Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle.

One slow and three fast myosin heavy chains have been described in typical skeletal muscles of the adult rat using immunocytochemical analysis. Electrophoretic isolation and immunochemical identification of these four isoforms has not been achieved. An electrophoretic procedure is described which, by altering the cross-linkage and polymerization kinetics of 5% polyacrylamide gels, allows resolution of these four distinct myosin heavy chains. Using specific monoclonal antibodies and double immunoblotting analysis, the identity and electrophoretic migration order of the myosin heavy chains was established to be: 2A less than 2X less than 2B less than beta/slow.

Morphometric analysis of phrenic motoneurons in the cat during postnatal development.

The dendritic geometry of 20 phrenic motoneurons from four postnatal ages (2 weeks, 1 and 2 months, and adult) was examined by using intracellular injection of horseradish peroxidase. The number of primary dendrites (approximately 11-12) remained constant throughout postnatal development. In general, postnatal growth of the dendrites resulted from an increase in the branching and in the length and diameter of segments at all orders of the dendritic tree. There was one exception. Between 2 weeks and 1 month, the maximum extent of the dendrites increased in parallel with the growth of the spinal cord; however, there was no increase in either combined dendritic length or total membrane surface area. In addition, there was a significant decrease in the number of dendritic terminals per cell (59.8 +/- 9.3 vs. 46.4 +/- 7.4 for 2 weeks and 1 month, respectively). The distance from the soma, where the peak number of dendritic terminals per cell occurred, ranged from 700-900 microns at 2 weeks and 2 months to 1,300-1,700 microns in the adult. The diameter of dendrites as a function of distance from the soma along the dendritic path increased with age. The process of maturation tended to increase the distance from the soma over which the surface area and dendritic trunk parameter (sigma d1.5/D1.5) remained constant. The three-dimensional distribution of dendrites was analyzed by dividing space into six equal volumes or hexants. This analysis revealed that the postnatal growth in surface area in the rostral and caudal hexants was proportionately larger than that in either the medial, lateral, dorsal, or ventral hexants. Strong linear correlations were found between the diameter of the primary dendrite and the combined length, surface area, volume, and number of terminals of the dendrite at all ages studied.

Sleep architecture and continuity measures of neonates with chronic lung disease.

Electroencephalographic (EEG) sleep studies of 25 preterm neonates with chronic lung disease (CLD) corrected to a fullterm postconceptional age were compared with recordings from two groups of neonates without CLD: a fullterm appropriate for gestational age group (9 patients) and a preterm group studied at a corrected term postconceptional age (15 patients). Electrographic/polygraphic studies were obtained using 21-channel EEG recordings. Scores were tabulated based on minute-by-minute visual analyses of sleep state, number and duration of arousals, body movements and rapid eye movements (REM). A significant reduction in the percentage of active sleep was noted in the CLD group compared to both control groups (31.15% vs. 47.01% and 52.9%, respectively). The mean percentage of indeterminate sleep was significantly increased in the study group as compared to both control groups (31.23% vs. 15.18% and 11.5%). In addition, significant differences were noted between the CLD group and the healthy preterm control group with respect to the number (0.29/minute vs. 0.13/minute) and duration (4.8 seconds vs. 2.94 seconds) of arousals as well as the total number of body movements (1.57/minute vs. 0.74/minute). These data suggest that neurophysiological organization of the immature brain, as reflected in neonatal sleep architecture and continuity measures, is adversely affected in neonates with CLD. EEG sleep architecture and continuity measures may be helpful in predicting the longitudinal outcome of infants with CLD as this group is at risk for adverse neurodevelopmental outcome.

Comparison of EEG sleep measures in healthy full-term and preterm infants at matched conceptional ages.

