21). Post-acclimation V (E) was significantly elevated in every time point in wt mice at FIO2=0.10 when compared to pre-acclimation values. In contrast, tg6 mice exhibited a non-significant increase in V (E) throughout acute hypoxia exposure. Changes in V (E) are associated with adjustments in tidal volume (V(T)). HVR and HVD were independent of EE in tg6 and wt mice before chornic hypoxia exposure. HVR was significantly greater in wt than in tg6 mice after chronic hypoxia. After acclimation, HVD decreased in tg6 mice. Chronic hypoxia exposure caused hct to increase significantly in wt mice, while only a marginal increase occurred in the tg6 group. Although pre-existent EE does not appear to have an effect on HVR, the observation of alterations on V(T) suggests that it may contribute to time-dependent changes in ventilation and in the acute HVR during exposure to chronic hypoxia. In addition, our results suggest that EE may lead to an early "blunting" of the ventilatory response.     

Carotid sinus nerve responses and ventilatory acclimatization to hypoxia in adult rats following 2 weeks of postnatal hyperoxia

Adult rats have decreased carotid body volume and reduced carotid sinus nerve, phrenic nerve, and ventilatory responses to acute hypoxic stimulation after exposure to postnatal hyperoxia (60% O2, PNH) during the first 4 weeks of life. Moreover, sustained hypoxic exposure (12%, 7 days) partially reverses functional impairment of the acute hypoxic phrenic nerve response in these rats. Similarly, 2 weeks of PNH results in the same phenomena as above except that ventilatory responses to acute hypoxia have not been measured in awake rats. Thus, we hypothesized that 2-week PNH-treated rats would also exhibit blunted chemoafferent responses to acute hypoxia, but would exhibit ventilatory acclimatization to sustained hypoxia. Rats were born into, and exposed to PNH for 2 weeks, followed by chronic room-air exposure. At 3-4 months of age, two studies were performed to assess: (1) carotid sinus nerve responses to asphyxia and sodium cyanide in anesthetized rats and (2) ventilatory and blood gas responses in awake rats before (d0), during (d1 and d7), and 1 day following (d8) sustained hypoxia. Carotid sinus nerve responses to i.v. NaCN and asphyxia (10 s) were significantly reduced in PNH-treated versus control rats; however, neither the acute hypoxic ventilatory response nor the time course or magnitude of ventilatory acclimatization differed between PNH and control rats despite similar levels of PaO2 . Although carotid body volume was reduced in PNH rats, carotid body volumes increased during sustained hypoxia in both PNH and control rats. We conclude that normal acute and chronic ventilatory responses are related to retained (though impaired) carotid body chemoafferent function combined with central neural mechanisms which may include brainstem hypoxia-sensitive neurons and/or brainstem integrative plasticity relating both central and peripheral inputs.     

缺氧性肺动脉和体动脉增压反应的研究——慢性间断缺氧的影响及其机制

雄性长白种(Landrace strain)幼猪10只,每天在低压舱内于4,000m模拟高原停留8小时,共30天。第31天再次缺氧时,其缺氧性肺动脉增压反应比急性缺氧组(11只)显著增强,但同时测得的全血量,血浆量和心输出量都无显著增加。仅血球量和红细胞压积增高,不过,都未达到足以影响肺动脉压的程度。股动脉血和肺动脉血的组织胺含量不仅未增加,反而下降。两组动物的肺血管阻力都增大,但前者更显著。两组动物的体动脉压在缺氧时都升高。在4,000m模拟高原,异搏停(钙离子拮抗剂)可致两组动物的肺动脉压和体动脉压明显下降,其作用可维持2小时以上。间接说明钙离子与缺氧性肺动脉和体动脉增压反应有关。     

Platelet-activating factor receptor and respiratory and metabolic responses to hypoxia and hypercapnia

