Karen J VanderWall

Bone morphogenetic protein-2 induces scar formation and skin maturation in the second trimester fetus.

&NA; Fetal mammals heal skin wounds through the second trimester of development without evidence of scar. We have investigated the role of bone morphogenetic protein 2 (BMP‐2), which is a member of the TGF‐&bgr; superfamily, in normal skin development and fetal wound healing. We first used RNA in situ hybridization to demonstrate that BMP‐2 was expressed at low levels in the developing hair follicles and in the epidermis of normal human fetal skin. We then created an in vivo model to test how exogenous BMP‐2 would affect fetal skin development and wound healing. Fifty micrograms of BMP‐2 was implanted into the subcutis of five 70‐day‐old fetal lambs through a fullthickness linear incision. The BMP‐2 was placed beneath the right half of the wound, whereas the left half served as an untreated control. In two of the five animals 1 &mgr;g of TGF‐&bgr; was placed into the same position in addition to the 50 &mgr;g of BMP‐2. Twenty days later (90 days gestation, term = 140 days) all the fetal wounds were examined for evidence of cellular hyperproliferation and scar formation. BMP‐2 induced massive dermal and epidermal growth when compared with controls. This finding was characterized by marked epidermal thickening and keratinization, a dramatic increase in the number of hair follicles, and more than 50 percent thickening of the dermis. The dermal thickening was the result of both increased cellularity and deposition of large irregular collagen bundles. Wounds treated with both BMP‐2 and TGF‐&bgr; healed also with an adult‐like pattern of scar formation. Surprisingly, the wounds with BMP‐2 alone healed with an equal pattern of scar, indicating that there was not an additive effect of combining BMP‐2 and TGF‐&bgr;. We conclude that BMP‐2 is a pleomorphic growth factor that induces cellular growth, maturation, and fibroplasia in both the dermis and epidermis. Further analysis of this growth factor in both fetal and adult wound healing may lead to important discoveries regarding the control of scar formation and fibrosis in many adult tissues. (Plast. Reconstr. Surg. 101: 12, 1998.)

Fetal Diaphragmatic Hernia: Echocardiography and Clinical Outcome

It is difficult to predict survival of fetuses diagnosed prenatally with congenital diaphragmatic hernia. Some studies suggest that left heart underdevelopment is associated with poor outcome, but fetal echocardiographic variables have not been conclusively proven to be good predictors of postnatal survival. The authors reviewed detailed fetal echocardiographic studies in twelve fetuses with congenital diaphragmatic hernia. Ten echocardiographic variables, including left and right ventricular width, left ventricular volume, and left ventricular mass, were examined from a four-chamber view, corrected for gestational age, and compared with normal data. The results of this study showed no significant differences between survivors and nonsurvivors in the ten variables analyzed. Although left heart dimensions and left ventricular volume in fetuses with congenital diaphragmatic hernia were below the expected normal range, these results did not predict postnatal outcome.

A New In Utero Model for Lateral Facial Clefts

The etiopathogenesis behind the formation of atypical craniofacial facial clefts remains unknown. To test the hypothesis that physical restricting forces such as amniotic bands can lead to the formation of these unusual clefts in the postorganogenesis period, we have modified a previously reported fetal lamb model of amniotic band syndrome to examine the effects of these bands on craniofacial development. Five 70-day gestation fetal lambs (term, 140 days) were exposed via a maternal hysterotomy. In each animal, an attempt was made to create a lateral craniofacial cleft by applying a 2–0 nylon suture as a constriction band to the growing face. The sutures were attached to either the zygomatic arch or the infraorbital rim externally and then looped circumferentially into the oral commissure. Each suture was positioned so as to create either a Tessier type 5 or a Tessier type 7 cleft. Four of five fetal lambs survived to term. Both types of lateral facial clefts were effectively produced using this model. In each group, the presence of an intraoral constriction band led to the formation of macrostomia, with an average 7.4-mm lateral displacement of the oral commissure. In addition to these soft tissue changes, each animal also had partial bony clefting (i.e., a bony groove) induced by the pressure of the restriction band across the growing facial skeleton. In the two lambs with the Tessier type 7 cleft, incomplete bony clefts developed across the zygomatic arch. In three animals with bands placed across the medial infraorbital rim, significant infraorbital and malar bony clefts formed similar to a classic Tessier type 5 facial cleft. No evidence of tissue necrosis, maceration, or ulceration was noted in any animal. These data present, for the first time, evidence that the constriction of craniofacial growth by external forces such as a swallowed amnionic band can lead to the development of lateral facial clefting involving both soft tissue and bony elements. These malformations are likely due to a combination of directly tethering normal tissue migration and an increase in local pressure, which produces cellular ischemia and apoptosis. Furthermore, our data demonstrate that these clefts can occur later in fetal development during a period of facial growth rather than during the period of primary facial morphogenesis.

