Tracheal ligation and mechanical ventilation do not improve the antioxidant enzyme status in the lamb model of congenital diaphragmatic hernia

Tracheal Ligation and Mechanical Ventilation Do Not Improve the Antioxidant Enzyme Status in the Lamb Model of Congenital Diaphragmatic Hernia By Pierina

Kapur,

Bruce A. Holm,

Michael

S. Irish, Alka Patel, and Philip

Buffalo, New

Background/Purpose: The antioxidant enzyme (AOE) system is the primary intracellular defense system of the lung against oxygen toxicity. The authors recently demonstrated depressed levels of catalase, glutathione peroxidase, and superoxide dismutase in congenital diaphragmatic hernia (CDH) lambs compared with controls. The aim of this study was to determine whether tracheal ligation (TL) or mechanical ventilation (recently shown to stimulate growth and surfactant metabolism, respectively) could induce an elevation in AOE activity. Methods: Four nonventilated lambs with surgically created CDH and TL and five CDH lambs ventilated for 4 hours were studied. Lung tissue was analyzed for AOE and the results compared with untreated CDH lambs. Results: Both ventilation above that of untreated

and TL failed CDH lambs.

to elevate

AOE

activity

T

HE PULMONARY ANTIOXIDANT enzyme system (AOE) is the primary intracellular defense system of the lung against oxygen toxicity. The damaging effects of oxygen are mediated by partially reduced, active oxygen species,1*2the most predominant of which are the superoxide anion, hydrogen peroxide, and the hydroxyl radical. In the healthy fetus, there is a rapid rise in pulmonary AOE activities before birth.3-5 In neonates with congenital diaphragmatic hernia (CDH), the oxidative stress normally occurring at birth is further enhanced by the high oxygen delivery of mechanical ventilation. Recently we have shown that, in addition to being surfactant deficient,6,7 the CDH neonate is further compromised by a deficiency in the AOE system (unpublished data). This deficiency, in conjunction with the high From the Buffalo Institute of Fetal Therapy, Children’s Hospital of Buffalo, Departments of Pediatric Surgery, Pediatrics, and Pharmacology and Toxicology, State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, Nk: Presented at the 45th Annual International Congress of the British Association of Paediatric Surgeons, Bristol, England, July 21-24, 1998. This work was supported by a grant from The National Institute of Health (HL 49977). Address reprint requests to Philip L. Glick, MD, The Buffalo Institute of Fetal Therapy, Department of Pediatric Surgery, The Children’s Hospital of Bgffalo, 219 Bryant St. Buffalo, NY 14222. Copyright Q 1999 by WB. Saunders Company

0022.3468/99/3402-0007$03.00/O 270

L. Glick

York

Conclusions: The data provide further evidence that TL does not improve lung metabolism or maturation. Mechanical ventilation, which often involves high oxygen delivery, is a necessary and often beneficial therapeutic modality. In the CDH neonate compromised not only by low baseline levels of the AOE, but also by an inability to induce enzyme synthesis in response to hyperoxia, mechanical ventilation may, by causing lung injury, be contributing to the high morbidity and mortality rate associated with CDH. J Pediatr Surg 34270-272. Copyright o 1999 by W.B. Saunders Company.

INDEX WORDS: Congenital diaphragmatic hernia, antioxidant enzyme, catalase, glutathione peroxidase, superoxide dismutase, tracheal ligation, mechanical ventilation.

oxygen delivery of mechanical ventilation, may be contributing to the high morbidity and mortality associated with CDH. Our goal in this study was to determine whether tracheal ligation (shown to stimulate lung growth)*-lo or mechanical ventilationl’ could induce an elevation in AOE activity in the fatal lamb model of congenital diaphragmatic hernia. These studies were approved by the animal care committee of The State University of New York at Buffalo. MATERIALS

AND

METHODS

The Animal Model At 78 days’ gestation, diaphragmatic hernias were surgically created in nine lamb fetuses as previously described.‘j Four lambs underwent a second operation at 120 days’ gestation, when tracheal ligation was performed.t2 All pregnancies were allowed to continue until term (140 days), at which time the lamb was delivered by cesarean section. Instrumentation At term, while on placental circulation, the head and upper torso of five fetuses were delivered and an endotracheal tube (inside diameter, 4 mm) was inserted and secured via a tracheotomy. Through the same incision, polyvinyl catheters were threaded into the right internal carotid artery and jugular veins for measurement of arterial blood gas levels and intravenous access.

