Nitrofen induces a redox-dependent apoptosis associated with increased p38 activity in P19 teratocarcinoma cells.

Nitrofen is a diphenyl ether herbicide that produces a spectrum of fetal abnormalities in rodents. To characterize the molecular mechanisms of nitrofen-mediated birth defects at the cellular level, we explored its effects on undifferentiated P19 teratocarcinoma cells. Nitrofen induces a time-dependent cell death of P19 cells that is associated with increases in TUNEL-positivity and caspase-3 cleavage suggesting that nitrofen induces P19 cell apoptosis. In addition, the increase in TUNEL-positive cells was inhibited with zVAD-fmk, suggesting that nitrofen induces a caspase-dependent apoptosis. Nitrofen treatment was associated with increased p38 MAP kinase activity, though pretreatment of cells with multiple p38 inhibitors did not affect nitrofen-mediated caspase-3 cleavage, suggesting caspase-3 cleavage is p38-independent. Nitrofen induced a dose-dependent increase in reactive oxygen species (ROS), which was accompanied by a decrease in the ratio of reduced/oxidized glutathione, indicating that nitrofen alters the cellular redox state of these cells. Furthermore, pretreatment of cells with N-acetyl cysteine gave a dose- and time-dependent reduction of caspase-3 cleavage, supporting the observations that caspase-3 cleavage is cell-redox-dependent. Therefore, nitrofen induces P19 cell apoptosis that is cell-redox-dependent and is associated with increases in p38 activity and ROS and may play a role in nitrofen-mediated birth defects.

Treatment of pain in acutely burned children.

The child with burns suffers severe pain at the time of the burn and during subsequent treatment and rehabilitation. Pain has adverse physiological and emotional effects, and research suggests that pain management is an important factor in better outcomes. There is increasing understanding of the private experience of pain, and how children benefit from honest preparation for procedures. Developmentally appropriate and culturally sensitive pain assessment, pain relief, and reevaluation have improved, becoming essential in treatment. Pharmacological treatment is primary, strengthened by new concepts from neurobiology, clinical science, and the introduction of more effective drugs with fewer adverse side effects and less toxicity. Empirical evaluation of various hypnotic, cognitive, behavioral, and sensory treatment methods is advancing. Multidisciplinary assessment helps to integrate psychological and pharmacological pain-relieving interventions to reduce emotional and mental stress, and family stress as well. Optimal care encourages burn teams to integrate pain guidelines into protocols and critical pathways for improved care.

Decreased mitogen activated protein kinase activities in congenital diaphragmatic hernia-associated pulmonary hypoplasia.

BACKGROUND/PURPOSE: The mechanisms that cause pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) currently are unknown. The authors proposed that the reduced size and immaturity of these lungs may be associated with differences in the levels of mitogen activated protein (MAP) kinase phosphorylation (extracellular signal regulated protein kinases, ERK-1 and -2). METHODS: ERK-1 activities were measured using immune-complex kinase assays on fetal whole-lung lysates obtained from both nitrofen and olive oil-treated (control) pregnant rats. In addition, ERK-1 and ERK-2 functional activities were estimated by semiquantitative Western blot analysis, using an antibody specific for the diphosphorylated (dp-ERK, activated) forms of the enzymes. RESULTS: ERK-1 activities, measured using immune-complex kinase assays, were reduced in CDH lungs compared with olive oil-treated controls (P <.02). In addition, dp-ERK-1 and dp-ERK-2 levels were found to be reduced in CDH lungs compared with controls (dp-ERK-1, P =.003; dp-ERK-2, P =.04), whereas ERK-1 and ERK-2 protein levels were unchanged. CONCLUSIONS: The lower values of ERK-1 activity and reduced amounts of dp-ERK-1 and dp-ERK-2 in lung tissue from CDH animals, suggests that ERK-1 and ERK-2 activities are reduced in pulmonary hypoplasia associated with CDH. The observed reduction in ERK-1 and ERK-2 activities implicates attenuated cell signaling upstream of the ERK-1 and -2 enzymes.

Tracheal resection and reanastomosis in the neonatal period.