Activation of the platelet-activating factor receptor (PAFR) regulates neural transmission. A PAFR blocker reduced the peak hypoxic (pHVR) but not hypercapnic ventilatory (HCVR) responses in rats [Am. J. Physiol. 275 (1998) R604]. To further examine the role of PAFR in respiratory control, genotype-verified PAFR -/- and PAFR +/+ adult male mice underwent hypoxic and hypercapnic challenges. HCVR was similar in the two groups (p-NS). However, pHVR was significantly reduced in PAFR -/- mice (38 +/- 13% baseline [S.D.]) compared to PAFR +/+ mice (78 +/- 16% baseline; P < 0.001, ANOVA), with reduced tidal volume recruitments during pHVR. In addition, hypoxic ventilatory depression was attenuated in PAFR -/- mice (P < 0.01), and was primarily due to attenuation of the time-dependent decreases in oxygen consumption during sustained hypoxia (P < 0.01). Thus, PAFR expression/function modulates components of the acute ventilatory and metabolic adaptations to hypoxia but not to hypercapnia. Imbalances in PAFR activity may lead to maladaptive regulation of the tightly controlled metabolic-ventilatory relationships during hypoxia.     

α2-Adrenoreceptor mediated sympathoinhibition of heart rate during acute hypoxia is diminished in conscious prostacyclin synthase deficient mice

Acute hypoxia increases ventilatory drive in conscious animals, resulting in tachycardia. Sustained hypoxia changes the initial chemoreflex ventilatory increase to secondary ventilatory depression, which then evokes a gradual secondary heart rate (HR) reduction. Prostacyclin (PGI₂) release is known to potentiate α₂-adrenoreceptor (α₂-AR) mediated inhibition of sympathoactivation during ischaemia and hypoxia. We examined whether α₂-AR mediated sympathoinhibition was responsible for limiting hypoxic heart rate increases during initial sympathoactivation, and subsequent secondary HR depression, and if PGI₂ is required for sympathoinhibition of HR. The responses of unrestrained PGI₂ synthase deficient (PGID) and wild type (WT) mice to acute hypoxia (10% O₂ for 30 min) were investigated by simultaneous telemetry, whole body plethysmography and open-flow respirometry. PGID mice exhibited potentiated (p < 0.007) after intraperitoneal vehicle injection (n = 8), but not so HR responses compared to WT mice during sustained hypoxia. Idazoxan (α₂-AR antagonist, i.p. bolus 3 mg/kg) pretreatment did not change hypoxic ventilatory response in either group, but significantly elevated hypoxic HR in WT mice only (p < 0.013). Sodium meclofenamate (cyclooxygenase inhibition, i.p. bolus 25 mg/kg) pretreatment eliminated the potentiated of PGID and caused significant basal hypotension that led to a transient hypertensive response to hypoxia. From these results, we suggest that α₂-AR activation is required for coupling HR to central inspiratory drive during acute hypoxia, and that PGI₂ is required to enhance the inhibition of sympathoactivation.     

Effect of domperidone on ventilation and polycythemia after 5 weeks of chronic hypoxia in rats

Chronically hypoxic humans and some mammals have attenuated ventilatory responses, which have been associated with high dopamine level in carotid bodies. Alveolar hypoventilation and blunted ventilatory response have been recognized to be at the basis of Chronic Mountain Sickness by generating arterial hypoxemia and polycythemia. To investigate whether dopamine antagonism could decrease the hemoglobin concentration by stimulating resting ventilation (VE) and/or hypoxic ventilatory response, 18 chronically hypoxic rats (5 weeks, PB=433 Torr) were studied with and without domperidone treatment (a peripheral dopamine antagonist). Acute and prolonged treatment significantly increased poikilocapnic ventilatory response to hypoxia (RVE ml/min/kg=VE at 0.1 FI(O(2))-VE at 0.21 FI(O(2))), from 506+/-36 to 697+/-48; and from 394+/-37 to 660+/-81, respectively. In addition, Domperidone treatment decreased hemoglobin concentration from 21.6+/-0.29 to 18.9+/-0.19 (P<0.01) in rats chronically exposed to hypobaric hypoxia. Our study suggests that the stimulant effect of D(2)-R blockade on ventilatory response to hypoxia seems to compensate the low hypoxic peripheral chemosensitivity after chronic exposure and the latter in turn decrease hemoglobin concentration.