- Email: [email protected]
Decreased pulmonary nitric oxide synthase activity in the rat model of congenital diaphragmatic hernia
Decreased Pulmonary Nitric Oxide Synthase Activity in the Rat Model of Congenital Diaphragmatic Hernia By Hratch L. Karamanoukian, Tracie Peay, John E. Love, Ehad AbdeI-Rahman, Paresh Dandonna, Richard G. Azizkhan, and Philip L. Glick
Buffalo, New York • Because nitric oxide (NO) dilates vascular smooth muscle cells, a deficiency of endogenous pulmonary nitric oxide production by nitric oxide synthase (NOS) has been suggested to be involved in the pathophysiology of pulmonary hypertension in congenital diaphragmatic hernia (CDH). Our aim was to determine whether experimentally induced CDH in rats results in a decrease in the synthesis of NO in the lungs. Adult Sprague-Dawley rats were fed 300 mg/kg of nitrofen at 10.5 days' gestation. CDH, control, and sham (dosed with nitrofen, but without CDH) lungs were homogenized at full term (22 days' gestation) for measurement of NOS activity using the 14C-L-arginine to 14C-L-citrulline conversion assay, Western blot analysis with anti-endothelial cell NOS (EC-NOS} monoclonal antibody (mAb) was performed, and NOS expression was measured by densitometry. NOS activity was highest in the pulmonary parenchyma of control rat lungs (0.420 _+ .020 f m o l / m i n / m g lung; n = 11), intermediate in sham lungs (0.370-+ 0.010 f m o l / m i n / m g lung; n = 14), and lowest in CDH lungs (0.300 -+ 0.04 f m o l / m i n / m g lung; n = 12). NOS activity in the CDH and sham lungs was significantly lower than that of control lungs (P < .05). There was no difference in pulmonary NOS activity between sham and CDH lungs. NOS protein expression by Western blot analysis paralleled the observation for NOS activity in all groups, with the highest concentrations in controls, intermediate expression in sham lungs, and lowest expression in CDH lungs. Both NOS expression and NOS activity are significantly decreased in CDH rat lungs. Pulmonary hypertension in this model may be attributable to a deficiency of endogenous NO. This is the first reported study to suggest that decreased NOS activity may result in pulmonary hypertension in CDH. Copyright © 1996by W.B. Saunders Company INDEX WORDS: Congenital diaphragmatic hernia, pulmo-
nary hypertension, nitric oxide synthase.
T
H E H I G H M O R T A L I T Y rate associated with congenital diaphragmatic hernia (CDH)
From the Buffalo Institute of Fetal Therapy, The Children's Hospital of Buffalo, and the Departments of Surgery, Pediatrics, and Medicine, State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, N Y Presented at the 1995 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, San Francisco, California, October 13-15, 1995. Supported in part by grants from the American Lung Association, the Women and Children's Health Research Foundation, and NIH HL 36543 and 49977. Address reprint requests to Philip L. Glick, MD, The Buffalo Institute of Fetal Therapy, Department of Pediatric Surgery, The Children's Hospital of Buffalo, 219 Bryant St, Buffalo, N Y 14222. Copyright © 1996 by W.B. Saunders Company 0022-3468/96/3108-0003503. 00/0 1016
has been shown to result from a combination of pulmonary hypoplasia and pulmonary hypertension. CDH lungs have a reduced number of conducting airways, alveoli, and preacinar pulmonary vessels. 1-4 In addition, the maturity of the lung appears retarded. 5 The results of morphological analyses have suggested that pulmonary hypertension is caused by abnormal muscularization of the acinar arteries and a decrease in the overall cross-sectional area of the pulmonary vascular bed, affecting both ipsilateral and contralateral lungs. 6-9 These morphological features also are prominent features of the fetal lamb and rat models of CDH. t°,11 Whole animal studies have shown the presence of pulmonary hypertension by the measurement of elevated levels of both pulmonary pressures and pulmonary vascular resistance in the newborn period. 12-14 Because nitric oxide (NO) dilates vascular smooth muscle cells, a deficiency of nitric oxide synthase (NOS) has been suggested to be involved in the pathophysiology of pulmonary hypertension in CDH. Studies in the fetal lamb model of CDH have demonstrated NOS in the pulmonary vessels by both immunohistochemistry and NADPH diaphorase activity, which indicates the presence of the NO-producing enzyme in CDH. 15The intriguing question remains: Is there a deficiency in the endogenous production of NO in the pulmonary vascular bed, and if so, does it result in pulmonary hypertension? The aim of the present study was to determine whether there is a decrease in NOS activity in the lung parenchyma and/or a deficiency of NOS expression in the nitrofeninduced rat model of CDH. MATERIALS AND METHODS
Nitrofen-Induced Rat CDH Model Adult Sprague-Dawley rats were bred at our laboratory animal center after controlled overnight matings. A positive smear was considered proof of pregnancy, and the day this observation was made was designated as day 0. The animals were provided with ad libitum feeding and water throughout pregnancy. At 10.5 days of pregnancy (full term, 22 days), 300 mg/kg body weight of nitrofen (2,4-dichlorophenyl-P-nitrophenyl; Dupliar, Weesp, Netherlands), dissolved in olive oil, was administered as a single dose via a gastric tube. Cesarean section was performed on day 21. Control animals were given the same dose of olive oil, without nitrofen. CDH, control, and sham specimens (dosed with nitrofen, but without a
JournalofPediatric Surgery, Vo131,No 8 (August), 1996: pp 1016-1019
DECREASED NITRIC OXIDE SYNTHASE IN CDH
1017
diaphragmatic hernia) were stored at -70°F until they were studied further.