BACKGROUND/PURPOSE: Severe congenital tracheal stenosis is rare. Most of these can be managed conservatively before elective repair. Focal tracheal stenosis has been treated with resection of the involved trachea and primary reanastomosis in older infants. The authors found no reports of repair of this lesion in neonates. Two patients are presented with severe respiratory failure on the first day of life that required extracorporeal life support (ECLS) who underwent successful tracheal resection and reanastomosis (TRR) during the first week of life. METHODS: A retrospective review was conducted. RESULTS: Both babies had severe pulmonary hypertension and carbon dioxide retention despite maximal therapy and were placed on ECLS shortly after transfer. One had an isolated stenosis of the upper trachea, and the other had agenesis of the right lung, esophageal atresia with tracheoesophageal fistula, and a tracheal stenosis at the end of a short trachea with a long, narrow left bronchus. Both underwent diagnostic studies and had surgical repair while on ECLS at day 3 and 7 of life without bleeding complications. They were weaned off ECLS 1 and 8 days after surgery. One patient was extubated and did well. The other was extubated transiently, but required a tracheostomy because of left mainstem bronchomalacia. Both are alive and well at 18 and 38 months of age, with no narrowing of the repairs. CONCLUSION: In the setting of severe respiratory failure requiring ECLS support, TRR can be performed safely and successfully in the neonate with focal tracheal stenosis.

Management of the high-risk pediatric burn patient.

BACKGROUND/PURPOSE: Inhalation injury, flame burn exceeding 30%, and age under 48 months all have been cited as independent risk factors for mortality; the combination of all 3 risk factors is unusual. The authors have experienced an overall reduction in mortality rate and chose to examine this high-risk group to define techniques useful in improving outcome in pediatric burns. METHODS: A review was done of children with all 3 risk factors over a recent 9-year interval. All were treated with a system of care emphasizing precise fluid repletion, early wound excision and closure, and avoidance of injurious pulmonary inflating pressures and concentrations of oxygen. Data are expressed as mean +/- SD. RESULTS: There were 26 children admitted with all 3 risk factors. Their average age was 2.1 +/- 1.1 years (range, 5 weeks to 3.7 years), and burn size was 61% +/- 21% (range, 30% to 98%) of the body surface. All required mechanical ventilation for an average of 28 +/- 4.5 days (range, 7 to 74 days). Two children underwent tracheostomy; all others were treated with protracted oral intubation. Inhaled nitric oxide (NO) was used in 3 children, all of whom were considered for extracorporeal membrane oxygenator (ECMO) support, although none went on to ECMO. Only 7 children (27%) never had any bacteremia. Ventilator-related pneumonia occurred in 8 children (31%). Total lengths of stay, including acute and rehabilitation hospitalizations, averaged 105 +/- 10 days (1.87 +/- 0.2; range, 0.66 to 4.8 days per percent burn). After exclusion of 1 child with a 98% third-and fourth-degree burn, pre-hospital cardiac arrest, and anoxic brain injury who had support withdrawn at 6 hours, all children survived to discharge; 23 followed up in our clinic currently are alive and well with no overt residual respiratory insufficiency. CONCLUSION: A high rate of survival can be expected in young children with large burns and inhalation injury.

Surgical treatment of congenital adrenal hyperplasia.

Unraveling of the genetics of CAH offers the possibility of earlier detection and prenatal treatment or, alternatively, blastocyst embryo selection and eventually in utero gene therapy. Endocrine, surgical, and anesthesia management after birth have improved, leading to a better outcome for these patients. In the authors' experience, early one-stage reconstructive surgery, although demanding, allows one to use all available tissue. Once mastered, the repair is actually technically easier than vaginal pull-through surgery in the adolescent. Patients go through childhood with a body image that is more concordant with normal. Neither the child nor the parents must suffer the anticipation of a major operative intervention at puberty that can cause great emotional stress and that may be more difficult. The authors have encountered situations in late adolescence in which it has been impossible to separate the urogenital sinus from below. Under these circumstances, one can consider a posterior sagittal approach in which the rectum is bivalved to allow one to approach the vagina from below in an attempt to separate it safely from the urethra and to mobilize it to the perineum. It is also feasible to consider fashioning a segment of sigmoid colon as a neovagina, realizing that mucosal drainage needs to be managed daily. The authors have also encountered the rare 46,XX patient raised as a male and committed to the male role. In these cases, the patient can be offered gonadectomy, followed by staged complex hypospadias repair, and surgery to remove Müllerian structures and, if possible, to preserve the vas, followed by prepenile scrotal repair and insertion of testicular prostheses. Children with CAH require a lifetime of care with surgical approaches that are age appropriate. These patients can lead a full and productive life. It is the physician's responsibility to make certain that these children reach their full potential with the least number of interventions, which should be designed and optimized to produce the best possible outcome.

