Fetal Lamb Model Research Articles

Developmental Alterations in the Fetal Lamb Bladder Subjected to Partial Urethral Obstruction in Utero

To identify the structural changes seen in the developing bladder subjected to outflow obstruction, a fetal lamb model was developed. With this model, which attempts to reproduce conditions such as those found in posterior urethral valves, quantitative biochemical parameters were used to evaluate the effects of partial outflow obstruction on detrusor cellularity and innervation in the developing bladder. Partial urethral obstruction was created in 9 fetal sheep (90 to 100 days gestation), 6 sham operated animals serving as controls. Fetuses were delivered at term and sacrificed. Bladders were removed and weighed. Mucosa free detrusor was assayed for DNA and choline acetyltransferase (ChAT) activity, indices of cell number and cholinergic innervation, respectively. In similar specimens of detrusor, cell ploidy and nuclear size were determined by flow cytometry and nuclear morphometry, respectively. The results showed that partial urethral obstruction in utero resulted in a significant increase in bladder weight (p < 0.05) and total detrusor DNA content (p < 0.01). With the increase in detrusor DNA content, there was a parallel increase in total detrusor ChAT activity. These results suggest that early development of the bladder is altered both on a cellular level (increase in cellularity) and a neural level (increase in cholinergic nerve density) under conditions of mechanical distension.

Fetal Compensatory Renal Growth Due to Unilateral Ureteral Obstruction

To determine whether fetal compensatory renal growth occurs in response to early gestational unilateral renal obstruction and to help elucidate the characteristics of this response, a fetal lamb model was developed in which unilateral ureteral obstruction was created at 60 days of gestation (term 135 to 140 days) and the effects of the obstruction were studied at varying periods thereafter. Kidneys were retrieved at 2 weeks (75 days in 3 cases), 5 weeks (95 days in 4) and 10 weeks (135 days in 9) after obstruction, weighed and preserved for histology and biochemical studies. Deoxyribonucleic acid (DNA), ribonucleic acid and protein were quantitated using standard assays. Morphometric studies to estimate glomerular number were performed using standard stereological methods.Contralateral kidney weight was increased compared to normals in 55 cases at all ages. The relative difference increased from 75 to 95 days. The fractional increase at 135 days (49.5%, p <0.001 versus normal) was not different from 95 days (46.4%, p = 0.87), indicating that growth rate acceleration occurred predominantly before 95 days. Total renal DNA was increased above normal at each time point, with an ultimate increment proportionate to weight. Protein concentration was unchanged, suggesting that weight increase was not due to changes in renal water. Protein/DNA ratios were unchanged, indicating a predominantly hyperplastic process. Ribonucleic acid/DNA ratios decreased, possibly indicating a high rate of proliferation. Total glomerular number at term did not increase with in utero compensatory renal growth.Unilateral ureteral obstruction created early in gestation produced compensatory renal growth of the intact kidney in the fetal lamb. This process demonstrates an early but transient growth rate acceleration, hyperplasia and no increase in total glomerular or nephron number. The occurrence of compensatory renal growth in the fetal kidney with minimal functional renal demand would suggest that its mechanisms may be independent of functional demand. This model is unique and well suited to study compensatory renal growth.

Summary of the Annual Meeting of the Section on Urology, American Academy of Pediatrics

The Section on Urology of the American Academy of Pediatrics met for 2½ days in conjunction with the 60th annual meeting of the American Academy of Pediatrics in New Orleans, LA, October 26 through 28, 1991. This conference remains the most important meeting in the United States for pediatric urology. The number and quality of papers presented continue to grow and there is a rich, international participation. The meeting was presided over by Chairperson Edward S. Tank, Jr, MD, of the University of Oregon Health Sciences Center, Portland, OR. The papers presented at this meeting that are of interest to the practicing pediatrician are summarized here according to topic. OBSTRUCTION Investigation of structural, biochemical, and functional changes in both the developing bladder and in the upper urinary tract were studied in the fetal lamb model of bladder outlet obstruction by Peters et al from Boston Children's Hospital and by Cendron et al from Johns Hopkins Hospital. Their findings included an increase in bladder weight due to hypertrophy and hyperplasia, increase in the cholinergic receptor density of the smooth muscle cells of the bladder, and alterations in the myosin heavy-chain isomer ratio. Following birth these alterations in growth and differentiation resulted in functional bladder abnormalities as measured by urodynamic studies. These abnormalities included an increase in bladder capacity, an increase in maximal intravesical pressure with filling, and an increase in postvoid residual urine volumes. Further study in this model should allow better understanding of the spectrum of bladder dysfunction resulting from in utero outflow obstruction of the bladder.

