Philippe Haouzi

H2S induced hypometabolism in mice is missing in sedated sheep.

On the basis of studies performed in mice that showed H(2)S inhalation decreasing dramatically the metabolic rate, H(2)S was proposed as a means of protecting vital organs from traumatic or ischemic episodes in humans. Hypoxia has in fact also long been shown to induce hypometabolism. However, this effect is observed solely in small-sized animals with high VO2 kg(-1), and not in large mammals. Thus, extrapolating the hypometabolic effect of H(2)S to large mammals is questionable and could be potentially dangerous. We measured metabolism in conscious mice (24 g) exposed to H(2)S (60 ppm) at an ambient temperature of 23-24 degrees C. H(2)S caused a rapid and large (50%) drop in gas exchange rate, which occurred independently of the change in body temperature. The metabolic response occurred within less than 3 min. In contrast, sheep, sedated with ketamine and weighing 74 kg did not exhibit any decrease in metabolic rate during a similar challenge at an ambient temperature of 22 degrees C. While a part of H(2)S induced hypometabolism in the mice is related to the reduction in activity, we speculate that the difference between sheep and mice may rely on the nature and the characteristics of the relationship between basal metabolic rate and body weight thus on the different mechanisms controlling resting metabolic rate according to body mass. Therefore, the proposed use of H(2)S administration as a way of protecting vital organs should be reconsidered in view of the lack of hypometabolic effect in a large sedated mammal and of H(2)S established toxicity.

Control of Breathing During Exercise

During exercise by healthy mammals, alveolar ventilation and alveolar-capillary diffusion increase in proportion to the increase in metabolic rate to prevent PaCO2 from increasing and PaO2 from decreasing. There is no known mechanism capable of directly sensing the rate of gas exchange in the muscles or the lungs; thus, for over a century there has been intense interest in elucidating how respiratory neurons adjust their output to variables which can not be directly monitored. Several hypotheses have been tested and supportive data were obtained, but for each hypothesis, there are contradictory data or reasons to question the validity of each hypothesis. Herein, we report a critique of the major hypotheses which has led to the following conclusions. First, a single stimulus or combination of stimuli that convincingly and entirely explains the hyperpnea has not been identified. Second, the coupling of the hyperpnea to metabolic rate is not causal but is due to of these variables each resulting from a common factor which link the circulatory and ventilatory responses to exercise. Third, stimuli postulated to act at pulmonary or cardiac receptors or carotid and intracranial chemoreceptors are not primary mediators of the hyperpnea. Fourth, stimuli originating in exercising limbs and conveyed to the brain by spinal afferents contribute to the exercise hyperpnea. Fifth, the hyperventilation during heavy exercise is not primarily due to lactacidosis stimulation of carotid chemoreceptors. Finally, since volitional exercise requires activation of the CNS, neural feed-forward (central command) mediation of the exercise hyperpnea seems intuitive and is supported by data from several studies. However, there is no compelling evidence to accept this concept as an indisputable fact.

Carotid chemoreceptor response to natural stimuli in the newborn kitten

The activities of carotid chemoreceptors at three levels of inspired PO2 (55, 145 and 690 Torr) and at two levels of inspired PCO2 (35 and 70 Torr in O2) were studied in 28 anesthetized, mechanically ventilated kittens aged 0-17 days. A biphasic response to hypoxia was found in 46% of them: the chemosensory activity increased to a peak within 30 sec after the initial response to hypoxia and thereafter declined slowly to a stable value. The steady-state single-fiber chemosensory activity at an inspired PO2 of 55 Torr was significantly lower in kittens less than 10 days old (mean +/- SE: 5.8 +/- 0.6 impulses.sec-1) than in the older ones (8.8 +/- 1.3 impulses.sec-1, P less than 0.03). The response curves to arterial PO2 were hyperbolic in both groups but the curve for the younger kittens was displaced to the left of the curve for the older ones. The response to hypercapnia was a progressive increase in chemosensory activity with little evidence of rapid or slow adaptation. The response to hypercapnia was significantly stronger in the older kittens than in the young ones. It is concluded that, in the kitten, the carotid chemoreceptor response to hypoxia may be biphasic. The responses to hypoxia and hypercapnia are already developed but are weak at birth and continue to develop further during the first weeks of postnatal life.

Femoral vascular occlusion and ventilation during recovery from heavy exercise

Ventilation and cardiac output subside gradually following cessation of exercise, which is commonly linked to the slow wash-out of materials from the recovering muscles. The effect of hindering the removal of the metabolic products of heavy cycle exercise on the kinetics of ventilation and gas exchange was studied in 5 subjects by occluding the femoral circulation with cuffs during the first 2 min of recovery (15 tests). Fifteen undisturbed recoveries served as controls. Compared to spontaneous recovery, circulatory obstruction induced an immediate (from the first breath) decrease in minute ventilation (VE), while end-tidal CO2 (PETCO2) as well as lactate and K+ in venous blood at forearm did not change significantly. A ventilatory deficit of 27 +/- 9 L was observed from the 2 min of occlusion. Following cuff deflation, VE rose 2-3 breaths after PETCO2 began to increase in every subject. The mechanisms of the normocapnic reduction of VE during occlusion, as well as the rise of ventilation following cuff release, are still unclear. However, these results argue against any significant role for hyperpnea-inducing intramuscular chemoreception, or point to muscular perfusion as a prerequisite of such a mechanism to operate.