A new in utero sheep model for unilateral coronal craniosynostosis.

&NA; Several animal models have been designed in the past to analyze the pathophysiology and management of craniosynostosis, very few of which were intrauterine. Those that were interuterine had problems with either a short gestation or limited availability that prevented most researchers from using them in treatment analysis. We desired to create a biologically sound intrauterine model of craniosynostosis, using an animal with a long gestation and an early calvarial bone formation, which was easy to manipulate in utero, that could be created by any researcher studying this disorder. Using biologic data available regarding growth factors thought to be involved in bone growth and cranial suture closure, we developed a new in utero fetal lamb model for the study of craniosynostosis. Ten 70‐day gestation fetal lambs (term gestation 140 days) received a midline coronal incision to expose both coronal sutures. The entire right coronal suture was then excised along with a 4‐mm bony margin. In each animal, the site was packed with 25 mg of demineralized sheep bone powder augmented with 50 &mgr;g of bone morphogenetic protein‐2 (BMP‐2) and 1 &mgr;g of poly‐transforming growth factor‐&bgr;. The scalp was closed, and the sheep were returned to the uterus until either 90 or 140 days of gestation. Complete fusion of the right coronal suture occurred in all fetuses by 90 days gestation. In every animal, rightsided frontal bone flattening and supraorbital rim elevation were evident. Histologic analysis showed bony synostosis at the suture site without evidence of suture regeneration. By 140 days, this isolated suture fusion led to marked craniofacial abnormalities including right supraorbital rim elevation, significant frontal bone flattening, a decrease in the anterior‐posterior length of the cranial vault, and flattening of the cranial base. In conclusion, we have developed a new model for the study of the secondary effects induced by the process of cranial suture fusion, which produces abnormalities seen in naturally occurring cases of isolated right coronal suture synostosis. In addition, this model confirms that isolated coronal suture fusion alone can lead to the multiple cranial and facial abnormalities seen with this disorder, even in the absence of associated cranial base suture fusions. (Plast. Reconstr. Surg. 101: 278, 1998.)

Experimental fetal transesophageal and intracardiac echocardiegraphy utilizing intravascular ultrasound technology

Fetal transesophageal and intracardiac echocardiography by utilizing ultrasound technology permits accurate definition of cardiac anatomy in fetal sheep. Because fetal transesophageal echocardiography is less invasive than intracardiac echocardiography, it has the potential to serve as a monitoring tool for currently developed open and fetoscopic fetal cardiac interventions.

Transesophageal echocardiography in fetal sheep. A monitoring tool for open and fetoscopic cardiac procedures.

AbstractBackground: Cardiac procedures in exteriorized fetuses or assisted by fetoscopy require monitoring capabilities not attended by conventional maternal transabdominal echocardiography. Methods: We, therefore, assessed the potential of fetal transesophageal echocardiography (TEE) utilizing an intravascular ultrasound catheter (IVUC) for fetal cardiac monitoring. We inserted a 10-F-10-MHz IVUC into the esophagus in 12 exteriorized fetal sheep and by a fetoscopic approach in 4 fetal sheep. Cardiac events were observed. Heart rate, cardiac rhythm, patency of the foramen ovale and ductus arteriosus, and the width of the branch pulmonary arteries could be assessed in all fetuses. Ventricular contractility could be assessed only in fetuses weighing less than 2.5 kg. Larger fetuses did not allow adequate imaging of the apical portion of the ventricles because of limited tissue penetration of the IVUC. Fetal TEE permitted placing small guide wires in the cardiac atria and left ventricle. Short-lived premature beats following intracardiac manipulations of these wires could be observed by fetal TEE in all cases. Results: At autopsy, no complications from IVUC insertion were observed in the exteriorized fetuses. Fetoscopic placement of the IVUC resulted in minor perioral skin erosion in two nonexteriorized fetuses. Conclusions: In conclusion, fetal TEE can be achieved with minor fetal injury and may provide useful information during open and fetoscopic cardiac procedures. Further improvements in IVUC design will permit the application of this technique to monitor human fetal cardiac procedures.