Continuous electroencephalogram (EEG) sleep studies were obtained on healthy full-term and preterm infants at matched conceptional ages. Studies were recorded under environmentally controlled conditions. Eighteen healthy preterm infants were matched to 18 full-term infants based on conceptional age, sex, race and socioeconomic class. The initial 3 hours of a 12-hour recording were simultaneously recorded on paper and computer. The visually scored data based on the paper recordings for sleep architecture and continuity measures were studied. Differences in each sleep organization for the preterm infants included the following: a longer ultradian sleep cycle (70 minutes vs. 53 minutes, p = 0.02) was noted. More abundant tracé alternant (34% vs. 28%, p = 0.02) and less abundant low-voltage irregular active sleep (13% vs. 17%, p = 0.05) were noted. Although no differences were observed for sleep latency and efficiency, the preterm infants had fewer numbers and shorter durations of arousals, fewer body movements and rapid eye movement (REM) (p < 0.01), particularly during quiet sleep. The extrauterine experience or the earlier birth of the preterm infant may influence specific sleep architecture and continuity measures when compared with the sleep of full-term infants who experienced a complete intrauterine gestation.

Comparisons of EEG sleep state-specific spectral values between healthy full-term and preterm infants at comparable postconceptional ages.

Differences in state-specific electroencephalographic (EEG) spectral values are described between groups of preterm and full-term neonates at comparable postconceptional term ages. Eighteen healthy preterm neonates of < or = 32 weeks gestation were selected from an inborn population of a neonatal intensive care unit. Twenty-four-channel recordings were obtained at a full-term age and compared with studies of 22 healthy full-term neonates. The initial three hours of each 12-hour study were recorded on paper from which EEG sleep state scores per minute were visually assessed. Six mean spectral values (i.e. total EEG, electromyogram, delta, theta, alpha and beta energies) were calculated from each corresponding minute of digitized data, which was also assigned one of six EEG sleep states. Each neonatal group displayed statistically significant differences among sleep-state segments for all spectral values. The alpha- and beta-range spectral values of the preterm group, compared to the full-term control group, were lower during all sleep state segments. Spectral values for the theta band were lower during both quiet sleep segments only, whereas spectral values for delta were lower during all sleep stages, except tracé-alternant quiet sleep. Significant differences in EEG spectral values were noted among states of sleep for both preterm and full-term infants of similar postconceptional term ages. These data also suggest differences in central nervous system maturation between neonatal populations. These findings strengthen our previously stated contention that there is a functional alteration in brain development of the preterm infant as reflected in sleep organization that results from a prolonged extrauterine experience and/or prematurity.

Computer classification of sleep in preterm and full-term neonates at similar postconceptional term ages.

A classification strategy of neonatal sleep is being developed by comparing visually scored minutes of 21 channels of electroencephalographic (EEG)/polygraphic recordings with the corresponding values for each physiological signal derived from either visual or computer analyses. Continuous 3-hour sleep studies on 54 preterm and full-term neonates at similar postconceptional term ages were acquired under environmentally controlled conditions using a computerized monitoring system. An on-line event marker program recorded behavioral observations. One of three EEG sleep states was assigned to each of 8,995 minutes by traditional visual analysis criteria. EEG spectral values, spectral and nonspectral cardiorespiratory calculations and behaviorally observed movements, arousals and rapid eye movement counts were submitted for discriminant analysis. Based on the total minutes known for each of three states (i.e. active, quiet and awake), linear combinations of all specified digitized parameters were formed into an arithmetic algorithm by use of discriminant analysis, which served as the basis of a state assignment for each minute. Fifty percent of the data were arbitrarily used as the training set to derive the state classification model. The remaining fifty percent of the data were used as the cross-validation "test sample" to determine the accuracy of the classification when compared to the visually analyzed score for each corresponding minute. Thirteen out of 32 physiological measures best predicted state of both preterm and full-term neonatal groups. For both groups, the correct classification for active sleep was 90.3%, quiet sleep was 97.4%, awake was 97% and the overall accuracy was 93.3%. However, the order of significance for specific variables differed between these two neonatal groups. Differences in the order of variables that predict sleep states between preterm and full-term infants may reflect adaptation of brain function of the preterm infant to prematurity and/or prolonged extrauterine experience.

Maternal smoking and infant respiratory distress syndrome.

