Effects of pentobarbital on proopiomelanocortin opioid products of neonatal piglets during normoxia and hypoxia.

Abstract Endogenous opioids have been shown to suppress physiological functions in the neonate. It has been suggested that anesthesia with barbiturates might enhance this suppression by influencing opioid systems directly. To explore this possibility, naive piglets, 2.2+/-0.8 (X+/-SD) days old, underwent one of five protocols: 1) normoxia (control); 2) 10% 0(2)/90% N(2) (hypoxia); 3) saline injection ip during normoxia (sham anesthesia); 4) pentobarbital sodium, 25 mg/kg ip, during normoxia (barbiturate anesthesia); and 5) pentobarbital sodium, 25 mg/kg ip, during hypoxia (combined hypoxia and barbiturate anesthesia). Following the inhalation of either gas mixture for at least 30 min, and precisely 30 min after an injection, blood, cerebrospinal fluid and a dorsal medullary slice containing the nucleus tractus solitarii were collected and processed for measurement by radioimmunoassay of opioid proopiomelanocortin products. These comprised beta-lipotropin (the precursor), beta-endorphin-like immunoreactivity (containing the active peptide beta-endorphin) and N-acetyl beta-endorphin (a deactivated peptide). The most striking result was seen in the cerebrospinal fluid: As compared to barbiturate anesthesia, peptide levels with all other treatments, including combined hypoxia and barbiturate anesthesia, were consistently higher. In the plasma, peptide levels after either combined hypoxia and barbiturate anesthesia or hypoxia alone were generally higher than those of their respective controls (sham anesthesia, control). Plasma levels of beta-endorphin-like immunoreactivity and estimated beta-endorphin with combined hypoxia and barbiturate anesthesia were also higher than those with barbiturate anesthesia. The latter pattern was reversed in the nucleus tractus solitarii, in which beta-endorphin-like immunoreactivity and estimated beta-endorphin levels were lower with combined hypoxia and barbiturate anesthesia than with barbiturate anesthesia alone, although no significant differences were achieved. These results suggest that pentobarbital may decrease the central neuronal release of active endorphins, and thus decrease the quantity of these ligands available for interaction with opioid receptors. Hypoxia, on the other hand, appears to increase such release even in the presence of pentobarbital. Thus, during a hypoxic insult, the suppressive influence of opioids on physiological functions would be enhanced regardless of the presence of barbiturate anesthesia.

Proopiomelanocortin opioids in brain, CSF, and plasma of piglets during hypoxia.

beta-Endorphin-like immunoreactivity (BELI), containing the biologically active beta-endorphin, its precursor beta-lipotropin (BLP), and deactivated product N-acetyl-beta-endorphin (ABE), were measured by radioimmunoassay in plasma, cerebrospinal fluid (CSF), and in a dorsal medullary slice containing the respiratory-related nucleus tractus solitarius (dmscNTS) of young and older piglets in normoxia and hypoxia. Significant increase with hypoxia occurred in the levels of BLP in the plasma and CSF and of BELI and ABE in the plasma of the young group. In the older group, such increases occurred in ABE levels of the dmscNTS, in BLP and ABE levels of the CSF, and in plasma BELI. Estimated levels of pure beta-endorphin were higher in the CSF of young piglets during both control and hypoxia. With hypoxia, these estimated levels increased significantly in the plasma of the young age group but showed only a borderline increase in the old group. It is possible that higher opioid levels in the CSF of young as compared with older neonates, enhanced by a greater opioid increase in their plasma during hypoxia, may help explain the suppressed respiratory response to hypoxia of the newborn.

Neurochemicals and respiratory control during development.