Current expectations for survival in pediatric burns.

BACKGROUND: Conventional wisdom and published reports suggest that children, particularly those younger than 48 months, have higher mortality rates after burns than young adults. However, coincident with refinements in resuscitation, operative techniques, and critical care, survival rates for children with burns seem to have improved. To document this change and to define current expectations, a review of deaths during two 7-year intervals separated by a decade was done. DESIGN: We examined the clinical course of children who died after admission for care of acute thermal burns during two 7-year intervals: calendar years 1974 to 1980 inclusive (group 1) and 1991 to 1997 inclusive (group 2). Dying children were stratified by total body surface area (TBSA) burned: small (0%-39%), midsize (40%-59%), and large (60%-100%) TBSA burns. Children who arrived with anoxic brain injury or in a moribund state with refractory shock were excluded from analysis (4 children in group 1 and 5 in group 2); 2 of these children in group 2 died and became solid organ donors. SETTING: Regional pediatric burn center. PATIENTS: Six hundred seventy-eight children in group 1 and 1150 children in group 2. MAIN OUTCOME MEASURE: Survival. RESULTS: In children with 0% to 39% TBSA burns, mortality was 0.6% in group 1 and 0% in group 2 (Fisher exact test, P = .04; chi2 test, P = .02). In children with 40% to 59% TBSA burns, mortality was 7.7% in group 1 and 0% in group 2 (Fisher exact test, P = .07; chi2 test, P = .047). In children with 60% to 100% TBSA bums, mortality was 33.3% ingroup 1 and 14.3% in group 2 (Fisher exact test, P = .04; chi2 test, P = .02). Although 59% of the children in group 2 were younger than 48 months, including 55% of those with 40% to 59% TBSA burns and 41% of those with 60% to 100% TBSA burns, there were no deaths in this age group. CONCLUSION: Survival rates after burns have improved significantly for children. At present, most children, even young children and children with large burns, should survive.

Diltiazem reduces pulmonary arterial pressures in recurrent pulmonary hypertension associated with pulmonary hypoplasia.

BACKGROUND/PURPOSE: Recurrent pulmonary hypertension in the neonatal population is an unusual event with dire consequences. Pulmonary hypertension seen in association with pulmonary hypoplasia may be refractory to conventional medical management. The effect of the calcium channel antagonist diltiazem was studied in five patients with severe pulmonary hypertension. METHODS: A retrospective review of the hospital records was performed to determine the efficacy of diltiazem for refractory pulmonary hypertension. All five patients experienced and did not respond to maximal conventional therapy, which included inhaled nitric oxide, intravenous nitrates, and extracorporeal membrane oxygenation (ECMO). Right ventricular pressures were determined by transthoracic echocardiograms and were used to document improvement in the pressure gradients. Statistical analyses were performed using a paired Student's ttest. A P value of less than .05 was considered significant. RESULTS: Diltiazem significantly reduced the right ventricular systolic pressure (RVSP) from 82 +/- 8.4 mm Hg to 58.4 +/- 7 mm Hg (P = .008). Two patients died; one had a large ventricular septal defect, and the other suffered multisystem organ failure secondary to sepsis. The surviving patients were weaned off diltiazem and did not experience recurrent pulmonary hypertension. CONCLUSIONS: In cases of pulmonary hypoplasia with recurrent pulmonary hypertension, diltiazem may be considered as a therapy. A multicenter prospective trial is advocated.

Tracheal ligation increases mitogen-activated protein kinase activity and attenuates surfactant protein B mRNA in fetal sheep lungs.