The relationship between maternal smoking and infant respiratory distress syndrome (RDS) was investigated among 550 premature (36 weeks or less) births delivered at the University of Washington Hospital from 1977 to 1980. Forty-five percent of the mothers were smokers. To avoid bias due to the reduced birth weight of infants of smokers, infants of smokers and nonsmokers were compared within small gestational age categories (two-week intervals) and not by birth weight categories. Infants of mothers who smoked had a reduced incidence of RDS for their gestation compared with infants of nonsmokers. The probability of RDS (adjusted for gestational age and method of delivery) was 25% for the infants of smokers versus 38% for the infants of nonsmokers (odds ratio = 0.55, P = .005), equivalent to approximately a 1.5-week acceleration in lung maturity for infants of smokers. The smoking effect was not explained by demographic differences between smokers and nonsmokers, nor by differences in the incidence of pregnancy complications between the two groups. This study adds support to the theory that adverse pregnancy conditions may lead to an acceleration in pulmonary maturity to allow earlier extrauterine adaptation.

Role of endogenous opiates in hypoxic ventilatory response in the newborn primate.

The effects of opiate receptor antagonism by naltrexone hydrochloride on the biphasic hypoxic ventilatory response in the infant Macaca nemestrina have been investigated. Minute ventilation, tidal volume, and respiratory frequency were measured in six animals from timed gestations before and during inhalation of a hypoxic gas mixture. All studies were completed in non-rapid-eye-movement sleep. Arterial blood gases were obtained during each stimulus period. All animals demonstrated the typical biphasic ventilatory response to acute moderate-severe hypoxemia. After the administration of naltrexone hydrochloride to block opiate receptors, the animals still manifested a biphasic hypoxic response that was no different than that noted prior to drug administration. Naltrexone hydrochloride had no effect on room air resting ventilation in any of the animals. Our data suggest that endogenous opiates play no physiological role in the acute ventilatory response to moderate-severe hypoxia in the newborn subhuman primate.

Expiratory abdominal muscle activity during ventilatory chemostimulation in piglets.

We examined abdominal muscle minute electromyographic (EMG) activity (peak moving time average EMG x respiratory rate) during eupnea, hyperoxic hypercapnia (8% CO2-40% O2-balance N2), and hypoxia (13% O2) in 12 anesthetized (0.5% halothane) newborn piglets. In addition, we assessed the role of vagal afferent pathways in the abdominal muscles' response to ventilatory chemostimulation by examining abdominal EMG activity (EMGab) before and after bilateral cervical vagotomy in five animals. Phasic expiratory EMGab was observed in 11 of 12 piglets during eupnea. Hypercapnia was associated with a sustained augmentation of minute EMGab (444 +/- 208% control). In contrast, hypoxia consistently augmented (1 min, 193 +/- 33% control) then diminished (5 min, 126 +/- 39% control) minute EMGab. Vagotomy resulted in a decline in peak moving time average EMGab by approximately one-half (48 +/- 18% control); the abdominal muscles' response to ventilatory chemostimulation, however, was qualitatively unchanged. We conclude that 1) expiration during eupnea in anesthetized newborn piglets is associated with phasic EMGab; 2) both hypercapnia and hypoxia augment minute EMGab; however, only hypercapnia is associated with sustained augmentation; and 3) although vagal afferents have a role in modulating the base-line level of EMGab, other extravagal mechanisms appear to determine the pattern of EMGab in response to ventilatory chemostimulation.

Contractile properties of the rat external abdominal oblique and diaphragm muscles during development.

