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Myeloperoxidase activity as a lung injury marker in the lamb model of congenital diaphragmatic hernia
Myeloperoxidase Activity as a Lung Injury Marker in the Lamb Model of Congenital Diaphragmatic Hernia By Amanda J. McCabe, Mark Dowhy, Bruce A. Holm, and Philip L. Glick Buffalo, New York
Purpose: The antioxidant system is the primary intracellular defense system of the lung against oxygen toxicity (neutrophil sequestration). The CDH lamb model antioxidant system is deficient. It is hypothesized that pulmonary neutrophil sequestration may play a part in the acute lung injury of CDH patients. Myeloperoxidase (MPO) is a major constituent of neutrophil cytoplasmic granules and its activity therefore is a direct measure of neutrophil presence and an indirect indicator of lung injury.
normalized to the protein content of the supernatant and expressed as units of MPO activity per milligram of protein.
Methods: Eight lambs had left-sided diaphragmatic hernias surgically created at 80 days’ gestation and were delivered by cesarean section at 140 to 145 days. Eight littermate lambs served as controls. Lambs were either killed before ventilation or were ventilated conventionally for 4 hours with 100% O2 and then killed. The lungs were dissected en bloc and snap frozen. The samples were homogenized, sonicated, freeze-thawed, and separated by density centrifugation. Supernatants were analyzed for myeloperoxidase (MPO) activity by spectrophotometry with -dianisidine dihydrochloride and hydrogen peroxide at 460 nm. The MPO activity was
Conclusions: Ventilation and hyperoxia leads to neutrophil accumulation in lung tissue, which is most pronounced in the CDH lung tissue. This is a further clue to the pathophysiology of iatrogenic lung injury in CDH. The myeloperoxidase assay may now be used to evaluate antenatal or postnatal antioxidant therapies for iatrogenic lung injury in CDH. J Pediatr Surg 36:334-337. Copyright © 2001 by W.B. Saunders Company.
S
have the ability to mediate a myriad of effects within the pulmonary vasculature and epithelium, which lead to lung inflammation and the sequestration of activated neutrophils.3 Fortunately, under normal conditions, the endogenous antioxidant enzyme system degrades these toxic compounds before cellular damage ensues. We previously have reported a significant deficiency of antioxidant enzymes in the lamb model of CDH.4 We further have suggested that the endogenous antioxidant clearance pathways in the CDH lung become overwhelmed by the high levels of oxygen delivery to the tissues.4 This then results in acute lung injury, loss of the honeymoon period, and contributes to the high morbidity and mortality rates associated with CDH. Myeloperoxidase (MPO) is a major constituent of neutrophil cytoplasmic granules. The total activity of MPO in a tissue is therefore a direct measure of neutrophil sequestration in that tissue.5 The purpose of this study, therefore, was to investigate whether total MPO activity could be used as an indirect indicator of acute lung injury in the CDH lamb model.
OME NEONATES with congenital diaphragmatic hernia (CDH) show adequate oxygenation and ventilatory parameters in the absence of maximal medical therapy. This phenomenon is termed the honeymoon period. It suggests that there is sufficient lung tissue to be compatible with life. Subsequent deterioration is thought to be caused by iatrogenic ventilatory barotrauma or oxygen-induced injury to the lungs. The ventilatory support of neonates with CDH inevitably involves the delivery of high concentrations of oxygen. Hyperoxia is linked to the generation of partially reduced active oxygen species, which include the superoxide anion (•O2), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH).1,2 These reactive oxygen species From the Buffalo Institute of Fetal Therapy (BIFT), The Children’s Hospital of Buffalo (Kaleida Health), Departments of Surgery, Pediatrics, and OBGYN, The State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, NY. Presented at the 47th Annual International Congress of the British Association of Paediatric Surgeons, Sorrento, Italy, July 18-21, 2000. Address reprint requests to Philip L. Glick, MD, FACS, FAAP, FRCS, Surgeon-in-Chief, Children’s Hospital of Buffalo, 219 Bryant St, Buffalo, NY 14222. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3602-0019$35.00/0 doi:10.1053/jpsu.2001.20709 334
Results: There was significantly more MPO activity in the CDH-ventilated lungs than controls similarly ventilated (3,203 ⫾ 665 versus 1,220 ⫾ 194, P ⫽ .001). There was no difference in MPO activity between the CDH and control lungs (318 ⫾ 57 v 348 ⫾ 61; P ⫽ .5). There was no difference between right and left lungs in any group.