BACKGROUND: Tracheal ligation has been shown to accelerate fetal pulmonary growth in normal and hypoplastic lungs. Our aim was to study the effects of tracheal ligation on established molecular markers of growth and differentiation [mitogen-activated protein (MAP) kinase] and maturity [surfactant protein B (SPB) and fatty acid synthase (FAS)]. MATERIALS AND METHODS: Tracheal ligation was performed on four 100-day-gestation fetal sheep, with four age-matched fetuses undergoing maternal laparotomy and hysterotomy as control. Lungs from surviving fetuses (n = 2 in each group) were harvested after 4 days and frozen in liquid nitrogen. Protein lysates were prepared, and MAP kinase enzymatic assays [extracellular signal regulated protein kinase (ERK)-1 and -2] and Western blots were performed. Total RNA was isolated, and a fetal sheep lung cDNA library was created. The sheep SPB and FAS genes were cloned and sequenced. Northern blots were performed with the new clones, normalizing to beta-actin. RESULTS: Tracheal ligation lungs contained a larger volume of fluid (40 ml) compared with age-matched controls (8 ml). MAP kinase enzymatic ERK-1 activity was increased and SPB mRNA expression was reduced in fetal lungs after tracheal ligation. Neither ERK-2 enzymatic activities and FAS mRNA nor ERK protein levels were affected by tracheal ligation, by Western blot analysis. CONCLUSION: Tracheal ligation-induced fetal lung growth may be mediated in part via the MAP kinase pathway. Expression of SPB mRNA is attenuated by tracheal ligation, whereas FAS, one of the key enzymes that synthesizes the lipid portion of surfactant, is not affected.

Extracorporeal membrane oxygenation for nonneonatal acute respiratory failure.

HYPOTHESIS: Extracorporeal membrane oxygenation (ECMO) is effective in nonneonatal acute respiratory failure under certain circumstances. DESIGN: Retrospective medical record review. SETTING: The intensive care unit of a tertiary care hospital. PATIENTS: Thirty-four nonneonatal patients (mean age, 22 years; range, 8 days to 56 years), with ratios of the PaO2 to the fraction of inspired oxygen persistently below 70, who were treated with ECMO after maximal ventilator therapy had failed (mean time of ventilator therapy, 6.9 days; range, 1-41 days). The mean ECMO duration was 304 hours (range, 56-934 hours). Patients were grouped into 7 categories based on their diagnosis: sepsis or sepsis syndrome (n = 3), bacterial or fungal pneumonia (n = 10), viral pneumonia (n = 5), trauma or burn (n = 2), inhalation injury without burn (n = 1), immunocompromised state (due to transplantation or chemotherapy) (n = 8), and acute respiratory failure of unknown origin (n = 5). MAIN OUTCOME MEASURE: Survival to hospital discharge following ECMO therapy. RESULTS: Overall survival was 53% (18 patients). All 6 patients (100%) with viral pneumonias or isolated inhalation injuries survived. Of 13 patients with bacterial pneumonia, sepsis, or sepsis syndrome not complicated by multiorgan failure, 10 (77%) survived. In contrast, all but 1 of the immunocompromised patients died. Survival in patients who were intubated for less than 9 days before ECMO was 64%, whereas survival fell precipitously to 22% for patients who experienced mechanical ventilation for 9 or more days before the implementation of ECMO. Finally, the proportion of patients who died while receiving ECMO therapy was greater when the ECMO duration exceeded 300 hours (62% vs. 38%; P<.05). CONCLUSIONS: Nonneonatal survival with ECMO therapy is strongly dependent on the diagnosis. Pre-ECMO intubation for less than 9 days had little effect on survival. Survival rates decreased when the length of time of receiving ECMO exceeded 300 hours.

Acute hand burns in children: management and long-term outcome based on a 10-year experience with 698 injured hands.

