Sensitivities of eupnea and gasping to alterations in temperature of in vivo and perfused rat preparations

Severe hypoxia or ischemia causes an alteration from eupnea to gasping. At body temperatures approximating 37 degrees C in vivo, the frequency of gasping is much less than that of eupnea. However in a perfused juvenile rat preparation, which is maintained at 30-31 degrees C, the frequency of eupnea and gasping is the same. We hypothesized that brainstem mechanisms responsible for the neurogenesis of eupnea and gasping might have different sensitivities to alterations in temperature. In both decerebrate adult rats in vivo and in a perfused juvenile rat preparation, eupnea and gasping had different frequencies at rectal or perfusate temperatures in excess of 34 degrees C, whereas, at lower temperatures, eupnea and gasping had identical frequencies in both preparations. These findings support the conclusion that different brainstem mechanisms underlie the neurogenesis of eupnea and gasping. In addition, these findings have implications for interpretation of results from in vitro mammalian preparations, which are examined at temperatures at which the frequency of eupnea and gasping are indistinguishable.     

Influence of levels of carbon dioxide and oxygen upon gasping in perfused rat preparation.

In vivo, the augmenting pattern of integrated phrenic nerve discharge of eupnea is altered to the decrementing pattern of gasping in severe hypoxia or ischaemia. Identical alterations in phrenic discharge are found in perfused in situ preparations of the juvenile rat. In this preparation, gasping was produced by equilibration of the perfusate with various levels of carbon dioxide and oxygen. The duration of the phrenic burst, the interval between bursts and the burst amplitude were not significantly different following equilibration with 21-6%O(2) at 5% CO(2) or with 0-9% CO(2) at 6% O(2), with the exception that the burst amplitude was significantly greater in hypercapnic-hypoxia (9% CO(2) at 6% O(2)). It is proposed that hypoxia-induced gasping results from the release of an endogenous pacemaker activity of rostral medullary neurons. This release is caused by cellular mechanisms that change the balance between membrane ionic currents. Moreover, these cellular mechanisms may be explicitly induced by alterations in the ionic and metabolic homeostasis.     

Comparison of phrenic motoneuron activity in eupnea and apneusis

Our purpose was to characterize activities of phrenic motoneurons during apneusis. In decerebrate, cerebellectomized, vagotomized, paralyzed and ventilated cats, we recorded activities of phrenic nerve and single phrenic fibers during eupnea and apneusis. Reversible apneusis was obtained by cooling the rostral pons with a fork thermode. Phrenic motoneurons were defined as 'early' or 'late' during eupnea. Early units commenced activity before or during the first 20% of neural inspiration. The onset of discharge of late units extended throughout the rest of inspiration. In apneusis, some late units ceased activity entirely; others commenced activity at the end of the rising phase of phrenic activity or during the apneustic plateau. Early units commenced activities at the same time as in eupnea and generally maintained the same discharge frequency. Hence, the ramp phase of phrenic discharge in apneusis is generated largely by activities of early motoneurons. Our results imply that the level of bulbospinal activity impinging upon the phrenic nucleus is reduced in apneusis. The integration of efferent activity within the phrenic nucleus is discussed.     

Lesions of regions for in vitro ventilatory genesis eliminate gasping but not eupnea

Medullary regions, termed `pre-inspiratory' and `pre-Bötzinger', are considered critical for the neurogenesis of rhythmic ventilatory activity of in vitro preparations of the neonatal rat. We examined the influence of destruction of neurons in these regions, by microinjections of kainic acid, upon eupnea and gasping in vivo. Decerebrate, vagotomized, paralyzed and ventilated rats of age 8–15 days were used; the phrenic nerve activity was recorded. Eupnea was not consistently altered following destruction of neurons in any region. However, in the majority of animals, anoxia-induced gasping was not observed following injections of kainic acid into the `pre-inspiratory' region, `pre-Bötzinger' complex or lateral tegmental field; the latter region is important for the neurogenesis of gasping in adults. Injections into other regions did not prevent the elicitation of gasping. These results do not support the possibility that neuronal activities which are responsible for respiratory rhythm generation in vitro underlie the neurogenesis of eupnea in vivo.     