• p<.05 vs Control
0.5
# p = NS vs Sham
e)
NOS Activity
0.4
Fragments of lung tissue (CDH, sham, and control) weighing approximately 50 mg were homogenized in distilled water (dd H20; 1 g in 2 vol) and were stored on ice before use. Incubations were initiated by the addition of 25 p.L of tissue homogenate to 125 IxL of 50 nmol/L Tris HC1 buffer (pH, 7.4) containing 1.25 mmol/L CaCI2, 1.0 mmol/L EDTA (pH, 7.5), 1.0 mmol/L NADPH, 20 p.mol/L L-arginine (Sigma Chemical Co, St Louis, MO), and 3.314 Ixmol/L 14C-L-arginine (1 ixCi/mL), which had been preincubated for approximately 4 minutes at 30° Fahrenheit. Reaction was terminated after 40 minutes by the addition of 2.0 mL of "stop" buffer containing 20 mmol/L Tris HC1 (pH, 5.5) and 4.0 mmol/L EDTA (pH, 7.5), followed by Vortex mixing. Individual incubation mixtures (2.15 mL) were added to columns packed with 1.0 mL of Dowex AG50WX-8 (Na ÷ form; Fluka AG, St Louis, MO) that had been preequilibrated with a minimum of 2.0 mL stop buffer. Each column was allowed to elute by gravity into collection vials. Conversion of 14C-L-arginine to 14C-L-citrulline was quantified by liquid scintillation spectroscopy.
Western Blot Analysis Sodium dodecyl sulfate-polyacrylamide (8%) electrophoresis (SDS-PAGE) was performed as described by Cleveland et al. ~6 Electrophoresis transfer of separated proteins to nitrocellulose membranes (Bio-rad Laboratories, Richmond, CA) was performed according to protocols previously described by O'Connor et al. 17 Nitrocellulose membranes were incubated in Tris-buffered saline (TBS) (10 mmol/L Tris, 150 mmol/L NaCI; pH, 7.4), containing 5% Blotto (Carnation nonfat dry milk), for 1 hour at room temperature to block nonspecific binding. Incubation with antiECNOS mAb (1:250 dilution) (Transduction Laboratories, Lexington, KY) in TBS containing 5% Blotto was performed overnight at room temperature with rocking. The membranes were washed in TBS containing 0.5% Tween-20 (TABS) and further incubated in 5% Blotto containing goat antimouse horseradish peroxidaseconjugated gamma-immunoglobulin (1:3000) (Bio-rad Laboratories, Richmond, CA) for 1 hour at room temperature. Blots were washed extensively in TABS (3 x 10 minutes), air-dried, incubated with Deepened (Boston, MA) chemiluminescence-developing reagents for 1 minute, and exposed to Deepened-sensitive film for 1 minute. The molecular weights of resulting bands were determined from the migration positions of prestained colored molecular size markers (Bio-rad Laboratories). The EC-NOS expression for each specimen was quantitated by densitometry readings according to the area method and was reported as ratios (compared with the ECNOS expression in control lungs) for each group.
Statistical Analysis
~_~0.3
o.2 'r'~
0.1
0
Control
Sham
CDH
n=11
n=14
n=12
Fig 1. Pulmonary nitric oxide synthase activity in the nitrofeninduced rat model of CDH.
and was lowest in the CDH lungs (0.300 _+ 0.04 fmol/min/mg lung; n = 12) (Fig 1). NOS activity in the CDH and sham lungs was significantly lower than that of control lungs (P < .05). Although NOS activity was higher than in sham lungs than in the CDH lungs, the difference was not significant. NOS protein expression by Western blot analysis paralleled the observation for NOS activity in all groups, with the highest expression in control lungs, intermediate expression in sham lungs, and the lowest expression in CDH lungs (Fig 2). This was confirmed by densitometry readings. EC-NOS was calculated from the area under the curve and was reported as a ratio for the various groups of animals (control, 1.0; sham, 018; CDH, 0.6).
A
B
C
D
KD
204 140 132"~
65
All data are expressed as mean _+ standard error (SE) of the mean. Comparisons between groups of animals were made using analysis of variance followed by the Student's t test. P values of less than .05 were considered to represent a significant difference between the compared values.
* 7#
.
42.6 .
RESULTS
NOS activity was highest in the pulmonary parenchyma of control rat lungs (0.420_ .020 fmol/ min/mg lung; n = 11). It was intermediate in the sham lungs (0.370 ___0.010 fmol/min/mg lung; n = 14)
Fig 2. Western blot analysis with anti-ECNOS mAb on 50-~g total lung homogenates from (A) sham, (B) CDH, (C) control rat and (D) mouse endothelial cell homogenate as a positive control.