We studied the in vitro contractile and fatigue properties of the rat external abdominal oblique (EAO) and costal diaphragm (DIA) muscles during postnatal development. Isometric twitch contraction (CT) and half-relaxation (RT1/2) times were longer in both the EAO and DIA muscles during the early postnatal period and decreased with age. In the first postnatal week, the CT and RT1/2 were longer in the EAO than the DIA muscle. At 14 days of age and thereafter, the CT and RT1/2 were shorter in the EAO than in the DIA muscle. Force-frequency relationships of the EAO and DIA muscles changed during postnatal development such that the relative force (percent maximum) generated at lower frequencies (less than 15 pulses/s) decreased with age. Moreover the relative force generated by the EAO muscle at lower frequencies was greater than that of the DIA muscle during the early postnatal period but less than that of the DIA muscle in adults. The specific force of both the EAO and DIA muscles increased progressively with age. There were no differences in specific force between the EAO and DIA muscles at any age. The fatigability of the EAO and DIA muscles was comparable during the early postnatal period and increased in both muscles with postnatal development. In adults the EAO muscle was more fatigable than the DIA muscle. We conclude that the contractile and fatigue properties of the EAO and DIA muscles undergo significantly different postnatal transitions, which may reflect their functional involvement in sustaining ventilation.

Effect of undernutrition on contractile and fatigue properties of rat diaphragm during development.

The present study was designed to assess the effects of combined pre- and postnatal undernutrition on the in vitro contractile and fatigue properties of the rat diaphragm during development. In vitro direct stimulation of costal diaphragm from control (CTL) and undernourished (UN) rats was done on postnatal days 1, 4, 14, 21, 30, 40, 50, and 60. Combined pre- and postnatal undernutrition resulted in stunted animal growth but did not alter the diaphragm-to-total body weight ratio. Twitch contraction time, half-relaxation time, and force-frequency relationships were not consistently affected by undernutrition. Specific twitch force and specific tetanic force were also unchanged in the UN group. Fatigue resistance was high and comparable in UN and CTL groups at days 1 and 4. At day 14 and thereafter, fatigue resistance declined but was consistently higher in the UN than in the CTL group. We conclude that combined pre- and postnatal undernutrition results in a significant increase in fatigue resistance of the diaphragm compared with CTL, whereas diaphragm muscle contractile properties are not appreciably affected by prolonged undernutrition.

Postnatal expression of myosin isoforms in an expiratory muscle--external abdominal oblique.

We studied the postnatal expression of heavy-chain (MHC) and native myosin isoforms in an expiratory abdominal muscle of the rat, the external abdominal oblique (EO). Moreover, we contrasted EO myosin expression with that of the costal diaphragm (DIA) to draw inspiratory vs. expiratory muscle comparisons during development. Examination of MHC gels demonstrated a mature phenotype of slow and adult fast myosin isoforms at an earlier age in the EO (day 60) than in the DIA [day > 115 (adult)]. The mature MHC phenotype of the EO was characterized by a preponderance of MHC 2B, whereas the DIA was characterized by approximately equal portions of MHC slow, MHC 2A, and MHC 2X. During early postnatal development, there was a delay in the expression of MHC 2A in the EO compared with the DIA. However, MHC 2B, expressed later in development in both muscles, was noted in the EO before the DIA. We conclude that 1) the EO mature myosin phenotype is characterized by a preponderance of fast myosin isoforms and 2) the EO and DIA muscles are subject to different temporal patterns of isoform expression during postnatal development.

The activity pattern of phrenic motoneurons during the aspiration reflex: an intracellular study.

Membrane potential trajectories and discharge characteristics were measured intracellularly in 29 phrenic motoneurons of anesthesized, paralyzed and artificially ventilated cats during hypercapnic respiration and the aspiration reflex. Fifteen 'active' cells discharged spontaneously during inspiration, and the remaining 14 'quiescent' cells exhibited no discharge in spite of strong central respiratory drive. The mean membrane potential of the quiescent cells during inspiration (-62 +/- 4 mV) was significantly lower than the threshold level determined for the active cells -52 +/- 4 mV). The mean axonal conduction velocity was slower for the active (60.4 +/- 8.7 m/s) than quiescent cells (67.4 +/- 6.9 m/s). All phrenic motoneurons discharged during the aspiration reflex with maximum instantaneous frequencies ranging from 6 to 357 Hz. No differences were found for the maximum discharge frequency during the reflex between the active and quiescent cells. Although there were differences in the slopes of the depolarization during inspiration between the groups of cells, no such difference existed in the slopes during the aspiration reflex. The threshold level for the first spike during the reflex was the same as that during inspiration but the level for successive spikes became progressively less negative while spike amplitude decreased and duration increased. Stimulation of the nasopharynx to elicit the aspiration reflex was found to alter the timing of the subsequent respiratory cycles.