Fetal cardiovascular physiology and therapy.

The ability to diagnose fetal cardiovascular malformations prenatally has been established during the past 14 years. This has naturally led to an increased interest in the possibility of providing timely therapy in the neonatal period and has raised the prospects for prenatal intervention. Before medical, catheter, and/or surgical interventions are performed, however, it is important to understand the normal and abnormal physiology of the fetal cardiovascular system. Significant insights have been gained into human fetal cardiovascular physiology utilizing data previously gleaned from fetal lamb models and correlating anatomic, pulsed Doppler, and color flow Doppler observations that have been made echocardiographically during the second and third trimesters of human fetal cardiac development. Regional blood flow distribution studies in the human have demonstrated a relative right-ventricular volume dominance in the human fetus of a somewhat lesser magnitude than previously described in the lamb. Observations of ventricular filling characteristics suggest a relative paucity of diastolic 'reserve' in the human fetal heart implying a relative sensitivity to acute volume overloading. The human fetal heart, like that of the fetal lamb, also appears to have a relatively modest amount of 'systolic reserve', making the heart particularly susceptible to acute ventricular afterload. Further studies have demonstrated the ability to recognize altered intrauterine shunt flow across the fetal ductus arteriosus and foramen ovale. As predicted in fetal lamb models, alterations in shunt flow accompany major malformation complexes including ventricular hypoplasia. Studies to date suggest that attempts at surgical intervention should be made very cautiously due to the inability to ascertain whether altered shunt flow patterns precede (cause?) or follow the development of structural abnormalities.

Effects of single and repeated marijuana smoke exposure on fetal EEG

The effect of single and repeated marijuana smoke exposure on fetal EEG was investigated in the chronic fetal lamb model using power spectral analysis. Maternal inhalation of marijuana smoke (n = 9) resulted in a significant reduction in total power and power distribution in the delta (1–4 Hz) band, and an increase in power distribution in the faster frequencies in the first h after smoke inhalation. These EEG changes were not observed following maternal inhalation of placebo smoke (n = 5), nor in animals with 3–5 prior exposures to marijuana smoke (n = 5). These results suggest that the effects of marijuana smoke exposure on fetal EEG is short-lived and tolerance develops rapidly with repeated exposure.

Midgestational excisional fetal lamb wounds contract in utero

Clinical observations and experimental data suggest that fetal wound healing is very different from adult wound healing. An understanding of the biology of scarless fetal wound healing has tremendous clinical potential for modulating postnatal wound problems. In this study, the fetal lamb model was used to assess excisional fetal skin wound contraction in utero. Full-thickness 9-mm punch biopsy wounds were created on fetal lambs at 100 days' gestation (term, 145 days). Half of the wounds remained exposed to amniotic fluid, whereas the other half were covered by a silastic patch to exclude amniotic fluid. Wounds were harvested 3, 7, or 14 days later and wound areas were calculated. Exposure to amniotic fluid retarded wound contraction significantly at 3 days, but by 14 days all wounds had completely contracted and reepithelialized. Myofibroblasts are an important cellular element of wound contraction. The presence of wound myofibroblasts was documented by both transmission electronmicroscopy and immunocytochemistry with antimuscle actin antibody. It is concluded that fetal lamb wounds contract in utero and exposure to amniotic fluid appears to retard fetal skin wound contraction only during the early healing process.