INDEX WORDS: Congenital diaphragmatic hernia, myeloperoxidase, antioxidants, acute lung injury.
MATERIALS AND METHODS
Experimental Design Four groups of animals were studied (4 in each group). In group 1 (CDH), a left-sided diaphragmatic defect was created at 80 days’ Journal of Pediatric Surgery, Vol 36, No 2 (February), 2001: pp 334-337
MYELOPEROXIDASE ACTIVITY AS A LUNG MARKER IN CDH
Table 1. Body Weight and Wet Lung Weights for Control and CDH Animals Control
Body weight (Kg) Wet weight (g)
4 ⫾ 0.4 124 ⫾ 14
335
measured by the Micro-Lowry technique.9 Activity was expressed as units of MPO activity per milligram of protein.
CDH
4 ⫾ 0.5 53 ⫾ 8
gestation, and the lamb was delivered at 140 days. In group 2 (CDH VENT), a left-sided diaphragmatic defect was created at 80 days’ gestation; the lamb was delivered at 140 days and ventilated for 4 hours. Group 3 (CONTROL) consisted of nonoperated littermates that were delivered at 140 days. Group 4 (CONTROL VENT) consisted of nonoperated littermates that were delivered at 140 days and ventilated for 4 hours.
Fetal Surgical Procedures These studies were approved by the Animal Care Committee of the State University of New York at Buffalo. A left-sided diaphragmatic hernia was created surgically via an open hysterotomy at 80 days’ gestation as previously described.6 This stage of gestation in the sheep corresponds to the pseudoglandular phase of lung development. The pathophysiologic and morphometric changes seen in this model mirror those seen in human babies with CDH.7,8 At 140 days’ gestation, the fetuses were delivered by cesarean section. Animals in groups 1 and 3 were killed immediately, the chest opened, and the lungs perfused in situ with normal saline via the pulmonary artery. The heart and lungs were removed en bloc, and the lungs were perfused once again. The lung tissue was then snap frozen in liquid nitrogen and stored at ⫺80°C. Animals in groups 2 and 4 were instrumented while on the placental circulation. The fetal head and neck were delivered, and carotid and jugular lines were inserted through a right transverse neck incision. The trachea was then isolated and an endotracheal tube (4 mm), was inserted. The umbilical cord was then double tied and the lamb delivered, dried, wrapped in plastic, and placed on a warming blanket beneath a radiant warmer. The lambs were then ventilated for 4 hours (Servo ventilator 900 c; Siemens, Elema, Sweden). The initial rate was 40 breaths per minute with a peak inspiratory pressure (PIP) of 30 cm H2O, and a positive end expiratory pressure (PEEP) of 4 cm H2O. The inspired oxygen concentration was 100%. The rate was adjusted in an attempt to normalize PCO2, the PIP to obtain maximal PO2, and the PEEP was kept constant. Tris Hydroxymethyl amino methane (THAM) was given to maintain a base deficit of less than ⫺5 meq. Sedation was with ketamine, 3 mg/kg intravenously. At the end of the study period the lambs were killed and the lung tissue further handled as described previously.
Statistical Analysis Results are expressed as mean ⫾ SEM. Data analysis was performed using a Student’s t test with SigmaStat for Windows, Version 2.0 (1995, Jandel, Chicago, IL). P values less than .05 were considered significant.