Postnatal development of phrenic motoneurons in the cat.

The postnatal growth of phrenic motoneurons in the cat was studied using retrograde transport of horseradish peroxidase (HRP). The mean somal surface area of these developing motoneurons increased 2.5 times from day 3 to adult while the mean somal volume increased four-fold. This change in mean somal surface area during postnatal development was found to be correlated with the change in mean axonal conduction velocity measured from phrenic motoneurons.

The postnatal growth of motoneurons at three levels of the cat neuraxis.

The postnatal growth of motoneuron cell bodies located in the brainstem, cervical and lumbosacral spinal cord was investigated using retrograde transport of horseradish peroxidase in kittens ages 2, 12, 30, 55, 82 and 114 postnatal days and in an adult. The motoneurons innervating an extrinsic tongue muscle, the genioglossus, reached their adult size by eight weeks after birth. In contrast, the phrenic motoneurons innervating the diaphragm achieved adult size by 12 weeks and the motoneurons innervating the medial gastrocnemius muscle continued to grow beyond the twelfth postnatal week. The sizes of these motoneurons relative to one another remained constant during periods of development.

Computer analyses of EEG-sleep in the neonate: methodological considerations.

Neonatal EEG interpretation can aid in the estimation of central nervous system maturation, as well as provide diagnostic and prognostic information of the high-risk infant. However, one cannot easily visualize the complex interrelationships coupling EEG and polysomnographic components of the EEG-sleep rhythm. This is particularly relevant for the preterm neonate, in whom a rudimentary sleep cycle has not yet been clearly delineated. Computer analysis can augment the information derived from the visual interpretation of scalp-generated EEG activity. Automated techniques for EEG-sleep analysis have only recently been applied to a neonatal population. Such studies have been limited to full-term rather than preterm infants and rely on conventional methods that assume stationarity of neurophysiologic signals. We describe a computer system that simultaneously compares behavioral and electrographic components of EEG-sleep in a manner that preserves the integrity of the signals over time, while investigating the time- and frequency-dependent relationships among signals. Strategies for on-line and off-line editing, data storage, and off-line signal processing are described. Computational algorithms regarding analyses of EEG power, motility, and cardiorespiratory data are being used to study the ontogeny of EEG-sleep in asymptomatic preterm and full-term neonates. Computer strategies are based on both principles of stationarity and nonstationarity of physiologic signals and are applied depending on the temporal resolution required for specific signal processing needs.

Effects of a clinically detectable PDA on pulmonary mechanics measures in VLBW infants with RDS.

We examined the effect of a clinically detectable patent ductus arteriosus (PDA) and its successful treatment with indomethacin on serial measures of pulmonary mechanics in 10 very-low-birthweight (VLBW) intubated infants with respiratory distress syndrome (RDS). Pulmonary mechanics were measured by the passive expiratory flow technique. Total respiratory system compliance (Crs) gradually improved as RDS resolved. However, a significant decrease in mean Crs was associated with the development of a clinically detectable PDA, ranging from 1.51 +/- 0.21 to 0.90 +/- 0.08 mL/cmH2O/m (P less than 0.05). We also noted an increase in mean Crs, from 0.90 +/- 0.08 to 1.49 +/- 0.21 mL/cmH2O/m (P less than 0.05), after successful treatment of a PDA with indomethacin. Total respiratory system resistance (Rrs) did not change. We conclude that a clinically significant PDA is associated with a decreased Crs and that successful treatment of a PDA with indomethacin is associated with an improvement in lung compliance. These findings imply that the development of a clinically detectable PDA and its subsequent treatment complicates the interpretation of pulmonary mechanics data in VLBW infants with RDS.

Genioglossal recruitment during acute hypoxia and hypercapnia in kittens.