Ontogeny of fetal sheep polymorphonuclear leukocyte phagocytosis

Premature infants and neonates are vulnerable to bacterial sepsis. This susceptibility may be due to the relative immaturity of their immune systems. To determine if neonates and, in particular, premature infants have decreased polymorphonuclear leukocyte (PMN) phagocytosis, we tested PMN phagocytosis of Staphylococcus aureus as a function of gestational age in the fetal lamb model. Because phagocytosis is made more efficient by the presence of opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after exposure to fetal and adult plasma. PMNs were isolated from fetal lambs at 104, 114, 124, and 141 days' gestation (term gestation for the fetal lamb is 145 days), as well as from 10-day-old neonatal sheep and adult sheep. Labeled S aureus were opsonized by incubation in either fetal or adult plasma, or left unopsonized for baseline values. Phagocytosis was measured as a percent of adult PMN phagocytosis after adult plasma opsonization. It was found that fetal PMN function is limited by two factors during the early third trimester: a primary defect in the ability of the PMN to phagocytose S aureaus despite adequate opsonization, and the diminished ability of autologous fetal plasma to opsonize bacteria. The defect in PMN phagocytosis disappears late in the third trimester, but the inability of the fetal plasma to opsonize effectively continues until after birth.

Mucosal morphology in experimental intestinal atresia: Studies in the chick embryo

Lesions identical to those observed in human intestinal atresia (IA) have been experimentally reproduced in several mammal models by either mesenteric vessels or intestinal wall injury. The recent availability of an avian model led us to investigate whether the same lesions could be studied at less expense. An intestinal loop within the body stalk hernia was coagulated on the 12th incubation day in 427 chick embryos (group 3), the lesions were studied in survivors 4 days later under light and scanning electronmicroscopy (EM), and the findings were compared with those in 12 control embryos (group 1) and 14 sham-operated ones (group 2). Types I or II atresias were obtained in 61 (73.4%) of the 83 survivors in group 3. Seromuscular layers were normal at both ends of the lesion; there was some flattening of villi at the proximal, dilated end, and hyperplasia with apparently lengthened, branched villi at the distal, unused one. Mucosal pattern under light microscopy was strikingly close to that observed in human IA and in the fetal lamb experimental model. On the other hand, scanning EM showed that in this model there were no villi as such, but rather prominent mucosal folds that were regularly arranged in a tire-rubber pattern in the normal intestine, flattened and distended in the dilated one, and again roughly normal in the distal unused gut. High-power magnification scanning EM showed that enterocytes were normal at all levels but, in agreement with previous findings in the fetal lamb model, that intercellular spaces were widened in the dilated portion adjacent to the obstruction. It is concluded that the apparent hyperplastic aspect of distal atretic intestine could result from mucosal crowding within the unused lumen rather than from true villous enlargement. This study suggests that a chick embryo IA fetal model allows further investigation of the processes of intestinal adaptation after fetal insult at much lower prices than other experimental models.

Etiology of intestinal damage in gastroschisis. I: Effects of amniotic fluid exposure and bowel constriction in a fetal lamb model

Gastroschisis is often complicated by damage to the herniated small bowel, resulting in motility and absorption disturbances and occasional intestinal necrosis and atresia. To study the pathophysiology of this process, a model of gastroschisis was developed in fetal lambs. At 80 days' gestation, the anterior abdominal wall was partially excised to create a small peritoneal cavity, and the small bowel was extruded through a Silastic ring to create a defect of uniform size. In one experimental group, a tie was placed around the herniated bowel at the level of the abdominal wall to provide gradual constriction as the fetus grew. In a second group, no tie was placed. Control animals had a simple laparotomy and no abdominal wall defect; some also had a constrictor placed around the base of the bowel. The animals were delivered near term, and the bowel was evaluated histologically and by an in-vitro bowel motility assay. Histologic examination showed normal ganglion cells in all groups and no evidence of ischemic injury. A fibrous peel was seen only in bowel exposed to amniotic fluid, with or without a constrictor. Lymphatic and venous dilation, smooth-muscle thickening, and focal mucosal blunting were seen in bowel subjected to chronic obstruction by a constrictor, regardless of whether it was exposed to amniotic fluid. Both constriction of the bowel and amniotic fluid exposure were associated with a decrease in motility; these two effects were independent and additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary Development in the Fetal Lamb with Severe Bladder Outlet Obstruction and Oligohydramnios: A Morphometric Study