RESULTS
Whole animal weights for both CDH and control lambs were comparable. The lung wet weights of the CDH lungs were significantly smaller than those of controls (Table 1). There were no significant differences in MPO activity between right and left lungs in any of the 4 groups. At birth, both CDH (group 1) and Control lungs (group 3) had minimal MPO activity (318 ⫾ 57 v 348 ⫾ 61, respectively). There was no significant difference between these 2 groups (P ⫽ .5). Four hours of ventilation resulted in a significant increase of MPO activity in both control (group 4) and CDH (group 2) lungs when compared with their nonventilated counterparts. After 4 hours of ventilation there was significantly more MPO activity in the CDH-ventilated lungs (group 2) than controls (group 4) similarly ventilated (3203 ⫾ 665 v 1220 ⫾ 194, respectively; P ⫽ .001; Fig 1). DISCUSSION
The current study investigated whether myeloperoxidase activity in lung tissue samples could be used as an indirect measure of acute lung injury in the CDH lamb
Myeloperoxidase Assay Myeloperoxidase was extracted from each homogenized lung tissue sample by suspending the material in 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co, St Louis, MO) in 50 mmol/L potassium phosphate buffer, pH 6.0, before sonication in an ice bath for 10 seconds. The samples were freeze-thawed 3 times, after which sonication was repeated. Suspensions were then centrifuged at 40,000g for 30 minutes and the resulting supernatant further assayed. Myeloperoxidase activity was assayed spectrophotometrically using the method of Bradley et al5: 0.1 mL of supernatant was mixed with 2.9 mL of 50 mmol/L phosphate buffer, pH 6.0, containing 0.167 mg/mL -dianisidine dihydrochloride (Sigma Chemical) and 0.0005% hydrogen peroxide (Sigma Chemical). The change in absorbance at 460 nm was measured using a spectrophotometer (U-2000; Hitachi Instruments, Webster, NY). MPO activity was then derived from the observed change in absorbance per minute. The MPO activity was normalized further to the total protein content of the supernatant, as
Fig 1. Lung MPO activities expressed as units per milligram of total protein. * P ⴝ .001, Control nonventilated versus control ventilated; ** P ⴝ .005, CDH nonventilated versus CDH ventilated; ‡ P ⴝ .001 CDH ventilated versus control ventilated.
336
MCCABE ET AL
model. The findings show that conventional ventilation and hyperoxia causes increased myeloperoxidase activity in both control and CDH lungs. This effect was significantly greater in the CDH lung samples. This assay, therefore, gives a useful direct measure of neutrophil sequestration in the lung tissue samples, and is an indirect indicator of acute lung injury being present after only 4 hours of ventilation and hyperoxia. The presence of neutrophils in the ventilated tissue offers further insight into the possible nature of the iatrogenic injury occurring in cases of CDH. Because the current study has used both conventional ventilation and hyperoxia, it is not possible to ascertain the degree of iatrogenic lung injury caused by each of the supportive measures. It would therefore be of interest to limit the effects of possible barotrauma by ventilating in a highfrequency oscillatory mode with and without hyperoxia. Hyperoxia, which is linked to the generation of various reactive oxygen species (ROS), is a necessary “evil” in the resuscitation and maintenance of CDH neonates. Under normal conditions, endogenous antioxidant clearance pathways handle these toxic compounds through a series of enzymatic and nonenzymatic pathways. However, in the setting of the CDH hypoplastic, surfactantdeficient lungs, it is conceivable that the clearance pathways for ROS first become depleted and then overwhelmed by the generation of these highly reactive radicals. Cellular responses that trigger organ injury then are initiated. ROS can induce several types of cellular damage directly (ie, lipid peroxidation, destruction of enzymes, and cleavage of DNA strands) as well as altering the activity of proteins involved in signal trans-
duction. Therefore, any attempts to manufacture and replace the scavenging enzyme system by the CDH lung are further hampered. The concept of antioxidant therapies have been investigated using recombinant enzymes such as superoxide dismutase and catalase10-12 as well as chemical scavengers such as N-acetylcysteine13 in various lung disorders. But these interventions have been limited by enzyme instability and inadequate intracellular delivery to the sites of ROS action. Partial liquid ventilation may offer another promising intervention, as demonstrated by Rotta and Steinhorn.14 They found that the degree of pulmonary neutrophil accumulation, as measured by MPO activity, in an experimental animal model of acute lung injury, was decreased when the animals received partial liquid ventilation. Perfluorocarbon used for liquid ventilation has been shown previously to decrease free radical release by alveolar macrophages15 and attenuates oxidative damage to lung tissue.16 Finally, gene therapy may become a fruitful focus for the development of novel treatments that aim to clear these toxic products at the earliest stage of iatrogenic injury.3 Despite the advances in antenatal diagnosis and neonatal care, the morbidity and mortality rate caused by CDH remain unacceptably high. Future investigations that focus on the potentiation of fetal lung growth and development in utero, or antioxidant therapies will require postnatal investigation. The MPO assay described now will offer a useful test for evidence of the early onset of acute lung injury in the CDH setting and will obviate the need for prolonged ventilatory studies.
