Pulmonary and pericardial limitations to diastolic filling of the left ventricle of the lamb.

To determine which tissues limit left ventricular (LV) filling during the early neonatal period we studied LV end-diastolic pressure-diameter relationships in two groups of halothane-anesthetized lambs (five 1-h-old lambs and six 2- to 17-day-old lambs). First, we assessed LV end-diastolic pressure-diameter relations over a range of 5-20 mmHg, when the chest and the pericardium were closed; second, after the chest wall and lungs had been retracted from the heart; and finally, after the pericardium was retracted. In the oldest lambs LV diameter increased significantly [3.5 +/- 0.3% (SE) at an end-diastolic pressure of 10 mmHg; P < 0.05] after retracting the chest wall and the lungs and when the pericardium was still intact. By contrast, retracting the chest wall and lungs did not significantly change LV diameter in the youngest lambs. In both age groups LV diameters were greatest (P < 0.05) after the pericardium was subsequently retracted (the increase was evident at all end-diastolic pressures and averaged 9.0% relative to the closed-chest, closed-pericardium series). These studies confirm that the thoracic tissues substantially limit LV filling in young lambs. Immediately after birth this limitation is almost exclusively related to the pericardium, whereas in older lambs the chest wall-lung combination and the pericardium each contribute.

Fetal lung growth. Influence of pulmonary arterial flow and surgery in sheep.

Aberrant pulmonary arterial flow has been associated with pulmonary hypoplasia, which is a common cause of death in newborns. The current experiments were designed to confirm whether interruption of postductal main pulmonary artery (MPA) flow causes pulmonary hypoplasia. In addition, the effect of fetal surgery on lung growth was investigated. Fetal sheep, instrumented at 107 to 115 d gestation (MPA ligated [n = 5], sham operated [n = 3], and monitored [no thoracotomy, n = 5]), and unoperated twin fetuses (140-d controls, n = 5) were used for analysis of lung growth at 136 to 140 d gestation. Morphometric measurement of lung tissue volumes and assays of DNA, protein, and saturated phosphatidylcholine (SPC) were used to assess lung growth. MPA ligation significantly decreased lung growth and maturation as indicated by decreased surface area and volumes of fine nonparenchyma, future airspace, and parenchymal tissue, and by decreased lung weights, total DNA, protein, and SPC content. There was a significant increase in the volume percentage of coarse nonparenchyma and a decrease in the volume percentage of future airspace. The pattern of lung growth after MPA ligation suggests that growth of peripheral airspace and parenchymal tissue components does not occur, resulting in a significant decrease in the amount of new tissue formed and delayed tissue maturation. Fetal surgery significantly changed only DNA, protein, and SPC content. Studies of the regulation of lung growth must consider the role of pulmonary arterial flow and the smaller, but significant effects of fetal surgery.

Changes in pericardial pressure during the perinatal period.

BACKGROUND: To determine how the tissues that surround the heart affect diastolic and systolic function during the perinatal period, we studied the pressure-diameter relation of the left ventricle in partially delivered fetal lambs. METHODS AND RESULTS: We anesthetized (1.5-2.0% halothane, balance O2) and ventilated six pregnant ewes (142-144 days of gestation) and then partially delivered each lamb by cesarean section. Each lamb was instrumented to record left ventricular anteroposterior diameters (endocardial ultrasonic transducers), pericardial pressure (liquid-containing balloon), and left ventricular pressure (transducer-tipped catheter). Left ventricular pressure-diameter relations were recorded under three conditions: initially, with a closed chest and closed pericardium (before ventilation); second, after interruption of the umbilical circulation and 1 hour of ventilation; and finally, when the lungs and the pericardium were retracted from the heart. Pericardial pressure (recorded at a common diameter, i.e., the maximal end-diastolic diameter recorded before ventilation) decreased by 48% after 1 hour of ventilation (p < 0.05). After ventilation, left ventricular anteroposterior diameters were 4-5% greater (p < 0.05) at each end-diastolic pressure compared (12.5, 15.0, 17.5, and 20 mm Hg). Thus, ventilation appeared to increase left ventricular diastolic compliance. Contractility also appeared to increase after ventilation when evaluated using ventricular stroke work as a function of end-diastolic pressure as preload. When we used a more appropriate measure of preload (i.e., transmural end-diastolic pressure), ventilation did not change left ventricular diastolic compliance or contractility. Thus, left ventricular systolic function increased because of an increase in preload. CONCLUSIONS: The tissues surrounding the fetal heart significantly augment pericardial pressure and limit left ventricular preload. The initiation of ventilation reduces pericardial pressure, increases left ventricular preload, and increases left ventricular systolic function. At birth, a decrease in pericardial pressure and the resulting increase in preload may help increase left ventricular output through the Frank-Starling mechanism.