The lethal feature of male newborns with severe bladder outlet obstruction and oligohydramnios is pulmonary hypoplasia. We report a fetal lamb model of bladder outlet obstruction created in the early second trimester, and morphometric analysis of the profound pulmonary hypoplasia that resulted. At 55 to 65 days of gestation 8 consecutive male fetuses underwent surgical clipping of the urethra and urachus near the abdominal wall. A sham operation was performed in 1 female fetus. The lambs were delivered by cesarean section 10 weeks later. The right lungs were immediately fixed at a distending pressure of 25cm. water. The results of the morphometric studies were compared to control animals from our earlier study of normal fetal lamb lung development; slides for both studies were scored in a blind, randomized fashion by 1 of us (S. G. D.). Although mean body weights for the groups were similar, mean right lung volume in the operated lambs was 63.8cM.3 compared to 158cM.3 in the control animals and 137cM.3 in the sham operated fetus (p less than 0.0005). Relative volume of respiratory tissue was 87 per cent in the operated animals, and 92 per cent in the controls and the sham operated fetus (p less than 0.0005). Microscopic morphometry revealed an increased relative volume of inter-alveolar tissue in the experimental animals (35 compared to 23 per cent in the normal lambs, p less than 0.05), with a corresponding decrease in relative airspace volume. Alveolar numerical density and alveolar surface density were not statistically different between the groups, although total alveolar number and surface area were substantially decreased in the operated lambs due to the reduced mean lung volume. Mean alveolar wall thickness was increased at 3.6μM. in the operated group versus 2.1μM. in the normal group (p less than 0.0005) and 2.8μM. in the sham operated fetus. Thus, the lung in the lamb with severe bladder outlet obstruction is hypoplastic and immature.

Fetal lamb coagulation system during normal birth.

The contribution of the birth process to the unique coagulation system in the young was investigated using the fetal lamb model. Specific components of the coagulation system were measured prior to and immediately following delivery of the lamb. The results show that the birth process itself does not contribute to the physiologically low levels of coagulation factors present in the newborn.

Fetal surgery for obstructive uropathy: rational considerations.

The widespread use of high resolution dynamic ultrasound imaging methods in obstetrics now permits recognition of structural and/or functional developmental anomalies of fetal genitourinary tract with some considerable accuracy. Detection of congenital obstructive uropathy in the human fetus may occur as early as 16 weeks gestation. In the fetal lamb model, experimental occlusion of the outflow tract results in progressive hydronephrosis, pulmonary hypoplasia, and oligohydramnios. The renal parenchymal changes vary with the fetal age at obstruction ranging from simple hydronephrosis with later obstruction to dysgenesis with earlier obstruction. The pulmonary damage, and to some extent the renal damage, may be halted or even reversed with release of obstruction. These advances in diagnosis and an understanding of the pathophysiology have prompted attempts at chronic in utero diversion therapy in the human fetus with obstructive uropathy yielding encouraging, but as of yet, unproven success. In this report, the methods for such therapy, the rationale for the therapy, and the results of the therapy is reviewed.