1018
KARAMANOUKIAN ET AL
DISCUSSION
Inhaled NO is a selective pulmonary vasodilator. In a lamb model of persistent pulmonary hypertension, it has been shown to improve oxygenation and survival. 18,19 It also has been shown to improve oxygenation in infants who have persistent pulmonary hypertension (PPHN).2°,21 Hopes were high that NO would be the "magic bullet" to treat pulmonary hypertension in CDH. Although initial reports were disappointing, several recent case reports indicate that inhaled NO may be efficacious in the treatment pulmonary hypertension in CDH. 2°-23 We have shown that newborn lambs with surgically created CDH respond well to inhaled NO therapy if certain criteria are met. 12 Inhaled NO is effective in decreasing pulmonary pressures only after the surfactant deficiency has been corrected with exogenous surfactant, which enables NO to reach the presumed "diseased" vascular bed in the microcirculation of the lung. 12 It appears that exogenous surfactant is required in the lamb model of CDH for delivery of inhaled NO to the terminal lung units, where it works synergistically with exogenous surfactant to decrease intrapulmonary shunting and pulmonary artery pressures.12 Similar efficacy has been achieved with the combination of inhaled NO and high-frequency oscillatory ventilation (HFOV) or perflurocarbon-associated gas exchange (PAGE). 21-23 The nitrofen-induced rat model of CDH appears to be a cost-effective, multiparous model for the study of the pathophysiology of CDH. z4,25 The nitrofen model produces global abnormalities of the heart, kidneys,
liver, brain, thyroid, bones, and adrenal glands.26 It has been shown to partially mimic the pathophysi01ogy of CDH, because it has both pulmonary hypopiasia and a surfactant deficiency.27 In contrast to the lamb model of CDH, there is scant data regarding pulmonary circulation. It has been difficult, if not impossible, to quantitate pulmonary and systemic hemodynamics in the nitrofen-induced model because of the newborn rat's small size ( < 3 g). Therefore, it remains unknown whether pulmonary hypertension is a pathophysiological feature in this widely studied model of CDH. it Nevertheless, the results of preliminary studies that used barium-injected pulmonary arterial radiographs have suggested that there is a decrease in the size of the vascular bed in the nitrofen model of CDH. n Therefore, it has been assumed, but not proven, that pulmonary hypertension is a pathophysiologicai feature of the rat model of CDH. Although preliminary data have shown that the NOS mRNA levels are identical for CDH and control rat pups at term, 28 our results suggest that, based on the preliminary pulmonary vascular morphometric studies, pulmonary hypertension in the nitrofen-induced rat model of CDH may be caused by a deficiency of endogenous NO production by the pulmonary vascular bed. This is the first reported study to suggest that decreased pulmonary NOS activity may result in pulmonary hypertension, and the first study to indicate that decreased pulmonary NOS activity may result in pulmonary hypertension in CDH.
REFERENCES 1. Geggle RL, Murphy JD, Langleben D, et al: Diaphragmatic hernia in the fetus: Arterial structural changes and persistent pulmonary hypertension after surgical repair. J Pediatr 107:457464, 1985 2. Bohn D, Tamura M, Perrin D, et al: Ventilatory predictors of pulmonary hypoplasia in congenital diaphragmatic hernia, confirmed by morphologic assessment. J Pediatr 111:423-431, 1987 3. Levin DL: Morphologic analysis of the pulmonary vascular bedl in congenital left-sided diaphragmatic. J Pediatr 92:805-809, 1979 41 Kitagawa M, Hislop A, Boyden EA, et al: Lung hypoplasia in congenital diaphragmatic hernia. A quantitative study of airway, artery, and alveolar development. Br J Surg 58:342-346, 1971 5~ George DK, Cooney TP, Chiu BK, et al: Hypoplasia and immaturity of the terminal lung unit (acinus) in congenital diaphragmatic hernia. Am RevRespir Dis 136:947-950, 1987 6. Adzick NS, Harrison MR, Outwater KM, et al: Correction of congenital diaphragmatic hernia in utero IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis. J Pediatr Surg 20:673-680, 1985 7. Nguyen L, Guttman FM, De Chadarevian JP, et al: The mortality of congenital diaphragmatic hernia. Is total pulmonary mass inadequate, no matter what? Ann Surg 198:766-769, 1983
8. Reale FR, Esterly JR: Pulmonary hypoplasia: A morphometrie study of the lungs of infants with diaphragmatic hernia, anencephaly, and renal malformations. Pediatrics 51:91-96, 1973 9. Hislop A, Reid L: Persistent hypoplasia of the lung after repair of congenital diaphragmatic hernia. Thorax 31:450-455, 1976 10. DiMaio M, Ting A, Gil J, et al: A morphometric study of lung hypoplasia in a second trimester lamb model of congenital diaphragmatic hernia (CDH). Am J Resp Crit Care Med 149:A727, 1994 11. Tenbrinck R, Gaillard D, Tibboel D, et al: Pulmonary vascular abnormalities in experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg27:862-865, 1992 12. Wilcox DT, Glick PL, Karamanoukian HL, et al: Pathophysiology Of congenital diaphragmatic hernia V. Effect of exogenous surfactant therapy on gas exchange and lung mechanics in the lamb congenital diaphragmatic hernia model. J Pediatr 124:289-293, 1994 13. Karamanoukian HL, Glick PL, Wilcox DT, et al: Pathophysiology of congenital diaphragmatic hernia VIII: Inhaled nitric requires exogenous surfactant therapy in the lamb modei of congenital diaphragmatic hernia. J Pediatr 30:1-4, 1995 14. O'Toole SJ, Karamanoukian HL, Morin III FC, et al:
DECREASED NITRIC OXIDE SYNTHASE IN CDH
1019
Surfactant decreases pulmonary vascular resistance and increases blood flow in the lamb model of congenital diaphragmatic hernia. J Pediatr Surg 31:507-511, 1996 15. Karamanoukian HL, Glick PL, Wilcox DT, et al: Pathophysiology of congenital diaphragmatic hernia X: Localization of nitric oxide synthase in the intima of pulmonary artery trunks of lambs with surgically created congenital diaphragmatic hernia. J Pediatr Surg 30:5-9, 1995 16, Cjeve DW, Fischer SG, Kirschner MW, et al: J Biol Chem 252:1102-1106, 1977 17, O'Connor CG, Ashman LK: Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated antiimmunoglobnlin for determination of the specificityof monoclonal antibodies to protein mixtures. J Immunol Methods 54:267-271, 1982 18. Zayek M, Cleveland D, Morin FC: Treatment of persistent pulmonary hypertension in the newborn lamb by inhaled nitric oxide. J Pediatr 122:743-750, 1993 19, Zayek M, Wild L, Roberts JD, et al: Effect of nitric oxide on the survival rate and incidence of lung injury in newborn lambs with persistent pulmonary hypertension. J Pediatr 123:947-952, 1993 20. Karamanoukian HL, Glick PL, Zayek M, etal: Inhaled nitric oxide in congenital hyp0plasia of the lungs due to diaphragmatic hernia or oligohydramnios. Pediatrics 94:715-718, 1994 21. Kinsella JP, Neish SR, Ivy DD, et al: Clinical responses to prolonged treatment of persistent pulmonary hypertension of the
newborn with low doses of inhaled nitric oxide. J Pediatr 123:103108, 1993 22. Karamanoukian HL, Glick PL, WilcoxDT, et al: Pathophysiology of congenital diaphragmatic hernia (CDH) VII: The efficacy of inhaled nitric oxide (INO) in the lamb CDH model. Am Rev Respir Dis 147:A223, 1993 (abstr) 23. WilcoxDT, Glick PL, Karamanoukian HL, et al: Pathophysiology of congenital diaphragmatic hernia XIII: Perflurocarbon associated gas exchange improves pulmonary mechanics, oxygenation, and allows nitric oxide delivery in the hypoplastic lung congenital diaphragmatic hernia lamb model. Crit Care Med 23:1858-1863, 1995 24. Wilcox DT, Holm BA, Karamanoukian HL, et al: Nitrofen induced diaphragmatic hernia in rats. J Pediatr Surg 28:757, 1993 25. Tenbrinck R, Tibboel D, Gaillard JLJ, et al: Experimental induced congenital diaphragmatic hernia in rats. J Pediatr Surg 25:426-429, 1990 26. Kluth D, Kangah R, Reich P, et al: Nitrofen induced diaphragmatic hernia in rats: An animal model. J Pediatr Surg 25:850-854, 1990 27. Suen HC, Catlin EA, Ryan DP, et al: Biochemical immaturity of lungs in congenital diaphragmatic hernia. J Pediatr Surg 28:471-477, 1993 28. Suen HC, Bloch KD, Donahue PK: Antenatal glucocorticoid treatment corrects the pulmonary immaturity of congenital diaphragmatic hernia. Pediatr Res 35:523-529, 1994
Discussion S. Adzick (Philadelphia, PA): T h e key points for n e w b o r n s with severe C D H are the small lungs and the various p u l m o n a r y vascular abnormalities that correlate with the d r e a d e d p u l m o n a r y hypertension p r o b l e m that we all face f r o m time to time. T h e authors have shown, for the first time, that there m a y be a decrease in p u l m o n a r y N O S that m a y account, in part, for some of these findings. I have three questions. H a v e you looked, during gestation, to see w h e n these changes begin to occur? It might be interesting not just to look at term but to start at 13 days and go right to term, which I guess is 22 days. Second, I think it would be interesting to show conclusively which of the isoforms of N O S are involved. With your W e s t e r n blot analysis, you have looked, at endothelial cell NOS, but it would be nice to look at the Other N O S isoforms. Last, I think it would be i m p o r t a n t to share with the g r o u p the p r o o f that there actually is p u l m o n a r y hypertension in the nitrofen rat m o d e l of C D H . T. Moore (Los Angeles, CA): I rise just to c o m m e n t on a recent r e p o r t in the S e p t e m b e r 21, 1996 issue of Nature, f r o m Massachusetts G e n e r a l Hospital. T h e title is " H y p e r t e n s i o n in K n o c k o u t Mice Lacking the
G e n e for Endothelial Nitric Oxide Synthase." T h e article supports the findings p r e s e n t e d here. H.L. Karamanoukian (response): D r Adzick, thank you for your comments. I believe that the point you have m a d e is a very important one. It is also possible that nitrofen affects the inducible N O S m o r e than the endothelial isoform of this enzyme. A n d that could be an important component. As to the final point that was m a d e regarding p u l m o n a r y hypertension, ! think this is one of the weaknesses of this model. W e have spoken and c o m m u n i c a t e d with Dick Tibboel regarding this question. His g r o u p is in the process of measuring pulmonary h e m o d y n a m i c s in this model, p e r h a p s by microp u n c t u r e techniques. I think these experiments have b r o u g h t us the closest thus far to determining w h e t h e r or not there is p u l m o n a r y hypertension in this model. I thank Dr M o o r e for his c o m m e n t s as well. I was not aware of the report f r o m Boston regarding the k n o c k o u t model of p u l m o n a r y hypertension in the mouse, but I am pleased that the study supports our conclusions.
