Liver disease after intensive care of premature baboons: histopathologic observations.

BACKGROUND AND OBJECTIVES: Cholestasis affects 50% of extremely low-birth-weight infants. Its etiology remains poorly understood and the extent of liver injury in these infants is unclear. The premature baboon model provides an opportunity to study neonatal liver disease. We characterize hepatic histopathologic changes in this model. METHODS: Archival tissue and data were obtained from the Southwest Foundation for Biomedical Research Primate Center, San Antonio, TX. Animals were selected based on history of antenatal steroid therapy and absence of sepsis or necrotizing enterocolitis with a protocol duration of at least 21 days and no early death (n = 45). Baboons had been treated per protocol in the neonatal intensive care unit (NICU). At necropsy, liver tissue was harvested and stored. Tissues from fetal gestational controls at similar ages were used for comparison (n = 28). Histologic changes were scored by consensus of 2 pathologists blinded to treatment group. Descriptive and comparative statistics were performed. RESULTS: Control fetal livers had extramedullary hematopoiesis (EMH) that decreased across the gestational range. There was evidence of hepatocyte iron storage and ongoing portal tract development. Livers of NICU-treated baboons had increased Kupffer cell hypertrophy and hemosiderosis. There was a shift away from erythroid EMH toward increased myeloid EMH. There was increased cholestasis, ductular proliferation, portal tract fibrosis, and steatosis in treated animals. CONCLUSIONS: We found pathologic changes in NICU-treated baboons comparable with findings reported in human infants. The baboon model of prematurity may be a useful tool to explore cholestasis and liver dysfunction in extremely low-birth-weight infants.

Association of novel markers of liver disease with neonatal liver disease in premature baboons, Papio sp.

Parenteral Nutrition (PN) Associated Liver Disease (PNALD) affects up to 60% of neonates; however, techniques for diagnosing and monitoring disease progression remain limited. The neonatal baboon model may provide a unique opportunity to identify serologic markers associated with this disease. The purpose of this study was to investigate if Hyaluronic Acid (HA), TIMP metallopeptidase inhibitor 1 (TIMP1), Amino-terminal Propeptide of Type-III Collagen (PIIINP) and Enhanced Liver Fibrosis (ELF) score associate with histological liver disease in neonatal baboons exposed to PN. Preterm baboons delivered via c-section at 67% gestation received PN for 14 days with or without Intralipid (PRT+IL, PRT-IL, respectively) or were sacrificed after birth (PRTCTR). Term baboons were sacrificed after birth (TERMCTR) or survived 14 days (TERM+14d). Serum HA, TIMP1, and PIIINP concentrations were measured by ELISA. A blinded pathologist assigned liver histological scores following necropsy. HA increased 9.1-fold, TIMP1 increased 2.2-fold, and ELF score increased 1.4-fold in PRT-IL compared to PRTCTR. ALT, AST, and GGT were within normal limits and did not vary between groups. A trend towards increased fibrosis was found in PRT-IL baboons. Microvesicular hepatocyte steatosis and Kupffer cell hypertrophy were elevated in PRT-IL vs PRTCTR. HA and TIMP1 were significantly elevated in preterm baboons with early histological findings of liver disease evidenced by hepatic steatosis, Kupffer cell hypertrophy and a trend towards fibrosis whereas traditional markers of liver disease remained normal. These novel markers could potentially be utilized for monitoring early hepatic injury in neonates.

Surfactant composition and function in a primate model of infant chronic lung disease: effects of inhaled nitric oxide.

Bronchopulmonary dysplasia, or chronic lung disease (CLD), of premature infants involves injury from hyperoxia and mechanical ventilation to an immature lung. We examined surfactant and nitric oxide (NO), which are developmentally deficient in premature infants, in the baboon model of developing CLD. Fetuses were delivered at 125 d gestation and were managed for 14 d with ventilation and oxygen prn without (controls) or with inhaled NO at 5 ppm. Compared with term infants, premature control infants had reduced maximal lung volume, decreased tissue content of surfactant proteins SP-A, -B, and -C, abnormal lavage surfactant as assessed by pulsating bubble surfactometer, and a low concentration of SP-B/phospholipid. NO treatment significantly increased maximal lung volume and tissue SP-A and SP-C, reduced recovery of lavage surfactant by 33%, decreased the total protein:phospholipid ratio of surfactant by 50%, and had no effect on phospholipid composition or SP content except for SP-C (50%). In both treatment groups, levels of SP-B and SP-C in surfactant were negatively correlated with STmin, with a 5-fold greater SP efficiency for NO versus control animals. By contrast, lung volume and compliance were not correlated with surfactant function. We conclude that surfactant is often dysfunctional in developing CLD secondary to SP-B deficiency. NO treatment improves the apparent ability of hydrophobic SP to promote low surface tension, perhaps secondary to less protein inactivation of surfactant, and improves lung volume by a process unrelated to surfactant function.

Effect of ductus ligation on cardiopulmonary function in premature baboons.