Resuscitation and Monitoring The four unventilated tracheal ligation lambs were immediately killed at the time of delivery with a lethal injection of potassium Journal

of Pediatric

Surgery,

Vol34,

No 2 (February),

1999: pp 270-272

TRACHEAL

LIGATION

AND

ANTIOXIDANTS

IN CDH

271

chloride. The ventilated lambs were placed on pressure-cycled ventilation (Servo 900C; Siemens-Elema, Life Support Systems Division, Solna, Sweden; peak inspiratory pressure [PIP] maximum, 28 cm HzO; positive end-expiratory pressure [PEEP], 4 cm H20; rate, 60 bpm; Fioz, lOO%), resuscitated, and monitored for 30 minutes, during which time arterial blood gases were analyzed for pH, pco2, ~02, and base deficit. Trishydroxymethylamino methane JTHAM) was given to maintain a base deficit of greater than -5 mEq. Anesthesia and paralysis were maintained with intravenous ketamine (8 mg/kg) and pancuronium bromide (0.1 mg/kg). After 4 hours, the ventilated group was killed. Left

Lung

Right

Lung

Lung Homogenate Preparation After death, the animals were weighed. The chest was opened and the lungs perfused in situ via the pulmonary artery until blanched. The heart and lungs were removed en bloc and the lungs perfused once again. Tissue samples were snap frozen in liquid nitrogen and stored at -70°C until analysis, when tissue pieces were thawed and weighed. Tissue was then minced and homogenized in 10 mL/g tissue of cold 50 mmol/L K2HP04 buffer (pH 7.8; catalase [CAT], glutathione peroxidase [GPX], or 0.25 moVL sucrose solution superoxide dismutase [SOD] with a glass and Teflon homogenizer. The homogenate was then centrifuged for 10 minutes to remove cellular debris.

Fig 1. Lung CAT activities expressed as units per gram of tissue. No significant differences were seen between ventilated CDH lambs or tracheal ligation CDH lambs compared with untreated CDH lambs (P > .05).

0.7, P = .27, left; 4.8 2 0.5 v 5.68 5 0.7, P = .36, right; Fig 2). Likewise, SOD activity remained low in both left (2.4 t 0.4~2.8 t 0.3,P = .41)andrightlungs(2.8 & 0.4 v 3.2 + 0.3, P = .44) compared with untreated CDH lungs (Fig 3).

Antioxidant Enzyme Analysis Catalase activity was assayed spectrophotometrically by the method of Bergmeyer, as described by Luck.13 Glutathione peroxidase activity was measured specrophotometrically using the technique of Beutler,14 and superoxide dismutase activity was assayed spectrophotometrically (BIOXYTECH SOD-525; OXIS International: Inc, Portland, OR).

Statistical Analysis All results were expressed as means t SEM. All animals were equally weighted statistically and significant differences were determined by unpaired t test. Statistical significance was determined at P less than .05. Results were compared with our recently obtained results from unventilated CDH lambs and control lambs (unpublished data).

RESULTS

Whole animal weights for both CDH and control lambs were comparable. No discernible differences in enzyme activities were seen between the right and left lungs of either CDH or tracheal ligation lambs. Mechanical ventilation failed to elevate CAT activity levels (306.7 ? 100.4, left lung; 420.9 t 72.4, right lung) above that of untreated CDH lambs (606.9 + 111.6, left lung, P = ,065; 658.3 + 111.4, right lung, P = .092; Fig 1). No improvement in GPX was noted, activity levels in both lungs remaining low in ventilated animals (4.7 5 0.9, left; 5.39 -+ 0.9, right) compared with unventilated CDH (left, 5.32 2 0.6, P = .56; right, 5.68 2 0.7, P = .77; Fig 2). SOD levels in ventilated CDH lambs (2.2 t 0.3, left; 2.8 I 0.3, right) also remained unelevated when compared with unventilated CDH lambs (2.8 + 0.2, P = .13, left; 3.2 ? 0.3, P = 0.34, right; Fig 3). Likewise, TL failed to elevate CAT activity levels above those of untreated CDH lambs (529.4 -t 133.4 v 606.9 2 111.6, P = .66, left; 607.0 i 164.4 v 658.3 +. 100.4, P = .79, right; Fig 1). GPX levels also remained low in TL animals compared with untreated CDH lambs (4.2 % 0.8 v 5.7 ?