Effects of external constraint on the fetal left ventricular function curve.

To determine if external ventricular constraint significantly limits fetal left ventricular (LV) stroke volume and can thus account for the plateau of the fetal ventricular function curve, we studied nine fetal lambs (142 to 144 days' gestation) after partial delivery by cesarean section (halothane anesthetic). LV stroke volume (electromagnetic flow probe), LV end-diastolic pressure, and external ventricular constraint (intrapericardial pressure [liquid-filled balloon]) were measured over a range of end-diastolic pressures under two conditions: with a closed chest and closed pericardium and with an open chest and open pericardium. Stroke volume recorded during open chest and open pericardium exceeded those recorded during closed chest and closed pericardium at any given end-diastolic pressure (p less than 0.01). Decreases in external ventricular constraint significantly increased LV transmural pressure (preload) and substantially increased fetal LV stroke volume. Thus the plateau of the fetal ventricular function curve was largely a result of external ventricular constraint limiting LV preload, not necessarily a result of myocyte immaturity.

Main pulmonary artery ligation reduces lung fluid production in fetal sheep.

Pulmonary hypoplasia is increasing as a cause of neonatal death. To understand the pathophysiology of pulmonary hypoplasia, the physiology of fetal lung growth must first be understood. Lung fluid production and fetal breathing are primary factors regulating lung growth. Interruption of pulmonary arterial flow also decreases fetal lung growth. To define the relationship of pulmonary arterial flow to other factors known to be important for fetal lung growth, breathing and lung fluid production were measured after postductal main pulmonary artery (MPA) ligation in fetal sheep. Surgical preparation at 107-116 d gestation included placement of vascular catheters and a tracheal catheter connected to an intrauterine collection bag for lung fluid. Five fetuses served as monitored controls (catheters only), 3 as sham operated controls (catheters and thoracotomy), and 7 had MPA ligation. MPA ligation significantly decreased lung weights at 131-140 d; mean dry weight (g): MPA ligation--6.7, sham--23.4, monitored--22.3. Mean rates of lung fluid production (mL/h) were also decreased (d gestation): 116-122 d: MPA ligation--2.2, sham--9.1, monitored--6.8; 123-129 d: MPA ligation--2.1, sham--9.1, monitored--6.2; 130-136 d: MPA ligation--1.5, sham--12.4, monitored--7.7. There were no differences between MPA ligated, sham, and monitored fetuses in the incidence or intensity of fetal breathing movements. Decreased lung fluid production after main pulmonary artery ligation is most likely due to decreased secretion of lung fluid. Pulmonary arterial flow in other models of pulmonary hypoplasia which decrease lung fluid production (i.e., oligohydramnios) should also be examined.

Breathing at birth and the associated blood gas and pH changes in the lamb.

We examined the relationship between the initiation of breathing at birth and the timing of delivery of the chest in a group of 13 lambs undergoing spontaneous unassisted delivery at term. In 8 of 11 lambs with a diaphragm electromyogram or intrapleural pressure signal the first breath occurred before chest delivery. The first breath was always followed by a period of irregular and often powerful inspiratory efforts. Some of these inspirations were followed by a forceful expiration. A regular respiratory rhythm never developed until the chest had delivered, suggesting that chest expansion is essential for the establishment of rhythm. Although PaO2 increased rapidly after birth, pHa declined and reached its lowest level approximately 20 minutes postnatally suggesting that considerable anaerobic metabolism occurs in the face of adequate arterial oxygenation. The level of respiratory activity in the first 30 min following birth did not appear to be related to arterial PO2, PCO2 or pH. Neither the appearance of the EMG activity of the first breath, nor blood samples taken from 2 lambs simultaneously with the first breath, suggested that the first breath was a gasp initiated by asphyxial blood gases. Our results therefore do not support the current hypothesis that the first breath is a gasp initiated by asphyxia accompanying delivery.