Buffalo, New York • Because nitric oxide (NO) dilates vascular smooth muscle cells, a deficiency of endogenous pulmonary nitric oxide production by nitric oxide synthase (NOS) has been suggested to be involved in the pathophysiology of pulmonary hypertension in congenital diaphragmatic hernia (CDH). Our aim was to determine whether experimentally induced CDH in rats results in a decrease in the synthesis of NO in the lungs. Adult Sprague-Dawley rats were fed 300 mg/kg of nitrofen at 10.5 days' gestation. CDH, control, and sham (dosed with nitrofen, but without CDH) lungs were homogenized at full term (22 days' gestation) for measurement of NOS activity using the 14C-L-arginine to 14C-L-citrulline conversion assay, Western blot analysis with anti-endothelial cell NOS (EC-NOS} monoclonal antibody (mAb) was performed, and NOS expression was measured by densitometry. NOS activity was highest in the pulmonary parenchyma of control rat lungs (0.420 _+ .020 f m o l / m i n / m g lung; n = 11), intermediate in sham lungs (0.370-+ 0.010 f m o l / m i n / m g lung; n = 14), and lowest in CDH lungs (0.300 -+ 0.04 f m o l / m i n / m g lung; n = 12). NOS activity in the CDH and sham lungs was significantly lower than that of control lungs (P < .05). There was no difference in pulmonary NOS activity between sham and CDH lungs. NOS protein expression by Western blot analysis paralleled the observation for NOS activity in all groups, with the highest concentrations in controls, intermediate expression in sham lungs, and lowest expression in CDH lungs. Both NOS expression and NOS activity are significantly decreased in CDH rat lungs. Pulmonary hypertension in this model may be attributable to a deficiency of endogenous NO. This is the first reported study to suggest that decreased NOS activity may result in pulmonary hypertension in CDH. Copyright © 1996by W.B. Saunders Company INDEX WORDS: Congenital diaphragmatic hernia, pulmo-
nary hypertension, nitric oxide synthase.
T
H E H I G H M O R T A L I T Y rate associated with congenital diaphragmatic hernia (CDH)
From the Buffalo Institute of Fetal Therapy, The Children's Hospital of Buffalo, and the Departments of Surgery, Pediatrics, and Medicine, State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, N Y Presented at the 1995 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, San Francisco, California, October 13-15, 1995. Supported in part by grants from the American Lung Association, the Women and Children's Health Research Foundation, and NIH HL 36543 and 49977. Address reprint requests to Philip L. Glick, MD, The Buffalo Institute of Fetal Therapy, Department of Pediatric Surgery, The Children's Hospital of Buffalo, 219 Bryant St, Buffalo, N Y 14222. Copyright © 1996 by W.B. Saunders Company 0022-3468/96/3108-0003503. 00/0 1016
has been shown to result from a combination of pulmonary hypoplasia and pulmonary hypertension. CDH lungs have a reduced number of conducting airways, alveoli, and preacinar pulmonary vessels. 1-4 In addition, the maturity of the lung appears retarded. 5 The results of morphological analyses have suggested that pulmonary hypertension is caused by abnormal muscularization of the acinar arteries and a decrease in the overall cross-sectional area of the pulmonary vascular bed, affecting both ipsilateral and contralateral lungs. 6-9 These morphological features also are prominent features of the fetal lamb and rat models of CDH. t°,11 Whole animal studies have shown the presence of pulmonary hypertension by the measurement of elevated levels of both pulmonary pressures and pulmonary vascular resistance in the newborn period. 12-14 Because nitric oxide (NO) dilates vascular smooth muscle cells, a deficiency of nitric oxide synthase (NOS) has been suggested to be involved in the pathophysiology of pulmonary hypertension in CDH. Studies in the fetal lamb model of CDH have demonstrated NOS in the pulmonary vessels by both immunohistochemistry and NADPH diaphorase activity, which indicates the presence of the NO-producing enzyme in CDH. 15The intriguing question remains: Is there a deficiency in the endogenous production of NO in the pulmonary vascular bed, and if so, does it result in pulmonary hypertension? The aim of the present study was to determine whether there is a decrease in NOS activity in the lung parenchyma and/or a deficiency of NOS expression in the nitrofeninduced rat model of CDH. MATERIALS AND METHODS
Nitrofen-Induced Rat CDH Model Adult Sprague-Dawley rats were bred at our laboratory animal center after controlled overnight matings. A positive smear was considered proof of pregnancy, and the day this observation was made was designated as day 0. The animals were provided with ad libitum feeding and water throughout pregnancy. At 10.5 days of pregnancy (full term, 22 days), 300 mg/kg body weight of nitrofen (2,4-dichlorophenyl-P-nitrophenyl; Dupliar, Weesp, Netherlands), dissolved in olive oil, was administered as a single dose via a gastric tube. Cesarean section was performed on day 21. Control animals were given the same dose of olive oil, without nitrofen. CDH, control, and sham specimens (dosed with nitrofen, but without a
JournalofPediatric Surgery, Vo131,No 8 (August), 1996: pp 1016-1019
DECREASED NITRIC OXIDE SYNTHASE IN CDH
1017
diaphragmatic hernia) were stored at -70°F until they were studied further.