OBJECTIVE: To document long-term results associated with an coordinated plan of care for acutely burned hands in children. SUMMARY AND BACKGROUND DATA: Optimal hand function is a crucial component of a high-quality survival after burn injury. This can be achieved only with a coordinated approach to the injuries. Long-term outcomes associated with such a plan of care have not been previously reported. METHODS: Over a 10-year period, 495 children with 698 acutely burned hands were managed at a regional pediatric burn facility; 219 children with 395 injured hands were followed in the authors' outpatient clinic for at least 1 year and an average of >5 years. The authors' approach to the acutely burned hand emphasizes ranging and splinting throughout the hospital stay, prompt sheet autograft wound closure as soon as practical, and the selective use of axial pin fixation and flaps. Long-term follow-up, hand therapy, and reconstructive surgery are emphasized. RESULTS: Normal functional results were seen in 97% of second-degree and 85% of third-degree injuries; in children with burns involving underlying tendon and bone, 70% could perform activities of daily living and 20% had normal function. Reconstructive hand surgery was required in 4.4% of second-degree burns, 32% of third-degree burns, and 65% of those with injuries involving underlying bone and tendon. CONCLUSIONS: When managed in a coordinated long-term program, the large majority of children with serious hand burns can be expected to have excellent functional results.

A new ventilator improves CO2 removal in newborn lambs with congenital diaphragmatic hernia.

OBJECTIVES: To demonstrate improved ventilation with intratracheal pulmonary ventilation (ITPV) in new-born lambs with congenital diaphragmatic hernia, using a new microprocessor controlled ITPV-specific ventilator. DESIGN: Prospective study, with each animal serving as its own control (paired data). SETTING: Large animal research laboratory. SUBJECTS: Diaphragmatic hernias were created surgically in seven fetal sheep on gestational day 100 (term = 145 days). INTERVENTIONS: Lambs (2.7 to 5.0 kg) were delivered by cesarean section anywhere between gestational days 136 and 140. Arterial and venous catheterizations, bilateral chest tube thoracostomies, and tracheostomies were performed while the lambs received placental bypass. Initially, congenital diaphragmatic hernia lambs were supported on conventional pressure control mechanical ventilation to achieve steady state with measurements of baseline vital signs, arterial blood gases, and ventilatory settings. ITPV was instituted while maintaining constant peak carinal pressures and oxygen saturations. Statistical comparisons were made using the paired t-test. MEASUREMENTS AND MAIN RESULTS: Postductal Paco2 decreased from 110+/-21 (SD) torr (14.7+/-2.8 kPa) to 52+/-24 torr (6.93+/-3.2 kPa; p= .0014) on ITPV. Simultaneously, pH improved from 7.04+/-0.07 to 7.31+/-0.15 (p = .0012) and minute ventilation increased from 0.66+/-0.40 to 4.00+/-1.35 L/min (p = .0016). Peak carinal pressures and postductal Pao2 were unchanged. CONCLUSIONS: ITPV significantly improved CO2 removal in newborn lambs with diaphragmatic hernias without increasing airway pressures or changing oxygenation. Based on these results, we are conducting human clinical trials.

Prenatal vitamin E treatment improves lung growth in fetal rats with congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypoplasia. To discover factors that would accelerate fetal lung growth, the authors developed models of hypoplasia, found that antioxidants improved lung growth in vitro, and then proceeded to in vivo studies. METHODS: Timed-pregnant rats were fed nitrofen (100 mg) on gestational day 9.5 (term, 22), and fetal lungs were harvested at day 13.5 and placed in organ culture in serum-free media with (n = 10) or without (n = 9) additional vitamin E (0.134 IU/mL). Camera lucida tracings were made daily on live, unstained lungs for 4 days, scanned, digitized, and analyzed for multiple growth parameters. Similar nitrofen-exposed rats were fed an optimized total dose of 150 IU vitamin E (n = 19) or olive oil (n = 13) from days 16.5 to 20.5, and fetal lungs were harvested at day 21.5, weighed and fixed for histology, or homogenized and biochemically analyzed. RESULTS: Vitamin E accelerated hypoplastic fetal lung growth in vitro as measured by area, perimeter, lung bud count, perimeter over square root area, and fractal dimension. In vivo vitamin E significantly increased lung weights, total DNA, and protein contents. CONCLUSIONS: Vitamin E accelerates hypoplastic fetal rat lung growth and complexity in vitro, and prenatal vitamin E treatment in vivo improves pulmonary hypoplasia in fetal rats with CDH.

Insulin-like growth factor-I gene expression in three models of accelerated lung growth.