During ontogeny, the central nervous system undergoes neuronal growth, regression, and remodeling. The development of neurotransmitter and modulator systems is a plastic process with individual temporal characteristics for each system. These characteristics include the synthesis, degradation, or uptake of neurochemicals and, largely independently, the appearance of their receptors. Message transmission during ontogeny is compounded by the variable development of these systems and by the coexistence and cofunction among these chemicals. Nine neurochemical systems are discussed: adenosine, gamma-aminobutyric acid, opioids, prostaglandins, serotonin, progesterone, substance P, thyrotropin-releasing hormone, and the catecholamines. The possible role of each of these in natural perinatal respiratory control is evaluated according to predetermined criteria. These include the presence of a substance system in respiratory-related regions, physiologically appropriate changes in its concentration in these regions, elicitation of respiratory effects by agonists and antagonists, and abolition with an antagonist of the effect of a substance during its presumed activation by a physiological process. It is suggested that excessive levels of suppressant neuromodulators or an imbalance among neurochemicals can partly explain the special features of respiratory control in the perinatal period.

Chronic prenatal cocaine retards maturation of state and of respiratory patterns in swine.

This study assessed effects of prolonged prenatal cocaine exposure on respiratory pattern and sleep-wake states in a postnatal porcine model. Yucatan miniature sows received 2 mg/kg cocaine intravenously four times daily during 0.66-1.0 gestation. At birth, cocaine-exposed litters were fostered to unexposed paired sows and their litters. Chronically instrumented piglets were studied at 3-9 (young) and 21-31 days (older). Sleep-wake states were determined from electrocorticogram, eye movements, submental electromyogram, and behavior, and respiratory patterns were determined from diaphragmatic and posterior cricoarytenoid electromyograms (EMGdi and EMGpca, respectively). Under baseline conditions, prenatal cocaine 1) increased the number of apneas expressed by silence of EMGdi or EMGpca and prolonged the duration of EMGpca-related apneas at both ages; 2) increased the number of periodic breathing episodes at both ages; 3) increased percent time of active sleep and decreased that of wakefulness at both ages; and 4) increased time in quiet sleep in the older animals, producing in them a sleep-wake distribution similar to that of the young neonates. Whereas the findings in the youngest piglets may have been influenced by persistent systemic cocaine, those in the older preexposed piglets, devoid of systemic cocaine, imply that chronic prenatal cocaine retards the postnatal maturation of state and respiratory pattern.

Occlusion pressure response to inspiratory flow-resistive loading in anesthetized swine.

Conscious animals subjected to inspiratory flow-resistive loading augment respiratory drive [as measured by airway occlusion pressure (P100)] independently of changes in chemical drive. Past studies of anesthetized subjects, however, have failed to demonstrate this response, and investigators have concluded that its presence depends on a state of consciousness. We tested the hypothesis that respiratory depression due to anesthesia or endogenous opioids rather than unconsciousness per se was responsible for this observation. Miniature piglets were anesthetized with ketamine and xylazine and subjected to hyperoxic CO2 rebreathing trials with and without added inspiratory resistance, before and after treatment with the opioid antagonist naltrexone. Before naltrexone there was a parallel leftward shift in the occlusion pressure vs. PCO2 relationship without a change in slope (delta P100/delta PCO2). After naltrexone there was a 45.5 +/- 15% increase in slope with loading. Addition of incremental doses of pentobarbital markedly reduced this increase in slope. We conclude that anesthetized animals can demonstrate flow-resistive load compensation in the form of augmented neuromuscular output not due to increased chemical drive. Failure to observe this response in past studies may reflect respiratory depression due to the anesthetic agents employed.

Microdialyzed adenosine in nucleus tractus solitarii and ventilatory response to hypoxia in piglets.