• p<.05 vs Control
0.5
# p = NS vs Sham
e)
NOS Activity
0.4
Fragments of lung tissue (CDH, sham, and control) weighing approximately 50 mg were homogenized in distilled water (dd H20; 1 g in 2 vol) and were stored on ice before use. Incubations were initiated by the addition of 25 p.L of tissue homogenate to 125 IxL of 50 nmol/L Tris HC1 buffer (pH, 7.4) containing 1.25 mmol/L CaCI2, 1.0 mmol/L EDTA (pH, 7.5), 1.0 mmol/L NADPH, 20 p.mol/L L-arginine (Sigma Chemical Co, St Louis, MO), and 3.314 Ixmol/L 14C-L-arginine (1 ixCi/mL), which had been preincubated for approximately 4 minutes at 30° Fahrenheit. Reaction was terminated after 40 minutes by the addition of 2.0 mL of "stop" buffer containing 20 mmol/L Tris HC1 (pH, 5.5) and 4.0 mmol/L EDTA (pH, 7.5), followed by Vortex mixing. Individual incubation mixtures (2.15 mL) were added to columns packed with 1.0 mL of Dowex AG50WX-8 (Na ÷ form; Fluka AG, St Louis, MO) that had been preequilibrated with a minimum of 2.0 mL stop buffer. Each column was allowed to elute by gravity into collection vials. Conversion of 14C-L-arginine to 14C-L-citrulline was quantified by liquid scintillation spectroscopy.
Western Blot Analysis Sodium dodecyl sulfate-polyacrylamide (8%) electrophoresis (SDS-PAGE) was performed as described by Cleveland et al. ~6 Electrophoresis transfer of separated proteins to nitrocellulose membranes (Bio-rad Laboratories, Richmond, CA) was performed according to protocols previously described by O'Connor et al. 17 Nitrocellulose membranes were incubated in Tris-buffered saline (TBS) (10 mmol/L Tris, 150 mmol/L NaCI; pH, 7.4), containing 5% Blotto (Carnation nonfat dry milk), for 1 hour at room temperature to block nonspecific binding. Incubation with antiECNOS mAb (1:250 dilution) (Transduction Laboratories, Lexington, KY) in TBS containing 5% Blotto was performed overnight at room temperature with rocking. The membranes were washed in TBS containing 0.5% Tween-20 (TABS) and further incubated in 5% Blotto containing goat antimouse horseradish peroxidaseconjugated gamma-immunoglobulin (1:3000) (Bio-rad Laboratories, Richmond, CA) for 1 hour at room temperature. Blots were washed extensively in TABS (3 x 10 minutes), air-dried, incubated with Deepened (Boston, MA) chemiluminescence-developing reagents for 1 minute, and exposed to Deepened-sensitive film for 1 minute. The molecular weights of resulting bands were determined from the migration positions of prestained colored molecular size markers (Bio-rad Laboratories). The EC-NOS expression for each specimen was quantitated by densitometry readings according to the area method and was reported as ratios (compared with the ECNOS expression in control lungs) for each group.
Statistical Analysis
~_~0.3
o.2 'r'~
0.1
0
Control
Sham
CDH
n=11
n=14
n=12
Fig 1. Pulmonary nitric oxide synthase activity in the nitrofeninduced rat model of CDH.
and was lowest in the CDH lungs (0.300 _+ 0.04 fmol/min/mg lung; n = 12) (Fig 1). NOS activity in the CDH and sham lungs was significantly lower than that of control lungs (P < .05). Although NOS activity was higher than in sham lungs than in the CDH lungs, the difference was not significant. NOS protein expression by Western blot analysis paralleled the observation for NOS activity in all groups, with the highest expression in control lungs, intermediate expression in sham lungs, and the lowest expression in CDH lungs (Fig 2). This was confirmed by densitometry readings. EC-NOS was calculated from the area under the curve and was reported as a ratio for the various groups of animals (control, 1.0; sham, 018; CDH, 0.6).
A
B
C
D
KD
204 140 132"~
65
All data are expressed as mean _+ standard error (SE) of the mean. Comparisons between groups of animals were made using analysis of variance followed by the Student's t test. P values of less than .05 were considered to represent a significant difference between the compared values.
* 7#
.
42.6 .
RESULTS
NOS activity was highest in the pulmonary parenchyma of control rat lungs (0.420_ .020 fmol/ min/mg lung; n = 11). It was intermediate in the sham lungs (0.370 ___0.010 fmol/min/mg lung; n = 14)
Fig 2. Western blot analysis with anti-ECNOS mAb on 50-~g total lung homogenates from (A) sham, (B) CDH, (C) control rat and (D) mouse endothelial cell homogenate as a positive control.