REFERENCES 1. Frank L, Bucher JR, Roberts RJ: Oxygen toxicity in neonatal and adult animals of various species. J Appl Physiol 45:669-704, 1978 2. Fridovich I, Freeman B: Antioxidant defences in the lung. Ann Rev Physiol 48:693-702, 1986 3. Engelhardt JF: Redox-mediated gene therapies for environmental injury: Approaches and concepts. Antioxidants and Redox Signaling 1:5-27, 1999 4. Kapur P, Holm BA, Irish MS, et al: Tracheal ligation and mechanical ventilation do not improve the antioxidant enzyme status in the lamb model of congenital diaphragmatic hernia. J Pediatr Surg 34:270-272, 1999 5. Bradley PP, Priebat DA, Christensen RD, et al: Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. J Invest Dermatol 78:206-209, 1982 6. Glick PL, Stannard VA, Leach CL: Pathophysiology of congenital diaphragmatic hernia II. The fetal lamb model is surfactant deficient. J Pediatr Surg 27:382-388, 1992 7. Adzick NS, Outwater KM, Harrison MR: 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 8. de Lorimier AA, Tierney DF, Parker HR: Hypoplastic lungs in fetal lambs with surgically produced diaphragmatic hernia. Surgery 62:12-17, 1967
9. Lowry DH, Rosebrough NJ, Farr AL, et al: Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193: 265-275, 1951 10. Turrens JF, Crapo JD, Freeman BA: Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase. J Clin Invest 73:87-95, 1984 11. Tang G, White JE, Gordon RJ, et al: Polyethylene glycolconjugated superoxide dismutase protects rats against oxygen toxicity. J Appl Physiol 74:1425-1431, 1993 12. Walther FJ, David-Cu R, Lopez SL: Antioxidant-surfactant liposomes mitigate hyperoxic lung injury in premature rabbits. Am J Physiol 269:L613-L617, 1995 13. Bernard GR: N-Acetylcysteine in experimental and clinical acute lung injury. Am J Med 91:54S-59S, 1991 14. Rotta AT, Steinhorn DM: Partial liquid ventilation reduces pulmonary neutrophil accumulation in an experimental model of systemic endotoxemia and acute lung injury. Crit Care Med 26:1707-1715, 1998 15. Smith TM, Steinhorn DM, Thusu K: A liquid perfluorochemical decreases in vitro production of reactive oxygen species by alveolar macrophages. Crit Care Med 23:1533-1539, 1995 16. Steinhorn DM, Papo MC, Fuhrman BP: Oxidative damage is reduced during partial liquid ventilation with Perflubron. Crit Care Med 24:A148, 1996
MYELOPEROXIDASE ACTIVITY AS A LUNG MARKER IN CDH
337
Discussion C. Stolar (New York, NY): I like this paper very much because it speaks to our theme song of avoiding ventilating babies at all costs to try and minimise lung trauma. I think you have shown us some very interesting biologic information to support that the ventilator is a lethal device. I am surprised that you saw no difference between the right and left lungs because they can be such different sizes, particularly in this model because the compliance is so different. Is this because they are symmetrically deficient in surfactant. Why would you not see a difference in one side versus the other? A.J. McCabe (response): That was the reason we chose to make comparisons with the actual protein content of the lung, so we haven’t actually had any difference between compressed lung and a lung that is well airated. M. Hollwarth (Graz, Austria): You showed in earlier studies that there is a reduction of the amount of catalase and glutathione peroxidase in the tissue that is significant according to your statistics. My question is, do you think
that this reduction of enzymes in the tissue is significant in terms of not enough antioxidant power? I think the total amount of SOD and catalase is so huge in a cell that a reduction of about one third of the amount does not have any effects on the capacity of free oxygen radicals scavenging. A.J. McCabe (response): Our study was carried out over 4 hours, and we think that the capacity of the cell was used up gradually. I think it would be interesting to do the same studies under a shorter duration of ventilation. P.D. Losty (Liverpool, England): What sort of antenatal therapeutic strategies are you proposing? A.J. McCabe (response): I think any intervention that improves lung growth and in particular lung maturation has an important role to play in limiting the amount of trauma that we give these lungs. We have shown that the antioxidant status is not improved by tracheal ligation, which does improve lung growth, but I think perhaps a dynamic occlusion system might give us the maturation which we need in these lungs.