We examined genioglossal and diaphragmatic EMG activities in one- and two-month-old anesthetized kittens during acute exposures to hypoxic (13% or 10% O2) and hyperoxic hypercapnic (8% CO2/50% O2/balanced N2) gas mixtures. Phasic genioglossal EMG activity, frequently characterized by a combined inspiratory-expiratory discharge pattern, was observed in 3 of 8 one-month-old vs. 7 of 7 two-month-old kittens during hypercapnia (Chi-square P less than 0.05). The percentage of kittens recruiting genioglossal activity during hypoxic exposures was similar at both ages (1 month, 75%; 2 month, 83%). Analysis of the breath-by-breath response during trials of hypoxia in which genioglossal recruitment was observed, however, revealed that the one-month-old kittens exhibited phasic genioglossal activity in only 40 +/- 27% of the stimulated breaths, compared to 63 +/- 26% for the two-month-old ones (P less than 0.05) at each level of hypoxia. In this regard, the genioglossal response to hypoxia in one-month-old kittens was frequently characterized by early and only transient recruitment (when diaphragmatic activity was at its peak), while genioglossal recruitment was more sustained in two-month-old animals. These data indicate that genioglossal activity in kittens is often recruited during exposures to hypercapnia and hypoxia, and suggest that such recruitment is more frequent with increasing postnatal age.

Postnatal expression of myosin isoforms in the genioglossus and diaphragm muscles.

We studied the expression of myosin heavy chain (MHC) and native myosin isoforms in the genioglossus (GG) and costal diaphragm (DIA) muscles of the rat during postnatal development using both denaturing and nondenaturing gel electrophoresis. Primary myotubes in both fast and slow muscles homogeneously express slow as well as embryonic myosin. Since the adult GG is comprised primarily of fast MHC isoforms, whereas the adult DIA is characterized by a mixture of MHC slow and fast isoforms, we hypothesized that the GG and DIA would be subject to different temporal patterns of MHC isoform expression during postnatal development. Native myosin and MHC gels demonstrated a persistence of neonatal MHC (MHC neo) on day 25 in the GG, whereas this isoform was not detected beyond day 21 in the DIA. The MHC phenotype in GG of the adult demonstrated a predominance of MHC 2X (35% +/- 8) and MHC 2B (45% +/- 10) with a smaller proportion of MHC 2A (19% +/- 5). In contrast, the MHC phenotype in adult DIA was characterized by approximately equal proportions of MHC slow (25% +/- 3), MHC 2A (34% +/- 10), and MHC 2X (31% +/- 12) with a small percentage of MHC 2B (9% +/- 7). These data suggest that postnatal regulation of MHC expression in the GG and DIA is muscle specific.

Costal and crural diaphragm, and intercostal and genioglossal electromyogram activities during spontaneous augmented breaths (sighs) in kittens.

Spontaneously occurring augmented breaths (sighs) are common in infants. The pattern of electrical activity of the inspiratory muscles of the thorax and upper airway during augmented breaths, however, has not been fully characterized in this less than fully mature age group. We therefore examined costal and crural diaphragm and external intercostal and genioglossal EMG activities during spontaneous augmented breaths (n = 46) in 10 anesthetized (1.35% halothane) 1-month-old kittens breathing room air. EMG responses were assessed by comparing the spontaneous augmented breaths (AB) to the five immediately preceding breaths (control). The peak moving time average EMG activity observed during the AB was 240 +/- 32% (mean +/- SD) of control for the costal diaphragm, 279 +/- 66% of control for the crural diaphragm, and 274 +/- 68% of control for the external intercostal muscle. The mean increase in EMG activity during the AB was not significantly different among these three muscle groups (P greater than 0.25). Genioglossal EMG activity during AB was observed in only 1 of 10 study animals. These results document that during AB in anesthetized kittens, activity of the thoracic inspiratory muscles (costal/crural diaphragm and external intercostal muscles) increase in parallel, suggesting that they are modulated in a uniform manner. The infrequent observance of genioglossal activity during AB suggests that either 1) halothane anesthesia depresses genioglossal activity more than diaphragmatic and intercostal activity during AB or 2) that genioglossal recruitment is not necessary to maintain upper airway patency during this period of heightened respiratory drive.