Rostral medullary respiratory neuronal activities of decerebrate cats in eupnea, apneusis and gasping.

Eupnea is generated by mechanisms within the pons and medulla. Following removal of pons or exposure to anoxia, gasping is elicited. Eupnea and gasping are markedly different ventilatory patterns. The genesis of gasping is dependent upon rostral medullary neuronal activities. To generate the gasp, these activities should commence before the phrenic burst. In decerebrate, vagotomized, paralyzed and ventilated cats, eupnea was altered to gasping in anoxia. Rostral medullary neuronal activities had inspiratory, expiratory and phase-spanning patterns in eupnea. During gasping, some inspiratory neuronal activities commenced before the phrenic gasp; these same neurons had commenced activities after the onset of the eupneic phrenic burst. Expiratory and phase-spanning neurons did not discharge. Neuronal activities which are consonant with a role in the neurogenesis of gasping had very different discharge patterns in eupnea. Results support the concept that medullary mechanisms for gasping are incorporated in the ponto-medullary circuit responsible for the neurogenesis and expression of eupnea.     

Comparison of activities of medullary respiratory neurons in eupnea and apneusis

We evaluated patterns of antidromic latencies of medullary respiratory neurons in eupnea and apneusis to define how afferent influences from the pneumotaxic center regulate their activities. Apneusis was reversibly produced by cold block in decerebrate, vagotomized, paralyzed and ventilated cats. Most neurons, which discharged during all of eupneic inspiration or expiration, maintained the same pattern in apneusis. However, those active during only portions of these phases or spanning both changed markedly with alterations in periods of discharge, including tonic patterns or cessations of activity. Such marked changes were observed for activities of all laryngeal expiratory neurons. Upon termination of eupneic discharge, most bulbospinal and laryngeal neurons had transient peaks of latencies, indicating hyperpolarizations; declines from these peak values were greatly reduced in apneusis. Moreover, reflecting depolarizations, latencies of some inspiratory and expiratory neurons declined during eupneic expiration and inspiration, respectively; these declines were much reduced in apneusis. We conclude that the pneumotaxic center influences medullary respiratory neuronal activities not only at end-inspiration, but throughout the entire respiratory cycle.     

Prenatal cigarette smoke exposure and postnatal respiratory responses to hypoxia and hypercapnia

Prenatal cigarette smoke (CS) exposure, in combination with hypoxia and/or hyperthermia can lead to gasping and attenuated recovery from hypoxia in 7 days old rat pups. We studied 95 unanesthetized spontaneously breathing 14 days old rat pups to investigate if the destabilizing effects of increased ambient temperature and prenatal CS exposure on respiratory control observed in 7 days old rats were still evident at day 14. This postnatal age was selected as it is beyond the analogous risk period for SIDS in human. Furthermore, we investigated if the breathing responses to hypercapnia are affected by prenatal CS exposure. Since high ambient (HA) temperature can lead to gasping and aberrant respiratory control, we recorded respiratory patterns at low (24-25°C) and high (29-30°C) ambient temperatures, and under hypoxic or hypercapnic states. No gasping was observed in 14 days old rat pups. During hypoxia, breathing frequency increased in the CS-exposed group under low and HA temperatures. Rectal temperature decreased only in the sham group in response to low ambient temperature hypoxia. At HA temperature, breathing frequency increased in both sham and CS-exposed groups during hypercapnia, however, it remained elevated during washout period only in the sham group. We demonstrate that prenatal CS exposure continues to have profound effects on respiratory and thermoregulatory responses to hypoxia and hypercapnia at day 14. The attenuated respiratory and thermoregulatory responses to acute hypoxia and hypercapnia on day 14 demonstrate a strong interaction between CS exposure, respiratory control, and thermoregulation during postnatal maturation.     