BACKGROUND/PURPOSE: We have learned previously that in utero tracheal ligation reverses the structural and physiological effects of surgically created congenital diaphragmatic hernia. In addition, we have discovered that postnatal lung growth similarly can be accelerated using liquid-based airway distension with perfluorocarbon. Another model of accelerated lung growth is that of compensatory growth seen after neonatal pneumonectomy. In all of these models, growth has occurred because of an increase in alveolar number rather than enlargement of preexisting alveoli. However, the molecular mechanisms underlying these processes remain unknown. The purpose of this study was to determine if gene expression could be altered by changes in physical forces in the prenatal and postnatal lung. METHODS: The three models of accelerated lung growth studied were the following: (1) The prenatal group, consisted of fetal lambs (n = 12) that underwent the surgical creation of a left diaphragmatic hernia at 90 days' gestation. Six of these animals also underwent simultaneous tracheal ligation. (2) The PFC group consisted of five neonatal animals that underwent isolation of the superior segment of the right upper lobe, with intrabronchial distension with perfluorocarbon to 7 to 10 mm Hg pressure for a 3-week period. (3) The postpneumonectomy group consisted of four neonatal animals that underwent left pneumonectomy. In the fetal study, lungs were retrieved at term (130 days), and in the postnatal study, lungs were retrieved 3 weeks after initial intervention. In all cases, RNA was extracted from snap-frozen lung samples and Northern blot analysis performed. RESULTS: Insulinlike growth factor-I, insulinlike growth factor-II, and vascular endothelial growth factor gene expression were analyzed by densitometry. Insulinlike growth factor-I gene expression was found to be decreased in association with experimental diaphragmatic hernia (P = .005), but restored to normal with tracheal ligation. Insulinlike growth factor-I gene expression was significantly increased in both postnatal models of accelerated lung growth (P = .022, P = .016). No significant differences were found in insulinlike growth factor-II or vascular endothelial growth factor gene expression. CONCLUSIONS: The authors conclude from these preliminary data that (1) insulin like growth factor-I gene expression is reduced in experimental fetal diaphragmatic hernia and restored to normal by tracheal ligation, and (2) insulinlike growth factor-I gene expression is increased in both the liquid-based airway distension and postpneumonectomy models of accelerated postnatal lung growth. The authors speculate that all of these manipulations exploit a natural pathway essential for normal lung growth.

Congenital diaphragmatic hernia--does the side of the defect influence the incidence of associated malformations?

BACKGROUND/PURPOSE: Patients with congenital diaphragmatic hernia (CDH) frequently have associated anomalies. Experiments in the nitrofen CDH model have shown differential embryonic cell death patterns in rodents suggesting unique mechanisms in the formation of right-sided (RCDH) or left-sided (LCDH) diaphragmatic hernia. These findings provide insight into the pathogenesis of CDH and may aid our understanding on the spectrum of associated anomalies commonly observed in humans. This study therefore set out to test the hypothesis that the side of the diaphragmatic defect in humans is related to the incidence and severity of coexistent organ malformations. METHODS: The medical and autopsy records of 301 CDH patients presenting to two institutions over a 23-year period were examined to analyze these factors. RESULTS: One hundred patients (33%) were found to have one or more associated anomalies. The incidence of multiple-RCDH (10%) versus LCDH (7.3%) and cardiac anomalies-RCDH (10%) versus LCDH (8.5%) was similar in both groups of patients. However, the hypoplastic heart syndrome was a unique feature in 5 of 22 patients (23%) with LCDH who had cardiac abnormalities. This cardiac anomaly may be related developmentally to LCDH. CONCLUSION: The cellular mechanisms underlying the genesis of this spectrum of abnormalities in humans and the nitrofen CDH model warrant further study to elucidate factors governing embryonic cell fate and phenotype expression.

Prenatal hormones alter antioxidant enzymes and lung histology in rats with congenital diaphragmatic hernia.