Vascular distension in muscles contributes to respiratory control in sheep

It has recently been proposed that afferent fibers from skeletal muscle could sense the state of the microvascular circulation, linking ventilation to the degree of peripheral perfusion or vascular distension (Huszczuk et al., Respir. Physiol., 91:207-226, 1993). Ventilatory and circulatory responses to manipulation of peripheral vascular pressures in the hind limbs of anaesthetized (sodium thiopental) sheep were examined. Inflatable balloons were placed at the caudal ends of the abdominal aorta and the vena cava (Vc). Aortic (Ao) occlusion induced a consistent normocapnic decrease in minute ventilation (VE). In contrast, VE increased significantly during vena cava obstruction, leading to hypocapnia. Small changes in systemic blood pressure were observed (+7 mmHg for Ao occlusion and -12 mmHg during Vc obstruction). Moreover, inflation of the caval balloon superimposed on a previously established Ao occlusion, preventing venous drainage of anastomotic inflow, resulted in a significant rise in distal vascular pressures with trivial changes in systolic blood pressure. This led to a gradual rise of VE, despite further reduction of the CO2 flux to the lungs. The subsequent deflation of the aortic balloon, exposing the hindlimb vasculature to aortic pressure, resulted in an even more profound hypocapnic hyperpnea. The concurrent arterial blood pressure changes were too small to possibly involve the ventilatory component of the arterial baroreflex. We therefore hypothesize, that perfusion-related afferent signals within the muscles could contribute to respiratory homeostasis by maintaining ventilation of the lungs commensurate with the circulatory state of the muscular apparatus.

Comparison of the metabolic and ventilatory response to hypoxia and H2S in unsedated mice and rats.

Hypoxia alters the control of breathing and metabolism by increasing ventilation through the arterial chemoreflex, an effect which, in small-sized animals, is offset by a centrally mediated reduction in metabolism and respiration. We tested the hypothesis that hydrogen sulfide (H(2)S) is involved in transducing these effects in mammals. The rationale for this hypothesis is twofold. Firstly, inhalation of a 20-80 ppm H(2)S reduces metabolism in small mammals and this effect is analogous to that of hypoxia. Secondly, endogenous H(2)S appears to mediate some of the cardio-vascular effects of hypoxia in non-mammalian species. We, therefore, compared the ventilatory and metabolic effects of exposure to 60 ppm H(2)S and to 10% O(2) in small and large rodents (20g mice and 700g rats) wherein the metabolic response to hypoxia has been shown to differ according to body mass. H(2)S and hypoxia produced profound depression in metabolic rate in the mice, but not in the large rats. The depression was much faster with H(2)S than with hypoxia. The relative hyperventilation produced by hypoxia in the mice was replaced by a depression with H(2)S, which paralleled the drop in metabolic rate. In the larger rats, ventilation was stimulated in hypoxia, with no change in metabolism, while H(2)S affected neither breathing nor metabolism. When mice were simultaneously exposed to H(2)S and hypoxia, the stimulatory effects of hypoxia on breathing were abolished, and a much larger respiratory and metabolic depression was observed than with H(2)S alone. H(2)S had, therefore, no stimulatory effect on the arterial chemoreflex. The ventilatory depression during hypoxia and H(2)S in small mammals appears to be dependent upon the ability to decrease metabolism.

H2S concentrations in the arterial blood during H2S administration in relation to its toxicity and effects on breathing

Our aim was to establish in spontaneously breathing urethane-anesthetized rats, the relationship between the concentrations of H2S transported in the blood and the corresponding clinical manifestations, i.e., breathing stimulation and inhibition, during and following infusion of NaHS at increasing rates. The gaseous concentration of H2S (CgH2S, one-third of the total soluble form) was computed from the continuous determination of H2S partial pressure in the alveolar gas, while H2S, both dissolved and combined to hemoglobin, was measured at specific time points by sulfide complexation with monobromobimane (CMBBH2S). We found that using a potent reducing agent in vitro, H2S added to the whole blood had little interaction with the plasma proteins, as sulfide appeared to be primarily combined and then oxidized by hemoglobin. In vivo, H2S was undetectable in the blood in its soluble form in baseline conditions, while CMBBH2S averaged 0.7 ± 0.5 μM. During NaHS infusion, H2S was primarily present in nonsoluble form in the arterial blood: CMBBH2S was about 50 times higher than CgH2S at the lowest levels of exposure and 5 or 6 times at the levels wherein fatal apnea occurred. CgH2S averaged only 1.1 ± 0.7 μM when breathing increased, corresponding to a CMBBH2S of 11.1 ± 5.4 μM. Apnea occurred at CgH2S above 5.1 μM and CMBBH2S above 25.4 μM. At the cessation of exposure, CMBBH2S remained elevated, at about 3 times above baseline for at least 15 min. These data provide a frame of reference for studying the putative effects of endogenous H2S and for testing antidotes against its deadly effects.