Cellular electrophysiologic changes and "arrhythmias" during experimental ischemia and reperfusion in isolated cat ventricular myocardium

The cellular electrophysiologic consequences of both regional and global experimental ischemia and reperfusion were studied in the isolated cat myocardium, using conventional microelectrode techniques. Oxygenated Tyrode's solution was perfused through the left anterior descending and circumflex coronary arteries, while the preparation was superfused with Tyrode's solution gassed with 95% nitrogen and 5% carbon dioxide. Electrophysiologic characteristics of endocardial muscle cells were normal during coronary perfusion. When perfusion was discontinued for 30 minutes, resting membrane potential was decreased by 21.6 +/- 4.1%, action potential amplitude was decreased by 29.1 +/- 8.6% and action potential duration was decreased by 54.1 +/- 12.5% (p less than 0.001). Ectopic activity occurred after 5 to 10 minutes of ischemia and was more frequent in regional than in global ischemia (p less than 0.05). Rapid ventricular activity was observed in only 5 (17%) of 29 preparations during ischemia, whereas it occurred in 24 (83%) of 29 preparations during reperfusion. Rapid ventricular activity began 5 to 40 seconds (mean 19) after the start of reperfusion, stopped spontaneously after a mean of 113 +/- 211 seconds and occurred after both regional and global ischemia. The cellular electrophysiologic changes induced by ischemia returned to baseline values within the next 5 minutes. Repeated ischemia and reperfusion runs reproduced the same electrophysiologic changes and rapid ventricular activity. Coronary perfusion with procainamide (20 mg/liter) aggravated the ischemic depressions of action potential amplitude and action potential duration and increased conduction delay during ischemia, but it did not prevent rapid ventricular activity induced by reperfusion. In contrast, verapamil (1 mg/liter) perfusion did not affect the changes in action potential variables during ischemia but prevented reperfusion-induced rapid ventricular activity. Perfusion with calcium ion (Ca2+)-free Tyrode's solution just before ischemia and during reperfusion slowed or prevented reperfusion-induced rapid ventricular activity, without affecting the action potential changes during ischemia. It is concluded that, in these isolated perfused ventricular muscle preparations, different mechanisms may be operative in ischemic and reperfusion arrhythmias and Ca2+ may play an important role in the development of arrhythmias during the reperfusion phase of ischemia/reperfusion sequences.     

Brainstem amino acid neurotransmitters and hypoxic ventilatory response

The ventilatory response to acute hypoxia in mammalian species is biphasic, an initial hyperventilatory response is followed by a reduction in ventilation within 2-3 min below the peak level (roll-off). Brain amino acid neurotransmitters also change during hypoxia. This study explores the role of neurotransmitters in anesthetized adult Sprague Dawley rats mechanically ventilated during 20 min of 10% O2 breathing. Phrenic nerve activity was recorded, and microdialysate concentrations of selected amino acids were determined at 3- to 5-min intervals in respiratory chemosensitive areas of the ventrolateral medulla (VMS) 1.25-2.00 mm below the surface. Phrenic nerve output was biphasic during hypoxia, concurrent with a rapid glutamate and gradual GABA increase. Taurine first decreased, then increased. In both intact and chemodenervated animals, time-dependent change in phrenic nerve activity during hypoxia was associated with corresponding changes in glutamate, GABA, and taurine concentrations, suggesting that cumulative effects of changes in the concentration of these three amino acids could account for response of the phrenic nerve to hypoxia.     

Adequacy of oxygenation of isolated perfused rat heart

Detailed arguments are presented for the adequacy of oxygenation of the isolated perfused rat heart preparation when subjected to a work load according to the model of Neely. Lactate discharge from a heart perfused with glucose does not equal hypoxia (although lactate output is a prime sign of hypoxia in the heart in situ where numerous other substrates are available). Adequacy of oxygenation does not mean normality of oxygenation. There is still an outstanding question as to whether the high oxygen tensions might not promote lipid peroxide formation, thereby sensitizing the hearts to the potentially harmful effects of fatty acids. Bearing in mind the above reservations, the isolated perfused heart model has made major contributions to the study of normal myocardial metabolic patterns. Such a rapid pace of advance would not have been possible without the use of the Krebs-Henseleit bicarbonate buffer medium.     

Is creatine kinase a target for AMP-activated protein kinase in the heart?