Prenatal administration of dexamethasone (Dex) and thyrotropin-releasing hormone (TRH) synergistically enhances lung maturity, but TRH suppresses the antioxidant enzyme activity. Prenatal hormonal therapy improves alveolar surfactant content and lung compliance in rats with congenital diaphragmatic hernia (CDH). In full term neonatal rats with CDH we studied the effects of prenatal Dex or Dex+TRH on antioxidant enzyme activity at birth, on survival, and on lung morphometry after 4 h of ventilation with 100% O2. CDH was induced by administration of 2,4-dichlorophenyl-p-nitro-phenylether (Nitrofen) on gestational day 10. Dex+TRH-treated CDH rats had lower activity of glutathione reductase after birth than did sham-treated CDH pups. Dex-treated and sham-treated pups had similar antioxidant enzyme activity. Hormonal treatment did not change survival during ventilation. The average airspace volume increased in Dex-treated CDH pups after ventilation, with a small synergistic effect after addition of TRH. On the basis of our findings, we speculate that prenatal administration of Dex is the best choice to improve lung maturity and airspace volume in CDH patients.

High-frequency intratracheal pulmonary ventilation: improved gas exchange at lower airway pressures.

PURPOSE: Conventional ventilation in the neonatal intensive care unit causes iatrogenic injury to fragile newborn lungs, especially those with preexisting pathology or prematurity. Intratracheal pulmonary ventilation (ITPV), developed by Dr Theodor Kolobow and associates at the National Institutes of Health (NIH), incorporates a continuous flow of humidified gas through a reverse thrust catheter positioned at the distal end of the endotracheal tube. In animal studies ITPV was shown to facilitate gas exchange at low peak pressures by reducing physiological dead space, facilitating exhalation, and enhancing CO2 elimination. The specific aims of this project were (1) to invent a new ITPV-specific ventilator; (2) to optimize gas exchange in a newborn animal model at low airway pressures using higher frequency ITPV; and (3) to demonstrate efficacy and improved ventilation at lower airway pressures in a prematurity model. METHODS: (1) A new ventilator had to be constructed. The first prototype is microprocessor driven, incorporating controls for flow, pressures, and concentrations of gases. The ventilator has the capability to vary Fio2, respiratory rate (0 to 15 Hz), and inspiratory-expiratory I:E ratio. (2) Prototype testing was performed. Newborn lambs (n = 3, 6 to 7 kg) underwent tracheotomy and placement of arterial and venous lines. Lambs were initially supported on conventional mechanical ventilation (CMV). Animals were allowed to achieve steady state with measurements of baseline vital signs, arterial blood gases, and ventilatory settings. ITPV was instituted at a rate of 100 breaths per minute and flow adjusted to achieve lower peak carinal pressures than obtainable on conventional ventilation. In a stepwise fashion, respiratory rate, I:E ratio, and ITPV flows were varied while initially maintaining Paco2 constant, and then allowing improvement. (3) These experiments were repeated in preterm lambs (n = 6, 1.8 to 3.6 kg). RESULTS: At the time of transition from CMV to ITPV (rate, 100, I:E, 1:3), gas exchange was maintained despite a documented drop in average peak carinal pressure for the newborn lambs from 28.3 cm H2O on CMV to 10.3 cm H2O on ITPV (P = .028). The average peak carinal pressure fell even further at higher ITPV rates with adjustments in I:E ratio. For the premature lambs, peak carinal pressures also fell significantly on ITPV (44 to 32 cm H2O, P = .002) with corresponding significant improvement in ventilation (Paco2 from 52.2 to 31.9 mm Hg, P = .029). CONCLUSIONS: (1) Our new ITPV ventilator operates at rates and I:E ratios previously unobtainable. (2) In newborn and premature lambs ITPV functions most effectively at higher rates with higher gas flow rates and with longer exhalation, providing significantly improved gas exchange at significantly lower peak carinal pressures. (3) ITPV may prove beneficial in achieving gas exchange in newborns while avoiding barotrauma. Based on these data, we have initiated human clinical studies of ITPV in newborns with congenital diaphragmatic hernia or prematurity to improve gas exchange and reduce barotrauma in the neonatal intensive care unit.

Prenatal glucocorticoids improve pulmonary morphometrics in fetal sheep with congenital diaphragmatic hernia.