Levels of adenosine, inosine, and hypoxanthine from the interstitial space at the nucleus tractus solitarii were measured by microdialysis in eight 20- to 25-day-old anesthetized spontaneously breathing piglets. Microdialyzed samples were collected every 30 min for 2 h after the insertion of the probe to ensure stability of purine levels and then during 30 min each of normoxia, hypoxia (10% O2-90% N2), and normoxia. The purines were separated by high-pressure liquid chromatography with ultraviolet detection and quantified at 254-nm wavelength. Tidal volume, breathing frequency, minute ventilation, mean arterial blood pressure, pH, and gas tensions were measured. Compared with control, adenosine levels during hypoxia increased by 40.7 +/- 5.5% and then tended to decline during the recovery from hypoxia, but the levels remained higher than in control. Ventilatory measures exhibited a modest biphasic pattern during hypoxia and resumed control values by 10 min after the removal of the hypoxia. The increased adenosine release during hypoxia provides additional evidence for the possible participation of adenosine in the central suppression of breathing during hypoxia.

Curtailed respiration by repeated vs. isolated hypoxia in maturing piglets is unrelated to NTS ME or SP levels.

In early development, respiratory disorders can produce recurring hypoxic episodes during sleep. To examine possible effects of daily repeated vs. isolated hypoxic hypoxia, cardiorespiratory functions and central, respiratory-related neuromodulator levels in 21- to 32-day-old, chronically instrumented, unsedated piglets were compared between a fifth sequential daily hypoxia and an isolated hypoxia (10% O2-90% N2 for 30 min). Diaphragmatic electromyographic activity, heart rate and arterial pressure, and pH and gas tensions were measured. In vivo microdialysis, via chronically implanted guides, served to sample interstitial substance P (SP) and methionine-enkephalin (ME) at the level of the respiratory-related nucleus tractus solitarii (NTS). Compared with an isolated hypoxia, repeated hypoxia resulted in 1) lower respiratory frequency (f), ventilation equivalent, and arterial pH, higher arterial PO2 during hypoxia, and lower f in recovery from hypoxia; and 2) increased SP concentrations but no change in ME concentrations. We conclude that, in these maturing swine, repeated vs. isolated hypoxic exposure curtails respiratory responses to hypoxia by a mechanism(s) unrelated to SP or ME levels at the NTS.

Met-enkephalin-like immunoreactivity in microdialysates from nucleus tractus solitarii in piglets during normoxia and hypoxia.

Met-enkephalin-like immunoreactivity in microdialysates from the respiratory-related nucleus tractus solitarii was determined simultaneously with ventilatory responses in seven, spontaneously breathing, developing swine under conditions of normoxia, hypoxia and recovery from hypoxia for 30 min each. Assayed levels of Met-enkephalin-like immunoreactivity in normoxia were 0.89 +/- 0.23 pg/microliters. These levels increased to 203.6 +/- 32.2% and 283.1 +/- 55.8% of control during hypoxia and recovery, respectively. Hyperventilation during hypoxia was not sustained, comprising brief stimulation followed by return to near-control level. Taken together, these results provide further evidence that opioid release may contribute to the suppression of ventilation in hypoxia during development.

Age-related mu-, delta-and kappa-opioid ligands in respiratory-related brain regions of piglets: effect of prenatal cocaine.

Neonates, as compared to older subjects, exhibit increased signs of relative respiratory suppression such as apnea, periodic breathing and only transient hyperventilatory response to hypoxia. Prenatal cocaine exposure exaggerates the respiratory pattern disturbances observed in infants. As endogenous opioids cause central suppression of breathing, we tested their possible involvement in these effects by assessing opioid content in respiratory-related brainstem regions of 2 to 5 (young) and 18 to 22 (older) day-old piglets, unexposed or preexposed to cocaine during 0.66 to 1.0 gestation. The selected ages represent distinct stages in the postnatal development of respiration. beta-Endorphin, methionine-enkephalin, dynorphin A and dynorphin B from the tractus solitarii, ambigualis, gigantoreticularis and parabrachialis medialis nuclei were separated by high performance liquid chromatography, then quantified by radioimmunoassays. Opioid content was higher in the brain regions of the young than of the older piglets, and increased after cocaine exposure in both age groups, but more in the young. These findings support the possible contribution of high opioid content to the relative suppression of respiratory function in early life, and to the exaggerated respiratory dysrhythmia observed in cocaine preexposed neonates.