By phosphorylating target proteins, AMP-activated protein kinase (AMPK) inhibits ATP-utilizing proteins and activates ATP-synthesizing proteins, thereby increasing ATP synthesis under conditions such as hypoxia and ischemia. It has been proposed that AMPK also phosphorylates and inhibits creatine kinase (CK), the enzyme which catalyzes the reversible transfer of a phosphoryl group between creatine and ADP. Here, we examine the hypothesis that AMPK inactivates CK activity under three conditions where [AMP] and AMP-dependent AMPK velocity increase: increased workload both in the isolated rat heart and in the living rat, hypoxia in the living rat heart and low-flow ischemia in the isolated red blood cell perfused rat heart. For the experiments varying workload in the isolated rat heart (both ejecting and isovolumic models), we also changed oxidizable substrate available to the isolated heart in order to vary the [AMP]/[ATP]. CK reaction velocity in the intact rat heart was directly measured using (31)P magnetization transfer. The metabolically active AMP and ATP pools were determined from (31)P NMR measurements and we calculate AMP-dependent AMPK velocity from the Michaelis-Menten relationship. We found that under normoxic conditions where [AMP] and AMPK velocity increase, the linear relationship between CK and AMPK velocities is positive, not inverse. Under conditions of low pO(2) (hypoxia and low-flow ischemia), CK velocity fell 2-4-fold while the increase in AMP-activated AMPK activity was modest. This analysis illustrates the complex nature of AMPK regulation in the heart.     

Background activity in pulmonary vagal C-fibers and its effects on breathing

Vagal cooling experiments suggest that the deep slow breathing observed after vagotomy results not only from loss of pulmonary stretch receptor feedback, but also from loss of some unidentified vagal input. To investigate this possibility we cooled the vagus nerves in anesthetized dogs. In dogs breathing spontaneously, the Hering-Breuer reflex was abolished at 7 degrees C, but average expiratory time was unchanged and lengthened only on cooling below 3 degrees C. In artificially ventilated dogs the pulmonary vagus nerves were cooled in the chest and phrenic activity was recorded. Entrainment of phrenic bursts to the ventilator cycle ceased at 7 degrees C, and expiratory pauses shortened; they lengthened again on cooling below 3 degrees C. Cervical vagotomy did not change breathing pattern after the pulmonary vagus nerves were cut. Recording of afferent impulses during cooling showed that at 5 degrees C or less pulmonary vagal input was confined largely to nonmyelinated fibers; at 3 degrees C, background activity in pulmonary C-fibers was still 78% of control whereas myelinated afferents were virtually silent. We suggest that in eupnea low frequency, background activity in pulmonary afferent C-fibers shortens expiratory time.     

Active glottal closure during anoxic gasping in lambs.

The present study was aimed at assessing laryngeal dynamics and their consequences during anoxic gasping in ketamine-sedated lambs. We first verified that the glottis was closed between gasps during anoxic gasping in seven chronically instrumented lambs, aged 11-15 days. Recording of glottal constrictor muscle electrical activity, subglottal pressure and lung volume, together with endoscopic observation, confirmed the presence of active glottal closure with maintenance of a high lung volume between gasps. Secondly, we tested whether maintenance of a high lung volume between gasps improved autoresuscitation efficiency. Six sedated lambs aged 8-11 days underwent two anoxic runs, including one with an open tracheostomy to prevent maintenance of a high lung volume. Access back to air was allowed for gasping. No significant difference was found in time to eupnea resumption, hemodynamic parameters or arterial blood gases. We conclude that a high lung volume is actively maintained by glottal closure between anoxic gasps in sedated lambs. Further studies are however needed to define the importance of laryngeal dynamics during gasping.     