RATIONALE: The role of the patent ductus arteriosus in the development of chronic lung disease in surfactant-treated premature newborns remains unclear. OBJECTIVE: To examine the effects of ductus ligation on cardiopulmonary function and lung histopathology in premature primates. METHODS: Baboons were delivered at 125 d, (term = 185 d) treated with surfactant, and ventilated for 14 d. Serial echocardiograms and pulmonary function tests were performed. Animals were randomized to ligation (n = 12) or no ligation (controls, n = 13) on Day 6 of life. Necropsy was performed on Day 14. RESULTS: Compared with nonligated control animals, ligated animals had lower pulmonary-to-systemic flow ratios, higher systemic blood pressures, and improved indices of right and left ventricular performance. The ligated animals tended to have better compliance and ventilation indices for the last 3 d of the study. There were no differences between the groups in proinflammatory tracheal cytokines (interleukin [IL] 6 and IL-8), static lung compliance, or lung histology. CONCLUSION: Although a persistent patent ductus arteriosus results in diminished cardiac function and increased ventilatory requirements at the end of the second week of life, ligation on Day 6 had no measurable effect on the histologic evolution of chronic lung injury in this 14-d baboon model.

Pulmonary and systemic nitric oxide metabolites in a baboon model of neonatal chronic lung disease.

We report on developmental changes of pulmonary and systemic nitric oxide (NO) metabolites in a baboon model of chronic lung disease with or without exposure to inhaled NO. The plasma levels of nitrite and nitrate, staining for S-nitrosothiols and 3-nitrotyrosine in the large airways, increased between 125 d and 140 d of gestation (term 185 d) in animals developing in utero. The developmental increase in NO-mediated protein modifications was not interrupted by delivery at 125 d of gestation and mechanical ventilation for 14 d, whereas plasma nitrite and nitrate levels increased in this model. Exposure to inhaled NO resulted in a further increase in plasma nitrite and nitrate and an increase in plasma S-nitrosothiol without altering lung NO synthase expression. These data demonstrate a developmental progression in levels of pulmonary NO metabolites that parallel known maturational increases in total NO synthase activity in the lung. Despite known suppression of total pulmonary NO synthase activity in the chronic lung disease model, pulmonary and systemic NO metabolite levels are higher than in the developmental control animals. Thus, a deficiency in NO production and biological function in the premature baboon was not apparent by the detection and quantification of these surrogate markers of NO production.

Inhaled NO improves early pulmonary function and modifies lung growth and elastin deposition in a baboon model of neonatal chronic lung disease.

Nitric oxide (NO) serves multiple functions in the developing lung, and pulmonary NO production is decreased in a baboon model of chronic lung disease (CLD) after premature birth at 125 days (d) gestation (term = 185d). To determine whether postnatal NO administration alters the genesis of CLD, the effects of inhaled NO (iNO, 5 ppm) were assessed in the baboon model over 14d. iNO caused a decrease in pulmonary artery pressure in the first 2d and a greater rate of spontaneous closure of the ductus arteriosus, and lung compliance was greater and expiratory resistance was improved during the first week. With iNO, postmortem pressure-volume curves were shifted upward, lung DNA content and cell proliferation were increased, and lung growth was preserved to equal that which occurs during the same period in utero. In addition, the excessive elastin deposition characteristic of CLD was normalized by iNO, and there was evidence of stimulation of secondary crest development. Thus, in the baboon model of CLD, iNO improves early pulmonary function and alters lung growth and extracellular matrix deposition. As such, NO biosynthetic pathway dysfunction may contribute to the pathogenesis of CLD.

Developmental changes in nitric oxide synthase isoform expression and nitric oxide production in fetal baboon lung.

Nitric oxide (NO), produced by NO synthase (NOS), plays a critical role in multiple processes in the lung during the perinatal period. To better understand the regulation of pulmonary NO production in the developing primate, we determined the cell specificity and developmental changes in NOS isoform expression and action in the lungs of third-trimester fetal baboons. Immunohistochemistry in lungs obtained at 175 days (d) of gestation (term = 185 d) revealed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal airway epithelium. In proximal lung, there was a marked increase in total NOS enzymatic activity from 125 to 140 d gestation due to elevations in nNOS and eNOS, whereas iNOS expression and activity were minimal. Total NOS activity was constant from 140 to 175 d gestation, and during the latter stage (160-175 d gestation), a dramatic fall in nNOS and eNOS was replaced by a rise in iNOS. Studies done within 1 h of delivery at 125 or 140 d gestation revealed that the principal increase in NOS during the third trimester is associated with an elevation in exhaled NO levels, a decline in expiratory resistance, and greater pulmonary compliance. Thus, there are developmental increases in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period.

Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease.

Nitric oxide (NO), produced by NO synthase (NOS), serves multiple functions in the perinatal lung. In fetal baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) are all primarily expressed in proximal respiratory epithelium. In the present study, NOS expression and activity in proximal lung and minute ventilation of NO standard temperature and pressure (VeNO(STP)) were evaluated in a model of chronic lung disease (CLD) in baboons delivered at 125 days (d) of gestation (term = 185 d) and ventilated for 14 d, obtaining control lung samples from fetuses at 125 or 140 d of gestation. In contrast to the normal 73% increase in total NOS activity from 125 to 140 d of gestation, there was an 83% decline with CLD. This was related to marked diminutions in both nNOS and eNOS expression and enzymatic activity. nNOS accounted for the vast majority of enzymatic activity in all groups. The normal 3.3-fold maturational rise in iNOS protein expression was blunted in CLD, yet iNOS activity was elevated in CLD compared with at birth. The contribution of iNOS to total NOS activity was minimal in all groups. VeNO(STP) remained stable in the range of 0.5-1.0 nl x kg(-1) x min(-1) from birth to day 7 of life, and it then rose by 2.5-fold. Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity over the first 2 wk of postnatal life. It is postulated that these alterations in NOS expression and activity may contribute to the pathogenesis of CLD.