Effects of age and clustered hypoxia on [(125)I] substance P binding to neurotachykinin-1 receptors in brainstem of developing swine.

This work focused on the postnatal development of substance P-bound neurotachykinin-1 (NK-1) receptors in the porcine brainstem using 2-3-, 6-11-, 16-18-, and 21-28-day-old piglets versus adult, and on alterations in these receptors after single and six-daily repeated clustered hypoxia using 6-11- and 21-28-day-old piglets. NK-1 receptor localization and densities were determined by quantitative autoradiography using mono-iodinated Bolton-Hunter substance P ([(125)I]BHSP). Slide-mounted brainstem sections, incubated in [(125)I]BHSP and then exposed to film, have shown [(125)I]BHSP binding throughout many brainstem nuclei and tracts, including the ambigual/periambigual (nAmb), dorsal motor vagal (dmnv), gigantocellular (nGC), hypoglossal (nHyp), medial parabrachial (nPBM), lateral reticular (nRL), raphe magnus (nRMg), raphe obscurus (nROb) and solitary tract (nTS) nuclei. NK-1 receptor densities decreased with age. As compared to normoxia, NK-1 receptor densities increased significantly after the six-daily hypoxia protocol in nAmb, dmnv, nHyp, nRL, nRMg, nROb, and nTS of both the young and older age groups. This increase may represent receptor upregulation as an adaptation to repeated hypoxia.

Mu- and delta-opioid receptor densities in respiratory-related brainstem regions of neonatal swine.

The piglet displays similar postnatal development in respiration and sleep-wake behavior to the human. To shed light on the possible influence of opioid systems on these functions, this study assessed the density of mu- and delta-opioid receptors in brainstems of 2-3 and 5-7 (young), 14-17 (intermediate) and 20-21 (older) day-old piglets, using quantitative autoradiography. Serial 10 microns sections from fresh-frozen brains were incubated with either mu-(125I-DAGO) or delta-(125I-DPDPE) opioid ligands. The binding characteristics of each receptor remained unchanged over the age-range studied. delta-opioid receptor density was minimal in the young piglets, and increased over the age-range studied in all brainstem regions. mu-opioid receptor density exceeded delta-opioid density in all brainstem regions in young and older piglets, and remained unchanged with age. We conclude that, as in other species, the development of delta-opioid receptors in swine lags behind that of mu-opioid receptors, and that the distribution of each in the piglet's brainstem is distinct. The present findings help explain the changing influence of the mu- and delta-opioid systems on breating and state during postnatal development.

Age-related electrocorticographic and respiratory adaptation to repeated hypoxia.

Severe hypoxia is known to produce depression in electrical brain activity and perturbation of respiratory pattern. In piglets undergoing chronic recording of brain and respiratory muscle activities, a depressed electrocorticogram (ECoG) was observed in response to rapidly induced (< 30 s), brief (10 min), and moderate hypoxia (10% O2 in 90% N2) in 16 out of 42 study sessions in young (3- to 11-day-old) animals only. Responses to hypoxia were monitored over 4 consecutive days. In five cases, the latency to the onset of the ECoG depression increased progressively over the 4 test days, and its duration decreased progressively. An associated respiratory gasping pattern also exhibited gradual remission over consecutive days. These changes in the responses to repeated hypoxia demonstrate adaptation of mechanisms underlying neuronal perturbation by oxygen deprivation.

Repeated prenatal cocaine increases met-enkephalin immunoreactivity in respiratory-related medulla of developing swine.