1018
KARAMANOUKIAN ET AL
DISCUSSION
Inhaled NO is a selective pulmonary vasodilator. In a lamb model of persistent pulmonary hypertension, it has been shown to improve oxygenation and survival. 18,19 It also has been shown to improve oxygenation in infants who have persistent pulmonary hypertension (PPHN).2°,21 Hopes were high that NO would be the "magic bullet" to treat pulmonary hypertension in CDH. Although initial reports were disappointing, several recent case reports indicate that inhaled NO may be efficacious in the treatment pulmonary hypertension in CDH. 2°-23 We have shown that newborn lambs with surgically created CDH respond well to inhaled NO therapy if certain criteria are met. 12 Inhaled NO is effective in decreasing pulmonary pressures only after the surfactant deficiency has been corrected with exogenous surfactant, which enables NO to reach the presumed "diseased" vascular bed in the microcirculation of the lung. 12 It appears that exogenous surfactant is required in the lamb model of CDH for delivery of inhaled NO to the terminal lung units, where it works synergistically with exogenous surfactant to decrease intrapulmonary shunting and pulmonary artery pressures.12 Similar efficacy has been achieved with the combination of inhaled NO and high-frequency oscillatory ventilation (HFOV) or perflurocarbon-associated gas exchange (PAGE). 21-23 The nitrofen-induced rat model of CDH appears to be a cost-effective, multiparous model for the study of the pathophysiology of CDH. z4,25 The nitrofen model produces global abnormalities of the heart, kidneys,
liver, brain, thyroid, bones, and adrenal glands.26 It has been shown to partially mimic the pathophysi01ogy of CDH, because it has both pulmonary hypopiasia and a surfactant deficiency.27 In contrast to the lamb model of CDH, there is scant data regarding pulmonary circulation. It has been difficult, if not impossible, to quantitate pulmonary and systemic hemodynamics in the nitrofen-induced model because of the newborn rat's small size ( < 3 g). Therefore, it remains unknown whether pulmonary hypertension is a pathophysiological feature in this widely studied model of CDH. it Nevertheless, the results of preliminary studies that used barium-injected pulmonary arterial radiographs have suggested that there is a decrease in the size of the vascular bed in the nitrofen model of CDH. n Therefore, it has been assumed, but not proven, that pulmonary hypertension is a pathophysiologicai feature of the rat model of CDH. Although preliminary data have shown that the NOS mRNA levels are identical for CDH and control rat pups at term, 28 our results suggest that, based on the preliminary pulmonary vascular morphometric studies, pulmonary hypertension in the nitrofen-induced rat model of CDH may be caused by a deficiency of endogenous NO production by the pulmonary vascular bed. This is the first reported study to suggest that decreased pulmonary NOS activity may result in pulmonary hypertension, and the first study to indicate that decreased pulmonary NOS activity may result in pulmonary hypertension in CDH.
REFERENCES 1. Geggle RL, Murphy JD, Langleben D, et al: Diaphragmatic hernia in the fetus: Arterial structural changes and persistent pulmonary hypertension after surgical repair. J Pediatr 107:457464, 1985 2. Bohn D, Tamura M, Perrin D, et al: Ventilatory predictors of pulmonary hypoplasia in congenital diaphragmatic hernia, confirmed by morphologic assessment. J Pediatr 111:423-431, 1987 3. Levin DL: Morphologic analysis of the pulmonary vascular bedl in congenital left-sided diaphragmatic. J Pediatr 92:805-809, 1979 41 Kitagawa M, Hislop A, Boyden EA, et al: Lung hypoplasia in congenital diaphragmatic hernia. A quantitative study of airway, artery, and alveolar development. Br J Surg 58:342-346, 1971 5~ George DK, Cooney TP, Chiu BK, et al: Hypoplasia and immaturity of the terminal lung unit (acinus) in congenital diaphragmatic hernia. Am RevRespir Dis 136:947-950, 1987 6. Adzick NS, Harrison MR, Outwater KM, et al: Correction of congenital diaphragmatic hernia in utero IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis. J Pediatr Surg 20:673-680, 1985 7. Nguyen L, Guttman FM, De Chadarevian JP, et al: The mortality of congenital diaphragmatic hernia. Is total pulmonary mass inadequate, no matter what? Ann Surg 198:766-769, 1983
8. Reale FR, Esterly JR: Pulmonary hypoplasia: A morphometrie study of the lungs of infants with diaphragmatic hernia, anencephaly, and renal malformations. Pediatrics 51:91-96, 1973 9. Hislop A, Reid L: Persistent hypoplasia of the lung after repair of congenital diaphragmatic hernia. Thorax 31:450-455, 1976 10. DiMaio M, Ting A, Gil J, et al: A morphometric study of lung hypoplasia in a second trimester lamb model of congenital diaphragmatic hernia (CDH). Am J Resp Crit Care Med 149:A727, 1994 11. Tenbrinck R, Gaillard D, Tibboel D, et al: Pulmonary vascular abnormalities in experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg27:862-865, 1992 12. Wilcox DT, Glick PL, Karamanoukian HL, et al: Pathophysiology Of congenital diaphragmatic hernia V. Effect of exogenous surfactant therapy on gas exchange and lung mechanics in the lamb congenital diaphragmatic hernia model. J Pediatr 124:289-293, 1994 13. Karamanoukian HL, Glick PL, Wilcox DT, et al: Pathophysiology of congenital diaphragmatic hernia VIII: Inhaled nitric requires exogenous surfactant therapy in the lamb modei of congenital diaphragmatic hernia. J Pediatr 30:1-4, 1995 14. O'Toole SJ, Karamanoukian HL, Morin III FC, et al:
DECREASED NITRIC OXIDE SYNTHASE IN CDH
1019
Surfactant decreases pulmonary vascular resistance and increases blood flow in the lamb model of congenital diaphragmatic hernia. J Pediatr Surg 31:507-511, 1996 15. Karamanoukian HL, Glick PL, Wilcox DT, et al: Pathophysiology of congenital diaphragmatic hernia X: Localization of nitric oxide synthase in the intima of pulmonary artery trunks of lambs with surgically created congenital diaphragmatic hernia. J Pediatr Surg 30:5-9, 1995 16, Cjeve DW, Fischer SG, Kirschner MW, et al: J Biol Chem 252:1102-1106, 1977 17, O'Connor CG, Ashman LK: Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated antiimmunoglobnlin for determination of the specificityof monoclonal antibodies to protein mixtures. J Immunol Methods 54:267-271, 1982 18. Zayek M, Cleveland D, Morin FC: Treatment of persistent pulmonary hypertension in the newborn lamb by inhaled nitric oxide. J Pediatr 122:743-750, 1993 19, Zayek M, Wild L, Roberts JD, et al: Effect of nitric oxide on the survival rate and incidence of lung injury in newborn lambs with persistent pulmonary hypertension. J Pediatr 123:947-952, 1993 20. Karamanoukian HL, Glick PL, Zayek M, etal: Inhaled nitric oxide in congenital hyp0plasia of the lungs due to diaphragmatic hernia or oligohydramnios. Pediatrics 94:715-718, 1994 21. Kinsella JP, Neish SR, Ivy DD, et al: Clinical responses to prolonged treatment of persistent pulmonary hypertension of the
newborn with low doses of inhaled nitric oxide. J Pediatr 123:103108, 1993 22. Karamanoukian HL, Glick PL, WilcoxDT, et al: Pathophysiology of congenital diaphragmatic hernia (CDH) VII: The efficacy of inhaled nitric oxide (INO) in the lamb CDH model. Am Rev Respir Dis 147:A223, 1993 (abstr) 23. WilcoxDT, Glick PL, Karamanoukian HL, et al: Pathophysiology of congenital diaphragmatic hernia XIII: Perflurocarbon associated gas exchange improves pulmonary mechanics, oxygenation, and allows nitric oxide delivery in the hypoplastic lung congenital diaphragmatic hernia lamb model. Crit Care Med 23:1858-1863, 1995 24. Wilcox DT, Holm BA, Karamanoukian HL, et al: Nitrofen induced diaphragmatic hernia in rats. J Pediatr Surg 28:757, 1993 25. Tenbrinck R, Tibboel D, Gaillard JLJ, et al: Experimental induced congenital diaphragmatic hernia in rats. J Pediatr Surg 25:426-429, 1990 26. Kluth D, Kangah R, Reich P, et al: Nitrofen induced diaphragmatic hernia in rats: An animal model. J Pediatr Surg 25:850-854, 1990 27. Suen HC, Catlin EA, Ryan DP, et al: Biochemical immaturity of lungs in congenital diaphragmatic hernia. J Pediatr Surg 28:471-477, 1993 28. Suen HC, Bloch KD, Donahue PK: Antenatal glucocorticoid treatment corrects the pulmonary immaturity of congenital diaphragmatic hernia. Pediatr Res 35:523-529, 1994
Discussion S. Adzick (Philadelphia, PA): T h e key points for n e w b o r n s with severe C D H are the small lungs and the various p u l m o n a r y vascular abnormalities that correlate with the d r e a d e d p u l m o n a r y hypertension p r o b l e m that we all face f r o m time to time. T h e authors have shown, for the first time, that there m a y be a decrease in p u l m o n a r y N O S that m a y account, in part, for some of these findings. I have three questions. H a v e you looked, during gestation, to see w h e n these changes begin to occur? It might be interesting not just to look at term but to start at 13 days and go right to term, which I guess is 22 days. Second, I think it would be interesting to show conclusively which of the isoforms of N O S are involved. With your W e s t e r n blot analysis, you have looked, at endothelial cell NOS, but it would be nice to look at the Other N O S isoforms. Last, I think it would be i m p o r t a n t to share with the g r o u p the p r o o f that there actually is p u l m o n a r y hypertension in the nitrofen rat m o d e l of C D H . T. Moore (Los Angeles, CA): I rise just to c o m m e n t on a recent r e p o r t in the S e p t e m b e r 21, 1996 issue of Nature, f r o m Massachusetts G e n e r a l Hospital. T h e title is " H y p e r t e n s i o n in K n o c k o u t Mice Lacking the
G e n e for Endothelial Nitric Oxide Synthase." T h e article supports the findings p r e s e n t e d here. H.L. Karamanoukian (response): D r Adzick, thank you for your comments. I believe that the point you have m a d e is a very important one. It is also possible that nitrofen affects the inducible N O S m o r e than the endothelial isoform of this enzyme. A n d that could be an important component. As to the final point that was m a d e regarding p u l m o n a r y hypertension, ! think this is one of the weaknesses of this model. W e have spoken and c o m m u n i c a t e d with Dick Tibboel regarding this question. His g r o u p is in the process of measuring pulmonary h e m o d y n a m i c s in this model, p e r h a p s by microp u n c t u r e techniques. I think these experiments have b r o u g h t us the closest thus far to determining w h e t h e r or not there is p u l m o n a r y hypertension in this model. I thank Dr M o o r e for his c o m m e n t s as well. I was not aware of the report f r o m Boston regarding the k n o c k o u t model of p u l m o n a r y hypertension in the mouse, but I am pleased that the study supports our conclusions.