Morphometric study of the role of pulmonary arterial flow in fetal lung growth in sheep.

Pulmonary hypoplasia has been associated with absent or hypoplastic pulmonary artery in four cases in humans. Despite these reports, the effects of decreased pulmonary arterial flow on fetal lung growth have not been adequately studied. This study defines the effects of left pulmonary artery (LPA) ligation on fetal lung growth in sheep by comparing morphometrically determined pulmonary volumes from LPA-ligated, sham-operated, and un-operated control fetuses. LPA ligation (n = 5) or sham operation (n = 4) was performed at 105- to 114-d gestation. lungs were intratracheally fixed for light microscopy. At 112 d (n = 4) and at 140 d (n = 4), unoperated control fetuses were similarly delivered. Absolute pulmonary volumes were then measured using standard stereologic methods. Normal growth of the left lung from 112 to 140 d resulted in significant increases in wet and dry wt, displacement volume, and volumes of future airspace and capillary contents. LPA ligation caused significant decreases in left lung wet and dry wt, displacement volume, and in absolute volumes of fine nonparenchymal, future airspace, parenchymal tissue, and capillary contents compared to sham-operated and 140-d controls. Parenchymal tissue volume was also less than in 112-d controls. In addition, lung wt, displacement volume, and future airspace volume were significantly decreased in sham-operated fetuses compared to 140-d controls. The effects of LPA ligation on bronchial collateral circulation and factors known to affect lung growth (i.e. lung fluid volume) remain to be determined. Clearly, during the canalicular and alveolar stage of fetal lung development, pulmonary arterial flow is necessary for normal lung growth.

Measurement of lung water dynamics and lung aeration in the perinatal period by use of digital X-ray fluoroscopy.

A method has been developed for the measurement of lung water dynamics and regional aeration of the lung in anesthetized newborn lambs by use of X-ray fluoroscopy, video recording, and digital image processing. After cesarean section and before the first breath fetal lambs under halothane-oxygen were placed on an X-ray table and connected to a volume-cycled respirator. X-ray fluoroscopy commenced before the initiation of respiration, and the images were recorded on video tape. X-ray transmission through the thorax increased as the lung was aerated. The enhanced transmission was compared with the values obtained from a calibration water wedge from which an equivalent path length through water can be estimated. In testing this method, it was demonstrated that X-ray transmission was linearly related to the wet lung weight-to-body weight ratio and to the product of the wet-to-dry weight ratio multiplied by anatomic thickness of frozen lung blocks. Calibrated values were also linearly related to this product and to the actual measured height of the fluid and tissue in the fluid-filled lung.

An evolutionary link for developing mammalian lungs.

Lungs of the human infant and those of other mammals are filled with fluid immediately prior to birth. Studies of the ionic composition of this fluid indicate that active ionic transport processes occur in the epithelial cells of the potential airspaces. The purpose of this study was to see if these active ion pumps were present in developing species other than mammals thus providing a possible evolutionary link to mammals. A series of samples of lung liquid, amniotic fluid, and plasma were taken from embryonic marine turtles gathered from clutches incubating in the beach at Mon Repos, Queensland, Australia during the summer of 1986-87. The concentrations of sodium, potassium and chloride ions and protein measured in these liquids indicated that active pumping processes similar to that seen in the mammalian lung were present in the developing lungs of these marine reptiles and further, circumstantial evidence was gathered to suggest that this liquid was partially reabsorbed prior to hatching. The results support the notion that processes responsible for the normal development of the human lung and lungs of other mammals are also present in the hollow lungs of marine turtles. Thus there is an evolutionary counterpart controlling lung development in more ancient species. It may be possible to generalize this observation to the development of hollow lungs of other species.

Lung aeration and lung water dynamics in artificially ventilated newborn lambs.