Repeated prenatal exposure to cocaine is associated with attenuated respiratory and arousal responses in infants and piglets. As the normal development of these functions is influenced by medullary opioid systems, the present study explored the possible contribution of the opioid systems to the attenuation induced by cocaine. Methionine-enkephalin (met-enkephalin), an endogenous opioid peptide, was delineated by immunocytochemistry in respiratory- and arousal-related medullary regions of relatively immature (6-7-day-old) and more mature piglets (20-21-day-old). The animals were either unexposed, or exposed prenatally to 2 mg/kg cocaine four times daily administered to the pregnant sows intravenously throughout the last third of gestation. At control, met-enkephalin was found in the neurons, fibers and terminals of the respiratory- and arousal-related medullary regions throughout the age range studied. Prenatal cocaine exposure increased met-enkephalin immunoreactivity in the respiratory-related hypoglossal and solitary tract nuclei of both age groups. These findings support a modulatory role of met-enkephalin in the normal development of respiratory control, and an involvement of this peptide in the attenuation of respiration by repeated prenatal exposure to cocaine.

Electrocorticographic activity during repeated vs continuous hypoxia in piglets.

To assess the effects on brain activity of repeated vs continuous hypoxia, 16, 10- to 22-day-old piglets were instrumented chronically for electrocortical and arterial pH and gas tension measurements. They inhaled 10% or 6% O2 in N2 for 21 min, either continuously, or during seven, 3 min exposures interrupted by 3 min recovery periods in air, all while behaving naturally within a sealed, temperature controlled, plexiglass box. An isoelectric electrocorticogram (ECoG) and/or seizures, related to the onset of hypoxia, occurred repeatedly in 6 of the 10 exposures to 6% repetitive hypoxia, only twice in 8 exposures to 6% continuous hypoxia, and never in 10% hypoxia. A frequency analysis of the ECoG, excluding all sections exhibiting isoelectric and seizure activity, revealed no changes with 10% hypoxia, but a shift towards the lower bands during both repetitive and continuous 6% hypoxia. The extent of these shifts was greater in records that also displayed isoelectric ECoG and/or seizures. The ECoG spectrum recovered at the end of the hypoxic exposure, but not when isoelectric ECoG and/or seizures coexisted. We conclude that repeated, frequent episodes of hypoxia are more detrimental than a prolonged single event, and may contribute to the occurrence of Sudden Infant Death.

Prenatal cocaine alters normoxic sleep-wake and diaphragmatic EMG patterns in piglets.

This study assessed in piglets the effects of prenatal cocaine administration on sleep-wake states (SWS) and respiratory parameters, utilizing diaphragmatic electromyogram (EMGdi) recordings during normoxia before and after hypoxia (0.10 F(I,O2), 10 min). We asked whether the respiratory effects were linked to a specific SWS, and whether there was a difference in respiratory measures between the two normoxic conditions. Unsedated, chronically instrumented 3-9- or 21-31-day-old piglets, representing distinct stages in developmental respiratory control, were used. In pre-hypoxic normoxia, prenatal cocaine enhanced sleep at the expense of wakefulness and increased EMGdi amplitude, slope, and area in both age groups regardless of SWS; after the hypoxia, the respiratory findings persisted in the young group, but disappeared in the older group [corrected]. In the young group and regardless of SWS, interbreath interval (ttot) and expiratory duration (ttot - tEMGdi[duration of EMGdi]) were shorter in the cocaine-exposed than in the unexposed piglets, and ttot, tEMGdi, and (ttot - tEMGdi) decreased from pre- to post-hypoxic normoxia. In the older group, ttot and (ttot - tEMGdi) differed among SWS, but were unaffected by drug treatment; tEMGdi was higher with cocaine exposure in pre-, but not in post-hypoxic normoxia, and two-thirds of the EMGdi measurements during post-hypoxic normoxia exhibited a similar magnitude in the drug-treated and untreated groups regardless of SWS. We conclude that 1) prenatal cocaine alters both SWS and EMGdi, but the EMGdi effects are independent of SWS; and 2) the similar EMGdi patterns in the older group after hypoxia, regardless of drug treatment, suggest that hypoxia and chronic prenatal cocaine might alter EMGdi by similar mechanisms.

Primary central alveolar hypoventilation in a child: early diagnosis during acute illness; trials with respiratory stimuli; studies related to endorphins.