The in Utero Correction of Unilateral Coronal Craniosynostosis

&NA; We performed the first in utero correction of a unilateral right coronal craniosynostosis using 70‐day gestation fetal lambs. The craniosynostosis was created in eight fetuses by excising their right coronal sutures, and then placing demineralized bone powder, transforming growth factor‐&bgr;, and bone morphogenetic protein‐2 into the defect. Twenty‐one days later, after suture fusion had occurred, four of the eight sheep were treated with a 4 mm × 12 mm strip craniectomy to open the entire synostosed right coronal suture. The edges of the excision were wrapped with 100‐&mgr;m‐thick Gore‐Tex (W. L. Gore & Associates, Flagstaff, Ariz.) sheets to prevent bony refusion. All eight lambs then progressed to term (140 days). The skulls of four normal, unoperated, term lambs were used as controls. At 140 days, all four treated lambs had a widely patent strip craniectomy site without any evidence of bone regeneration. This in utero correction led to a marked improvement in craniofacial morphology of three of four animals when compared with the uncorrected controls with significant (p < 0.01) correction in orbital position, skull length, and shape of the frontal bone. This was in sharp contrast to the uncorrected animals, which had marked orbital elevation, compression of the anteroposterior length of the cranial vault, frontal bone flattening, and shortening of the cranial base. The fourth corrected animal also showed evidence of improvement but had some abnormal calvarial changes secondary to the development of horns, which displaced the calvaria in a downward vector. We conclude that the in utero correction of craniosynostosis is feasible and provides a significant benefit by decreasing the severity of many of the associated deformities seen with this disorder. (Plast. Reconstr. Surg. 101: 287, 1998.)

Nitroglycerin attenuates the bowel damage of necrotizing enterocolitis in a rabbit model.

Endogenous enteric nitric oxide has multiple functions. Enteric nitric oxide may be diminished in the premature infant and may therefore predispose the immature intestine to injury. The aim of this study was to determine if the infusion of a nitric oxide donor (nitroglycerin) would attenuate intestinal damage in a rabbit model of necrotizing enterocolitis. Transmural injection of rabbit intestinal loops with an acidified solution of casein and calcium gluconate simulates certain aspects of necrotizing enterocolitis. After injection of acidified casein solution into rabbit intestinal loops, twelve rabbits were randomly divided into two groups: six received maintenance fluids only and six received maintenance fluids and a nitroglycerin infusion adjusted to maintain mean arterial pressure 10 mm Hg below baseline (range, 2 to 12 micrograms/kg/min). After 3 hours, the rabbits were killed, and the intestinal tissue graded histologically. Intestinal damage in the nitroglycerin-treated rabbits was significantly less than that of untreated controls (mean histological grade of 0.39 v 1.48, P < .001). In this rabbit model of necrotizing enterocolitis, infusion of the nitric oxide donor nitroglycerin significantly attenuates intestinal damage. We speculate that enteric nitric oxide deficiency, as may exist in the preterm infant, predisposes the intestine to necrotizing enterocolitis.

Cyclic GMP Relaxes the Internal Anal Sphincter in Hirschsprung's Disease

In Hirschsprungs disease (HD), the aganglionic colon and internal anal sphincter (IAS) fail to relax. Aganglionic colon of HD patients relaxes in response to exogenous nitric oxide (NO), whereas the IAS from HD patients does not relax. The authors hypothesized that the failure of IAS relaxation is caused by a local deficiency of cyclic guanosine monophosphate (cGMP), the final metabolite in NO-mediated smooth muscle relaxation. To test this hypothesis, the authors measured the isometric tension of smooth muscle strips taken from the IAS and aganglionic colon of patients with HD before and after exposure to cGMP and compared this with ganglionic colon and IAS from normal controls. In HD patients both the IAS and aganglionic colon relaxed in response to cGMP (P < .05). The amount of relaxation observed in both the aganglionic colon and IAS was comparable to that measured in the normal controls. The observation that exogenous cGMP relaxes the IAS, whereas exogenous NO does not, suggests that mechanisms for relaxation may be different than those in the aganglionic colon and may explain persistent IAS dysfunction after resection of aganglionic colon. The defect of the IAS in HD may be the inability of the NO/cGMP pathway to induce smooth muscle cell relaxation rather than a defect in the smooth muscle cell.

Effects of nitroglycerin and indomethacin on fetal-maternal circulation and on fetal cerebral blood flow and metabolism in sheep.

OBJECTIVE Our purpose was to evaluate the effects of maternal administration of nitroglycerin and indomethacin on maternal and fetal hemodynamics and on fetal cerebral blood flow and metabolism in sheep. STUDY DESIGN Invasive vascular and fetal carotid flow monitoring was established in 12 gravid ewes. Isotonic sodium chloride solution, nitroglycerin, and indomethacin were infused maternally, and maternal and fetal heart rate, blood pressure, blood gas values, fetal carotid blood flow, and flow variability were measured. Fetal cerebral uptake of oxygen, glucose, and lactate were calculated. RESULTS Nitroglycerin infusion caused a significant increase in maternal and fetal heart rate and a significant decrease in maternal and fetal mean arterial pressure at a dosage of 10 microram/kg per minute, without a change in blood gas values. Neither drug had any effect on fetal carotid blood flow, flow variability, or cerebral substrate metabolism. CONCLUSION Maternal administration of nitroglycerin and indomethacin caused no adverse maternal or fetal circulatory changes and did not alter fetal carotid blood flow or substrate metabolism.