DISCUSSION

The current study was designed to determine whether tracheal ligation or mechanical ventilation could elevate the low AOE activity levels seen in CDH lambs. Our data provide further evidence that despite the fact that TL has been shown to stimulate lung growth in CDH,8-10TL does not improve lung metabolism or maturation.‘2J5 We also have added support to our initial hypothesis that lung AOE activity levels are lower in both ventilated and unventilated CDH lambs compared with controls and that the AOE system is a requisite to decrease iatrogenic lung injury during ventilation in CDH. This finding is corroborated by the findings of Sluiter et a1,16who demonstrated that before and after birth, the total lung levels of CAT and GPX activities in rats with nitrofen-induced CDH are lower than those in lungs from non-CDH rats. Immediately after birth, the neonate is exposed to substantial oxidative stress, which, unless the lung is 8

T i

y;9 sz B

6 5 4I

Len Lung

Right

Lung

Fig 2. Lung GPX activities expressed as units per gram of tissue. No significant differences were seen between ventilated CDH lambs or tracheal ligation CDH lambs compared with untreated CDH lambs (P > .05).

KAPUR

Fig 3. Lung SOD activities expressed as units per gram of tissue. No significant differences were seen between ventilated CDH lambs or tracheal ligation CDH lambs compared with untreated CDH lambs

(P > .051.

preadapted to handle it, would be expected to cause early and serious lung damage. In the healthy fetus, there is a rapid rise in pulmonary antioxidant enzyme activities before birth.3-5 It has been shown that the pulmonary AOE system and the surfactant system share a chronologically similar late gestational pattern of development, the lung markedly increasing both surfactant content and its AOE activity during the final 10% to 15% of gestation.5s17 Results of recent studies from our laboratory have demonstrated that mechanical ventilation stimulates surfactant synthesis and secretion in CDH lambs.” Given the similarities between the surfactant and AOE systems in terms of development, mechanical ventilation could

ET AL

have been expected to also stimulate AOE activity. However, our results have not shown this to be the case. Tracheal ligation has not been shown to improve lung maturation in past studies,12J5 and it was hypothesized that TL would not stimulate AOE activity in the lamb model of CDH. The results of the current study support this hypothesis and provide further evidence that, although TL may cause lung growth, it does not stimulate lung maturation. When the newborn is exposed suddenly to high levels of inspired oxygen with low levels of AOE, serious oxygen toxicity can result. Mechanical ventilation, which often involves high oxygen delivery, is necessary and often beneficial, but in the surfactant-deficient CDH neonate compromised by low baseline levels of the AOE and an inability to induce enzyme synthesis in response to hyperoxia, mechanical ventilation with high O2 levels may cause lung injury and contribute to the high morbidity and mortality rates associated with CDH. This could also help to explain the phenomenon of the “honeymoon period” so often seen in CDH. If this is true, exogenous antioxidant therapy could be used in the future to reduce the high morbidity and mortality rates associated with oxygen toxicity in CDH. ACKNOWLEDGMENTS The authors thank Theresa Czwojdak and June Sokolowski for their technical assistance, and Dr Sadis Matalon for his advice on the enzyme analysis.

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10. Nardo L, Hooper S, Harding R: Lung hypoplasia can be reversed by short-term obstruction of the trachea in fetal sheep. Pediatr Res 38:690-696, 1995 11. Irish MS, O’Toole SJ, Sharma A, et al: Ventilatory stratch enhances surfactant synthesis in the fetal lamb model of congenital diaphragmatic hernia. American Thoracic Society, San Francisco, CA. Am 3 Respir Crit Care Med 28: 1997 12. O’Toole SJ, Sharma A, Karamanoukian HL, et al: Tracheal ligation does not correct surfactant deficiency associated with congenital diaphragmatic hernia. J Pediatr Surg 31:546-550, 1996 13. Luck H: Catalase, in Bergmeyer H (ed): Methods of Enzymatic Analysis. New York, NY, Academic, 1963, pp 885-888 14. Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. New York, NY, Grune and Stratton, 1975, pp 71-75 15. O’Toole SJ, Karamanoukian HL, Irish MS, et al: Tracheal ligation: The dark side of in utero CDH treatment. J Pediatr Surg 32:407-410, 1997 16. Sluiter W, Bos AP, Siluin F, et al: Nitrofen-induced diapbragmatic hernias in rats: Pulmonary antioxidant enzyme activities. Pediatr Res 32:394-398, 1992 17. Yam J, Frank L, Roberts RJ: Oxygen toxicity: Comparison of lung biochemical responses in neonatal and adult rats. Pediatr Res 12:115-119,1978