Primary central alveolar hypoventilation (PCAH) has been described in adults, children, and infants. It is usually diagnosed clinically at an advanced stage when the secondary effects of chronic hypoxia and hypercapnia become evident. We report here PCAH in its early stage in a 7-year-old child, whose disease appeared as acute respiratory failure during an acute illness. When the acute illness subsided, the salient features of PCAH remained and were studied in some detail. We also describe the child's response to respiratory stimuli, and some results related to endorphins, including his plasma endorphin levels and response to a trial of naloxone.

Respiratory responses of piglets to hypercapnia during postnatal development: effects of opioids.

Resting respiratory and cardiovascular functions and the response to CO2 rebreathing were compared between 2.5 +/- 0.7 (mean +/- SE) and 34.1 +/- 1.9 day old piglets, before and after the opioid antagonist naltrexone (1 mg/kg IV). At rest, tidal volume, both absolute and per m2, inspiratory and expiratory time, absolute minute ventilation, and mean arterial pressure increased with age, and breathing frequency, minute ventilation per m2, and heart rate decreased, all of these with as well as without naltrexone. During hypercapnia, the pattern, but not the quantitative aspects of breathing changed with age. At rest, naltrexone produced hyperventilation in the young, but not in the older group. During hypercapnia, naltrexone had a sparse effect in both ages. We conclude that, in the anesthetized piglet, ventilatory functions at rest undergo change with postnatal age, but breathing responses to hypercapnia exhibit maturation in pattern only and not in magnitude. Whereas resting ventilation of young piglets is modulated by endogenous opioids, hypercapnia may activate opioids to a limited extent and in a manner unrelated to age.

Prenatal cocaine raises mu-opioid receptor density in piglet cardiorespiratory medulla.

Repeated prenatal exposure to cocaine attenuates arousal and cardiorespiratory functions in neonates. This study explored the possible role of brainstem mu- and delta-opioid systems in these effects. Medullary sections were obtained from 6 to 7 (young) and 20 to 21-day-old (older) piglets either unexposed or exposed prenatally to a 2-mg/kg intravenous cocaine hydrochloride dose, injected to the pregnant sow four times a day during the last third of gestation. Mu- and delta-opioid receptor binding was assessed by quantitative autoradiography using, respectively, 125I-DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol) and 125I-DPDPE (Tyr-D-Pen-Gly-pCl-Phe-D-Pen). At control, delta-, but not mu-opioid, receptor density increased with postnatal age. In contrast, cocaine-induced mu-, but not delta-opioid, receptor density increased 1) in the dorsal motor vagal (dmnX) and facial (nF) nuclei, and, at borderline significance level, in the cardiorespiratory-related gigantocellular reticular nucleus (nRG) of the young, and 2) in the spinal trigeminal nucleus and tract (nSp5), and in the cardiorespiratory-related medial solitary tract (nTSm) and lateral reticular (nRL) nuclei of both age groups. These findings support a possible participation of the mu-opioid system in the attenuation of arousal and cardiorespiration after repeated prenatal exposure to cocaine.

Prenatal cocaine alters open-field behavior in young swine.

Yucatan minisows received 2 mg/kg cocaine i.v. 4 times daily during the last third of gestation. Their piglets were fostered at birth to paired, unexposed sows with their litters, and studied at age 2 to 9 (young group) and 22 to 29 days (older group). Three to 5 exposed and unexposed piglets of each age group were videotaped together for 30 min on 5 consecutive days in an open-field environment. For each piglet, 41 behaviors were scored, timed, summed and clustered into 9 behavioral categories. With age, and independently of drug exposure, piglets spent more time in ingestion, immobility while alone and play/aggression, and less time in group locomotion. For the first 4 test days, the young exposed piglets spent more time in group immobility and less time in individual locomotion and rooting than their age-matched controls. In contrast, the older exposed and unexposed piglet groups did not differ in any of these behavioral clusters. These results suggest that prenatal cocaine exposure in neonatal swine may transiently affect responses to spatial novelty.