PURPOSE: Prenatal glucocorticoids reverse pulmonary immaturity in rodents with pharmacologically induced congenital diaphragmatic hernia (CDH). The authors applied quantitative stereologic morphometric techniques to test whether these effects could be reproduced in large animals (sheep) with surgically created CDH. METHODS: Diaphragmatic hernias were created surgically in fetal lambs at gestational day 80. The fetuses were treated with intravenous cortisol (n = 6) or normal saline control (n = 5) from days 133 to 135. Lungs distended at 15 cm pressure from each group were harvested at day 136, processed histologically, and studied by brightfield microscopy at 400 x using a 42-point equidistant counting grid. Ten morphometric parameters (Mean +/- SEM) were measured by point-counting 60 fields/lung, and analysis of variance was performed. RESULTS: The CDH-cortisol-treated lungs showed striking significant maturational improvements when compared with lungs of CDH-normal saline controls by seven of ten morphometric parameters. CONCLUSIONS: (1) Prenatal glucocorticoids accelerate lung maturity in fetal lambs with CDH by seven quantitative morphometric parameters. (2) The observation that prenatal glucocorticoid therapy improves measures of maturity for both CDH rodent and sheep models encourages proceeding with a Phase I human clinical trial in ultrasound-confirmed CDH.

Toward understanding the developmental anatomy of congenital diaphragmatic hernia.

The pathway of abnormal embryonic development leading to congenital diaphragmatic hernia (CDH) is incompletely understood. Using a nitrofen-induced model of left CDH in rats, sequential stages of development were analyzed by scanning electron microscopy. Abnormal development patterns were observed in the cells comprising the posthepatic mesenchymal plate and the adjacent liver. The septum transversum did not appear to be involved. In this article, the authors theorize that a disturbed "balance of cell growth" is responsible for the creation of the diaphragmatic defect.

Prenatal glucocorticoid therapy reverses pulmonary immaturity in congenital diaphragmatic hernia in fetal sheep.

OBJECTIVE: To assess the feasibility of conducting clinical trials of prenatal steroid therapy for congenital diaphragmatic hernia (CDH) in humans, the authors tested whether prenatal glucocorticoid, currently the standard treatment to minimize respiratory distress syndrome in premature infants, might improve the pulmonary immaturity in severe CDH in a large animal model. SUMMARY BACKGROUND DATA: The authors have used the nitrofen-induced rat model of CDH, which demonstrates immature lungs by biochemical, morphometric, and molecular biologic criteria. They also have shown that the lethally immature lungs of the full-term CDH rats can be improved by biochemical, morphometric, physiologic, and molecular criteria by treating the mothers with parenteral steroids at doses extrapolated from the current therapy used to accelerate lung development of premature human babies. METHODS: During a 3-year period in 88 fetal sheep, 1) left-sided diaphragmatic hernias were created surgically at varying gestational ages (day 78-90; term = 142-145 days) and size to maximize severity (n = 45), 2) placement and design of indwelling fetal intravenous catheters were optimized (n = 13), and 3) timing and dosage of cortisol administration were determined (n = 17). As a result, diaphragmatic hernias were created on day 80, intravenous catheters were placed on day 120, and twice-daily intravenous cortisol injections (n = 8) or saline as the control (n = 5) were administered (days 133-135). Lambs were delivered on day 136 via cesarean section to avoid steroid-induced abortion; vascular access was obtained, and the fetuses were ventilated at standard settings. Physiologic data were collected, and lungs were harvested for biochemical and histologic analysis. RESULTS: Significant improvements were measured in postductal arterial oxygen pressure ([PaO2] 38 +/- 6 mmHg after cortisol therapy compared with 20 +/- 3 mmHg for saline controls; p = 0.002) and in dynamic compliance (0.42 +/- 0.05 mL/cm H2O vs. 0.29 +/- 0.01 mL/cm H2O; p = 0.01). Lung glycogen levels in the right lung of the cortisol group were significantly better than controls (4.6 +/- 0.3 mg/g lung vs. 6.8 +/- 0.4 mg/g; p = 0.002), as were protein/DNA levels (8.3 +/- 0.9 mg/mg vs. 14.5 +/- mg/mg; p < 0.05). Striking morphologic maturation of airway architecture was observed in the treated lungs. CONCLUSIONS: Prenatal glucocorticoids correct the pulmonary immaturity of fetal sheep with CDH by physiologic, biochemical, and histologic criteria. These data, combined with previous small animal studies, have prompted the authors to initiate a prospective phase I/II clinical trial to examine the efficacy of prenatal glucocorticoids to improve the maturation of hypoplastic lungs associated with CDH.