Correction of congenital diaphragmatic hernia in utero IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis

Infants born with congenital diaphragmatic hernia (CDH) often have specific pathologic abnormalities of the pulmonary microcirculation that result in high pulmonary vascular resistance and extrapulmonary right-to-left shunting after birth. In an attempt to make an animal model with similar vascular changes, we created CDH in fetal lambs at 60 to 63 days gestation, repaired some at 100 to 113 days gestation, and subsequently performed morphometric analysis of the pulmonary vasculature. Creation of CDH at this early gestational age resulted in a high fetal mortality rate. In the unrepaired CDH lambs, the pulmonary vascular abnormalities were more severe in the left lung. Similar to human CDH, diaphragmatic hernia in the fetal lamb resulted in a decrease in the total size of the pulmonary vascular bed, a decrease in the number of vessels per unit area lung, and increased muscularization of the arterial tree. Fetal surgical repair of CDH restored the pulmonary arterial bed towards normal.

Problems of and possibilities in fetal surgery

Recent developments and improvements in prenatal diagnostic methods, and in particular antenatal ultrasonography, have made intrauterine detection of fetal abnormalities possible. Most defects are best treated after birth, only a few disorders are potentially amenable to surgical treatment in utero. Studies in animal models have helped us to define the pathophysiology, to recognize the interference of the abnormality with organ development and to determine whether elimination of the anomaly might allow fetal development to proceed normally. In-utero repair of congenital diaphragmatic hernia has been practiced on a fetal lamb model but has not yet been attempted in human beings. Treatment of fetal hydrocephalus has been investigated in sheep and rhesus monkeys, and the pathophysiology and intrauterine treatment of fetal hydrocephalus has been studied in fetal lambs and monkeys, but the question of the reversibility of renal dysplasia has not been resolved. Animal models have been described for possible intrauterine treatment of skeletal abnormalities like spina bifida. But more knowledge of embryology and of the pathophysiology of the malformation is needed, as well as about the efficacy and feasibility of this method before it can be attempted in human beings. Prerequisites for fetal surgery include the selection of those fetuses who might benefit from intrauterine treatment, counselling of the family concerned, and a highly experienced multidisciplinary team including a perinatal obstetrician, an ultrasonographer, a pediatric surgeon and a neonatologist. In human beings intrauterine treatment has been performed in erythroblastosis fetalis, urinary tract obstruction and hydrocephalus with encouraging results. There are many ethical, legal and social questions which cannot be answered; new guidelines and rules will have to be laid down.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acidosis on fetal and maternal blood coagulation: a fetal lamb model.

The effects of fetal acidosis (mean pH 6.93) on fetal and maternal blood coagulation were measured. Test results from 10 fetal lambs and mother ewes (127 +/- 2 days mean gestation) before and after fetal lactic acid infusions were compared to test results from eight control fetal lambs and mother ewes (127 +/- 3 days mean gestation) before and after control glucose infusion. Significant changes found in acidotic fetal lambs not seen in control fetuses included an increase in the white blood cell count (mean 2800/mm3 before to 3600/mm3 after acidosis; p = 0.0009), a shortening of the thrombin time (mean 17.8 s before to 11.2 s after acidosis; p = 0.0001), and decreases in the activities of factor V (mean 57% before to 37% after acidosis; p = 0.0014) and factor IX (mean 35% before to 29% after acidosis; p = 0.0128). There was also a reduction in the concentration of fibrinogen (mean 147 mg/100 ml before to 125 mg/100 ml after acidosis; p = 0.0492) but no significant changes in the levels of fibrin monomer, fibrinogen/fibrin degradation products, or antithrombin III. In vitro exposure of five different fetal whole blood samples to a pH of 6.9 for 2 h at 37 degrees C did not result in significant changes in any of the coagulation factor activities. A significant decrease in the level of factor V was also found in the mother ewes of the acidotic fetuses (mean 141% before to 113% after acidosis; p = 0.006) and a decrease in the level of maternal factor IX approached significance (mean 119% before to 102% after acidosis; p = 0.0564).(ABSTRACT TRUNCATED AT 250 WORDS)