Breathing pattern in rats with chronic section of the superior laryngeal nerves

In a first set of experiments we registered the integrated afferent activity of the superior laryngeal nerve (SLN) in adult anaesthetized rats. The activity increased with positive upper airway pressure (Pua); with progressively more negative Pua, the SLN activity at first declined then increased again. A second set of adult rats underwent bilateral section of the SLN (SLN denervated) or a sham operation (controls). Both groups appeared to recover promptly from the operation and 6 days later their resting breathing pattern was recorded by the barometric method. SLN denervated rats had a shorter inspiratory time (80%), hence higher frequency and mean inspiratory flow, than controls. During hypoxia (10 min at 10% O2) both groups hyperventilated with an almost identical pattern. The rats were then again anaesthetized and the right vagus cut in an attempt to reduce the afferent component from the lower airways, which may have masked the SLN regulatory contribution. One week after this second operation both SLN denervated and controls breathed more deeply and slowly than before vagotomy, but the pattern was not significantly different between the two groups, either in normoxia or hypoxia. Finally, the rats were anaesthetized and integrated diaphragm activity recorded during spontaneous breathing and the first effort against closure of the nostrils. With both vagi cut, the duration of the occluded effort was slightly longer in SLN denervated than in controls. These results suggest that in adult awake rats laryngeal afferent activity tends to decrease mean inspiratory flow. However, this regulatory contribution is small during eupnea and insignificant during hypoxic hyperventilation.     

Release of endogenous catecholamines in the ischemic myocardium of the rat. Part B: Effect of sympathetic nerve stimulation

The contribution of centrally originating sympathetic activity to the myocardial extracellular accumulation of noradrenaline during the early phase of ischemia has been assessed in a perfused (Langendorff) rat heart preparation isolated except for its sympathetic innervation. A 10-minute electrical stimulation (4 Hz, 5 V) of the left cervicothoracic ganglion during normal perfusion causes the overflow of 177.5 +/- 13.7 pmol noradrenaline/g heart, whereas such stimulation during ischemia liberates only 21.5 +/- 3.6 pmol/g (collected during reperfusion). When neuronal reuptake is blocked by desipramine, corresponding values are 321.5 +/- 22.5 pmol/g (normal flow) and 151.8 +/- 22.4 pmol/g (ischemia). After combined blockade of neuronal uptake, extraneuronal uptake, and alpha 2-receptors, nerve stimulation liberates 674 +/- 22 pmol/g during normal flow and 206 +/- 24.3 pmol/g during ischemia. These results suggest that, in vivo, centrally originating neural activity would not lead to substantial accumulation of noradrenaline within the extracellular space of the ischemic myocardium. This failure of accumulation is due to both a functioning neuronal uptake of noradrenaline and a failure of neurotransmission.     

Pulmonary C-fibers elicit both apneusis and tachypnea in the rabbit.

The effects of phenylbiguanide (PBG) on phrenic nerve and pulmonary C-fibers were studied in anesthetized spontaneously breathing rabbits with unilateral vagotomy. Right atrial injections of PBG at low (10 micrograms/kg) and high (100 micrograms/kg) dose resulted in a shallow tachypnea and apneusis followed by tachypnea, respectively, and these effects were blocked by procaine treatment of the vagus nerve. Also, the injection of PBG (100 micrograms/kg) still evoked the rapid shallow breathing preceded by apneusis in carotid chemoreceptor-denervated animals. Vigorous stimulation of pulmonary C-fibers by PBG (100 micrograms/kg) coincided with apneusis and the response was followed by a more modest increase in activity associated with tachypnea. Administration of PBG (10 micrograms/kg) into the right atrium caused an increase in pulmonary C-fiber activity associated with tachypnea. However, a small dose of PBG injected into the aortic circulation had no effect on the C-fiber activity but did inhibit respiration. These results suggest that the stimulation of pulmonary C-fibers via PBG injection can produce both inspiratory apnea and tachypnea.     

Long term facilitation of phrenic motor output

Episodic hypoxia or electrical stimulation of carotid chemoafferent neurons elicits a sustained, serotonin-dependent augmentation of respiratory motor output known as long term facilitation (LTF). The primary objectives of this paper are to provide an updated review of the literature pertaining to LTF, to investigate the influence of selected variables on LTF via meta-analysis of a large data set from LTF experiments on anesthetized rats, and to propose an updated mechanism of LTF. LTF has been demonstrated in anesthetized and awake experimental preparations, and can be evoked in some human subjects during sleep. The mechanism underlying LTF requires episodic chemoafferent stimulation, and is not elicited by similar cumulative durations of sustained hypoxia. Meta-analysis of phrenic nerve responses following episodic hypoxia in 63 experiments on anesthetized rats (conducted by four investigators over a period of several years) indicates that phrenic LTF magnitude correlates with peak phrenic responses during hypoxia and hypercapnia, but not with the level of hypoxia during episodic exposures. Potential mechanisms underlying these relationships are discussed, and currently available data are synthesized into an updated mechanistic model of LTF. In this model, we propose that LTF arises predominantly from episodic activation of serotonergic receptors on phrenic motoneurons, activating intracellular kinases and, thus, phosphorylating and potentiating ionic currents associated with the glutamate receptors that mediate respiratory drive.     