To the present lung liquid dynamics in the immediate neonatal period have been measured mainly by gravimetric techniques. This paper explores lung aeration and lung water dynamics in seven fetal lambs between 139 and 142 days of gestation delivered by caesarean section and ventilated on a constant-volume respirator. After caesarean section and instrumentation, fetal lambs were quickly transferred to a warm X-ray table and connected to a volume-cycled respirator. X-ray fluoroscopic images of the chest commenced before the first breath and were recorded on video tape. After 1 h of ventilation, measurements were made of pulmonary blood volume, and lung samples were taken for wet weight and dry weight analysis. Fluoroscopic image brightness was calibrated by comparison with images obtained from a water wedge, which extended across the X-ray field. Thus image brightness was related to "equivalent water path length" through the thorax. Within a defined lung field, image brightness increases as the amount of lung water in the path of the X-ray beam is reduced. This occurs rapidly at first and then more slowly over the remaining hour. There was considerable variation between the reduction of liquid in the lung fields examined within the one animal, as well as the absolute amount of fluid that had been cleared during the 1st h.

Lungs of the gecko Rhacodactylus leachianus (Reptilia: Gekkonidae): a correlative gross anatomical and light and electron microscopic study.

The lungs of the New Caldeonian gecko Rhacodactylus leachianus were examined by means of gross dissection and light and electron microscopy. This tropical species, which is the largest living gecko, possesses two simple, single-chambered lungs. Right and left lungs are of similar size and shape. The lung volume (27.2 ml.100 g-1) is similar to that of the tokay (Gekko gecko) but differs in that the gas exchange tissue is approximately homogeneously distributed, and the parenchymal units (ediculae) are very large, approximately 2 mm in diameter. The parenchymal depth varies according to the location in the lung, being deepest near the middle of the lung and shallowest caudally. Scanning and transmission electron microscopy reveal an unusual distribution of ciliated cells in patches on the edicular walls as well as on the trabeculae. Secretory cells are very numerous, particularly in the bronchial epithelium, where they greatly outnumber the ciliated cells. The secretory cells form a morphological continuum characterized by small secretory droplets apically and large vacuoles basally. This continuum includes cells resembling type II pneumocytes but which are devoid of lamellar bodies. Type I pneumocytes similar to those of other reptiles cover the respiratory capillaries, where they form a thin, air-blood barrier together with the capillary endothelial cells and the fused basement laminae. The innervation, musculature, and vascular distribution in R. leachianus are also characterized. Apparent simplification of the lungs in this taxon may be related to features of its sluggish habits, whereas peculiarities of cell and tissue composition may reflect demands of its mesic habitat.

Single breath airway occlusion measurements of compliance and resistance utilizing a mechanical ventilator.

A simple manipulation of the outflow valve of a Baby Bird MKII ventilator was used to measure passive lung mechanics by the airway occlusion technique. The results showed satisfactory reproducibility and comparison with other techniques at intra and interstudy level. This technique may have important clinical ramifications as it allows measurements of passive lung mechanics to be made without the need for cumbersome valves or handling of the airway. This convenience could also allow for concomitant plethysmographic lung volume assessments.

Effect of changing lung liquid volume on the pulmonary circulation of fetal lambs.

During fetal life the lung develops as a liquid-filled structure with low blood flow compared with postnatal life. We studied the effects of liquid expansion of the fetal lung by measuring vascular conductance in perfused lungs in situ and arterial diameters in excised lungs of fetal lambs. Pulmonary vascular conductance invariably rose as the lung was deflated from its initial volume; maximal deflation to residual volume increased conductance 122%. With reexpansion, conductance fell progressively, culminating in cessation of flow at lung volumes of twice the initial volume. These changes persisted after vagotomy and thoracic sympathectomy and therefore were mechanical in character. Lung expansion from residual volume initially expanded 300- to 500-micron arteries but compressed arteries greater than 1,500 micron. Further expansion reduced the caliber of all arteries. Thus increasing lung liquid volume progressively constricts the pulmonary circulation in the fetus. Because the fetal pulmonary vascular resistance-lung volume relationship differs from that of the U-shaped form found in adult lungs, concepts based on the adult pulmonary circulation are not appropriate for liquid-filled fetal lungs.

State-related changes in lung liquid secretion and tracheal flow rate in fetal lambs.