Intrauterine Cardiothoracic Surgery: The Fetal Lamb Model

We have developed fetal lamb models of congenital cardiothoracic lesions that have been allowed to progress through birth for physiological study. Simulated lesions, simulated repairs, actual lesions, and actual repairs have been performed in this model. Sixty-two fetal lambs comprised the study group, including 48 in which models were created and 14 controls. Models included pulmonary stenosis, aortic stenosis, and diaphragmatic hernia. Gestational age ranged from 90 to 120 days (0.6 of normal gestation). In each pregnant ewe, laparotomy and hysterotomy were performed under general anesthesia, with care taken to avoid placental vessels. The foreleg was exposed, the appropriate anterior chest wall was isolated, and a thoracotomy was done. Thoracic or cardiac procedures then were performed under controlled transplacental anesthesia and perfusion. Following completion of the procedure, the fetal thoracotomy was closed, sterile antibiotic solution was placed in the amniotic sac, and the hysterotomy and laparotomy were closed. Subsequently the fetus either was allowed to progress to birth and infant study or underwent subsequent intrauterine repair and then was allowed to progress to birth and neonatal study. This fetal lamb model provides reproducible anatomical and pathophysiological lesions to facilitate the development of techniques for repair of such lesions in early infancy. Further, it offers the potential for developing methods of intrauterine cardiothoracic surgical repair.

Neonatal adaptation: sympatho-adrenal response to umbilical cord cutting.

The present studies were designed to assess the individual effects of delivery and umbilical cord cutting on the stimulation of the sympatho-adrenal system during parturition. Pregnant ewes with time-dated singleton pregnancies were used in an acutely exteriorized fetal lamb model with an intact umbilical circulation. We observed a minimal, transient elevation in plasma catecholamines (CAT) coincident with the operative procedures and delivery. Subsequent cord clamping was observed to evoke a rapid and marked increase of both norepinephrine and epinephrine (E), maximal at 5 min and persisting over the 4-hr study period. Animals could be grouped on the basis of the observed CAT responses, severity of postpartum acidosis, the extent of free fatty acid (FFA) mobilization and degree of postpartum hypothermia. A blunted FFA response and slower correction of hypothermia were observed in the more acidotic animals despite higher CAT concentrations. One group of four animals had high peak CAT concentrations, 32,000 pg/ml norepinephrine and 35,000 pg/ml E, a deep nadir in pH of 6.88 +/- 0.09, a 2-hr delay in maximal FFA mobilization and slower correction of hypothermia. The other group of four animals had peak norepinephrine of 2800 pg/ml and E of 1100 pg/ml, a nadir in pH of 7.09 +/- 0.08, maximal plasma FFA concentration by 1 hr after cord cutting and a higher nadir in body temperature 35.7 versus 32.5 degrees C. The results demonstrate that umbilical cord cutting itself is a potent stimulus for fetal CAT release and FFA mobilization. Acidosis is capable of markedly augmenting E release in the mature fetus and obtunding chemical thermogenesis.

Development of blood coagulation--a fetal lamb model.

To study the normal development of blood coagulation factor activities in a growing fetus while avoiding the effects of labor and delivery, a chronic fetal lamb model was developed in which serial blood samples from 10 fetuses were studied during the third trimester of pregnancy and 24 hr after birth. Under operating room conditions with sterile technique, a polyethylene catheter to which heparin had been bound to both internal and external surfaces was inserted into the femoral artery of the fetus. The catheter was brought out through a skin pouch to the side of the ewe and enclosed in a zip lock bag. Blood samples were withdrawn from the catheter three times each week for measurement of coagulation factor activities. Levels of coagulation factor activities at birth in noncatheterized animals were not different from those found in catheterized animals except for factor IX activity which was 12% higher in the catheterized animals (0.02 less than P less than 0.05). The patterns of development for each of the coagulation factors were similar in all 10 animals studied. Fibrinogen, prothrombin, and factor VII show a decrease in activity early in the last trimester of pregnancy. Both factors VIII and IX show a significant increase in activity (23% factor VIII and 12% factor IX) associated with the process of delivery. The levels of coagulation factor activities at birth in the lamb relative to adult sheep normals are similar to those found in humans with the exception of factor XIII. Factor XIII is at normal levels in the newborn lamb and is reported to be at levels approximately 50% of the adult level in human infants.