Hydrogen sulfide oxidation and the arterial chemoreflex: Effect of methemoglobin

Endogenous H(2)S has been proposed to transduce the effects of hypoxia in the carotid bodies (CB). To test this hypothesis, we created a sink for endogenously produced H(2)S by inducing ∼10% methemoglobinemia via the injection of 250 mg of sodium nitrite in spontaneously breathing anaesthetized sheep. Methemoglobinemia has been shown to catalyze the oxidation of large quantities of sulfide in the blood and tissues. We found that the presence of metHb completely abolished the ventilatory stimulation induced by 10 mg NaHS (i.v.), which in control conditions mimicked the effects of breathing 6-7 tidal volumes of nitrogen, confirming the dramatic increase in the oxidative power of the blood for sulfide. The ventilatory responses to hypoxia (10% O(2)), nitrogen and hyperoxia were in no way depressed by the metHb. Our results demonstrate that the ventilatory chemoreflex is not depressed in the presence of a high oxidative capacity for sulfide and challenge the view that H(2)S transduces the effects of hypoxia in the CB.

Control of arterial PCO2 by somatic afferents in sheep

The ventilatory response to electrically induced rhythmic muscle contractions (ERCs) was studied in six urethane–chloralose‐anaesthetized sheep, while arterial oxygen and carbon dioxide pressure (P  a,O 2 and P  a,CO 2) and perfusion pressure were maintained constant at the known chemoreception sites. With cephalic P  a,CO 2 held constant, the response to inhaled CO2 was virtually abolished (0.03 ± 0.04 l min−1 Torr−1). During low‐current ERC, which doubled the metabolic rate ( increased from 192 ± 23 to 317 ± 84 ml min−1, P < 0.01), followed the change in closely (from 5.24 ± 1.81 to −9.27 ± 3.60 l min−1, P < 0.01) in the absence of any chemical error signal occurring at carotid and central chemoreceptor level (Δcephalic P  a,CO 2=−0.75 ± 1 Torr). Systemic P  a,CO 2 decreased by −2.47 ± 1.9 Torr (P < 0.01). Both heart rate and systemic blood pressure increased significantly by 18.6 ± 5.5 beats min−1 and 7.0 ± 9.3 mmHg, respectively. When the CO2 flow to the central circulation was reduced during ERC by blocking venous return ( decreased by 102 ± 45 l min−1, P < 0.01), ventilation was stimulated (from 11.99 ± 4.11 to 13.01 ± 4.63 l min−1, P < 0.05). The opposite effect was observed when the arterial supply was blocked. Finally, raising the CO2 content and flow in the systemic blood did not significantly stimulate ventilation provided that the peripheral and central chemoreceptors were unaware of the changes in blood CO2/H+ composition. Our results support the existence of a system capable of controlling blood P  a,CO 2 homeostasis when the metabolism increases independently of peripheral and central respiratory chemoreceptors. Information from the skeletal muscles related to the local vascular response provides the central nervous system with a respiratory stimulus proportional to the rate at which gases are exchanged in the muscles, thereby coupling ventilation to the metabolic rate.

Murine models in critical care research.

Introduction:Access to genetically engineered mice has opened many new opportunities to address questions relevant to the pathophysiology and treatment of patients in critical conditions. However, the results of studies in mice cannot disregard the unique ability of small rodents to adjust their temperature and high metabolic rate and the corresponding respiratory and circulatory requirements in response to hypoxia. Point of View:Studies performed in mice on questions related to metabolic, circulatory, and respiratory regulation should always be considered in light of the ability of mice to rapidly drop their nonshivering thermogenesis-related metabolism. As an example, it has been recently argued that a moderate level of inhaled hydrogen sulfide may have a potential benefit in patients in coma or shock or during an anoxic or ischemic insult, as this toxic gas dramatically reduces the metabolic rate in resting mice. However, acute hypometabolism has long been described in small mammals in response to hypoxia and is not specific to hydrogen sulfide. More importantly, mice have a specific metabolic rate that is 15–20 times higher than the specific metabolic level of a resting human. This difference can be accounted for by the large amount of heat produced by mice through nonshivering thermogenesis, related to the activity of uncoupling proteins. This mechanism, which is essential for maintaining homeothermia in small mammals, is virtually absent in larger animals, including in adult humans. Accordingly, no direct metabolic effect of hydrogen sulfide is observed in large mammals. We present the view that similar reasoning should be applied when the circulatory or respiratory response to hypoxic exposure is considered. This leads us to question whether a similar strategy could occur in mice in critical conditions other than hypoxia, such as in hypovolemic, septic, or cardiogenic shock. Conclusion:Mouse models developed to understand the mechanisms of protection against hypoxia or ischemia or to propose new therapeutic approaches applicable in critical care patients should be understood in light of the specificity of the metabolic, respiratory, and circulatory responses of mice to a hypoxic insult, since many of these adaptations have no clear equivalent in humans.