The volume of liquid in the fetal lungs depends on the rate of liquid secretion (Vs) across the pulmonary epithelium and the rate of flow out of the trachea (Vtr). We measured Vs, by an isotope-dilution technique, and Vtr, with a bubble flowmeter, during low-voltage (LV) and high-voltage (HV) electrocortical activity. In nine chronically instrumented fetal lambs, Vtr was greater during the transition to and at LV (16.98 +/- 1.98 ml/h, mean +/- SE, n = 23) than values during the transition to and at HV (8.69 +/- 0.8 ml/h). A pronounced peak in Vtr of 22.3 +/- 1.8 ml/h (n = 197) occurred at the transition to LV and early in the LV state. Ten minutes or more into LV, Vtr had declined to 10.3 +/- 1.8 ml/h (n = 235). Vtr remained low throughout the HV state. Vs values were not significantly different throughout the LV (11.83 +/- 1.34 ml/h, n = 216) and the HV (13.61 +/- 2.34 ml/h, n = 174) states. Diaphragmatic burst rate during LV (146.9 +/- 6.7 bursts/5 min, n = 432) was greater than during HV (26.5 +/- 4.6 bursts/5 min, n = 348), but burst rate was not correlated with Vtr. In summary, Vtr reaches a peak during the early part of LV when breathing commences and Vs remains constant throughout the behavioral cycle. As a result, lung liquid volume increases slightly during HV and decreases by a similar amount in the early part of LV.

Decline in lung liquid volume before labor in fetal lambs.

The volume of liquid in the fetal lung depends on the amount of liquid secreted across the pulmonary epithelium and the amount flowing through the trachea. Lung liquid volume (V1) and secretion rate Vs) were determined using an indicator-dilution technique, while tracheal flow rate (Vtr) was measured simultaneously with a bubble flowmeter. Least-squares regression analysis showed that in 10 chronically instrumented fetal lambs, V1 increased from 51.0 ml at 119 days to 104.6 ml at 135 days (V1 = -347.65 + 3.35 X days; 95% confidence limits on slope: 1.89-4.81) before declining to 70.2 ml at 142 days gestation (V1 = 768.8 - 4.92 X days; 95% confidence limits on slope: -2.55 to -7.30). Similarly Vs increased from 7.4 ml/h at 119 days to 16.8 ml/h at 133 days (Vs = -72.35 + 0.67 X days; 95% confidence limits on slope: 0.21-1.14), before declining to 7.1 ml/h at 142 days (Vs = 159.07 - 1.07 X days; 95% confidence limits on slope: -0.56 to -1.57). Vtr did not change significantly with gestation. We conclude that V1 increases until 135 days gestation, after which it falls substantially. This fall in volume, which occurs well before the onset of labor, results predominantly from the decline in Vs.

Phasic respiratory activity in the fetal lamb during late gestation and labour.

We quantified the respiratory activity of 9 fetal lambs using computer-analysis of the diaphragmatic electromyogram (EMG) obtained during 2 h recording sessions interspersed over the last 13 days of gestation. The fetuses delivered unassisted at an average gestational age of 145 days (term = 147 days). During the last 2 h of labour the number of phasic EMG bursts (breaths) averaged 3% of the peak recorded earlier in the study. This decline in breathing began at least 2 days before labour and resulted predominantly from the fetus spending an increasing proportion of time in apnoea. Respiratory rate within epochs of breathing also fell significantly 1 day before labour, and the proportion of time spent in the low voltage electrocortical state declined once labour commenced. No significant change occurred in arterial PO2, PCO2 or pH over the study period. We conclude that fetal respiratory activity falls well before the onset of labour, largely as a result of increased apnoea, and that the decline does not result from the development of a progressive hypoxaemia associated with labour.

An improved perfusion technique for fixation of sheep myocardium.

Although sheep have been widely used as models in the study of cardiac physiology, corresponding morphologic and morphometric data are scanty. For meaningful correlation of morphometric data with physiological information, it is desirable that fixation of the heart occur under controlled conditions. This paper describes a technique for in situ, retrograde aortic perfusion fixation of sheep myocardium under conditions of controlled pressure and minimal wastage of fixative. This is achieved by the application of snares around the brachiocephalic trunk and aortic arch, which are tightened at the start of the perfusion. These isolate the ascending aorta and the coronary vasculature from the remainder of the circulation and allow fixation of the whole heart at a controlled pressure. The method produces good fixation and contrast for transmission electron microscopy and is applicable to late-gestation fetuses, lambs, and adult sheep.