Lipoate ameliorates ischemia-reperfusion in animal models

Ischemia and reperfusion were studied in isolated working rat hearts and in exarticulated rat hind limbs. Free radicals are known to be generated in ischemia/reperfusion and to propagate complications. To reduce reperfusion injury, conditions were ameliorated including the treatment with antioxidants, lipoate or dihydrolipoate. In isolated working rat hearts, cardiac and mitochondrial parameters are impaired during hypoxia and partially recover in reperfusion. Dihydrolipoate, if added into the perfusion buffer at 0.3 microM concentration, keeps the pH higher (7.15) during hypoxia, as compared to controls (6.98). This compound accelerates and stabilizes the recovery of the aortic flow. With dihydrolipoate, ATP synthesis is increased, ATPase activity (ATP hydrolysis) reduced, intracellular creatine kinase activity maintained and thus phosphocreatine contents are higher than in controls. For exarticulated rat hind limbs, the dihydrolipoate group contained 8.3 microM in the modified reperfusate. Recovery of the contractile function was 49% vs. 34% in controls and muscle flexibility was maintained whereas it decreased by 15% in the controls. Release of creatine kinase from cells was significantly lower with dihydrolipoate. Lipoate/dihydrolipoate effectively reduced reperfusion injury in isolated working rat hearts and in exarticulated rat hind limbs after extended ischemia. Finally, the compound was successfully applied in an in vivo pig hind limb model.     

Biological and binding activities of pituitary hormones from the ostrich, Struthio camelus

Biological and binding activities of adenohypophysial hormones purified from the ostrich (ost), Struthio camelus, were compared to those of the corresponding hormones derived from mammalian and other avian species. The potency of ostrich prolactin was comparable to those of other avian preparations and slightly less active than the ovine hormone when tested in the pigeon crop-sac assay, but ostrich growth hormone (GH) was more potent than several other avian preparations and was comparable to mammalian GH in the rat tibia bioassays. Marked discrepancies were evident in the activities of both ostrich gonadotropins (ostGn) when they were tested in a variety of in vivo and in vitro bioassays and radioreceptor assays (RRAs). Both the ostrich follicle-stimulating hormone (ostFSH) and luteinizing hormone (ostLH) were among the most potent tested thus far in in vivo bioassays for total gonadotropin in a lizard and a cockerel; a high sialic acid content may account for these high potencies. OstFSH was also the most potent nonmammalian Gn tested in two FSH specific mammalian bioassays, an in vivo (ovarian augmentation) and an in vitro (cAMP production) rat bioassay; in fact, ostFSH behaved more like a mammalian than an avian hormone in these assays. However, the binding activity of ostFSH was not unlike that of other avian FSH preparations when tested in either mammalian or nonmammalian FSH-RRA systems. OstLH was more potent than other avian preparations in an in vitro mammalian bioassay, but not in avian or amphibian LH bioassays; species specificity was pronounced among these LH assays. Binding activities of ostLH in mammalian and avian LH-RRAs were generally consistent with potencies in the two species of bioassays. However, a marked discrepancy was apparent in the behavior of ostLH in FSH-RRAs. Although ostLH had very low FSH activity when tested by radioimmunoassay, by bioassay, or by FSH-RRA with avian gonads, it was equipotent to ostFSH in competing for FSH-binding sites on the mammalian gonad; in this respect it was more like turkey than chicken LH. The ability of ostLH to antagonize the biological activities of ostFSH in the stimulation of cAMP by rat seminiferous tubules confirms that ostLH binds to the same functional receptors as FSH on the rat testis, even though it does not induce the characteristic physiological response associated with FSH.