Purpose: The antioxidant system is the primary intracellular defense system of the lung against oxygen toxicity (neutrophil sequestration). The CDH lamb model antioxidant system is deficient. It is hypothesized that pulmonary neutrophil sequestration may play a part in the acute lung injury of CDH patients. Myeloperoxidase (MPO) is a major constituent of neutrophil cytoplasmic granules and its activity therefore is a direct measure of neutrophil presence and an indirect indicator of lung injury.
normalized to the protein content of the supernatant and expressed as units of MPO activity per milligram of protein.
Methods: Eight lambs had left-sided diaphragmatic hernias surgically created at 80 days’ gestation and were delivered by cesarean section at 140 to 145 days. Eight littermate lambs served as controls. Lambs were either killed before ventilation or were ventilated conventionally for 4 hours with 100% O2 and then killed. The lungs were dissected en bloc and snap frozen. The samples were homogenized, sonicated, freeze-thawed, and separated by density centrifugation. Supernatants were analyzed for myeloperoxidase (MPO) activity by spectrophotometry with -dianisidine dihydrochloride and hydrogen peroxide at 460 nm. The MPO activity was
Conclusions: Ventilation and hyperoxia leads to neutrophil accumulation in lung tissue, which is most pronounced in the CDH lung tissue. This is a further clue to the pathophysiology of iatrogenic lung injury in CDH. The myeloperoxidase assay may now be used to evaluate antenatal or postnatal antioxidant therapies for iatrogenic lung injury in CDH. J Pediatr Surg 36:334-337. Copyright © 2001 by W.B. Saunders Company.
S
have the ability to mediate a myriad of effects within the pulmonary vasculature and epithelium, which lead to lung inflammation and the sequestration of activated neutrophils.3 Fortunately, under normal conditions, the endogenous antioxidant enzyme system degrades these toxic compounds before cellular damage ensues. We previously have reported a significant deficiency of antioxidant enzymes in the lamb model of CDH.4 We further have suggested that the endogenous antioxidant clearance pathways in the CDH lung become overwhelmed by the high levels of oxygen delivery to the tissues.4 This then results in acute lung injury, loss of the honeymoon period, and contributes to the high morbidity and mortality rates associated with CDH. Myeloperoxidase (MPO) is a major constituent of neutrophil cytoplasmic granules. The total activity of MPO in a tissue is therefore a direct measure of neutrophil sequestration in that tissue.5 The purpose of this study, therefore, was to investigate whether total MPO activity could be used as an indirect indicator of acute lung injury in the CDH lamb model.
OME NEONATES with congenital diaphragmatic hernia (CDH) show adequate oxygenation and ventilatory parameters in the absence of maximal medical therapy. This phenomenon is termed the honeymoon period. It suggests that there is sufficient lung tissue to be compatible with life. Subsequent deterioration is thought to be caused by iatrogenic ventilatory barotrauma or oxygen-induced injury to the lungs. The ventilatory support of neonates with CDH inevitably involves the delivery of high concentrations of oxygen. Hyperoxia is linked to the generation of partially reduced active oxygen species, which include the superoxide anion (•O2), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH).1,2 These reactive oxygen species From the Buffalo Institute of Fetal Therapy (BIFT), The Children’s Hospital of Buffalo (Kaleida Health), Departments of Surgery, Pediatrics, and OBGYN, The State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, NY. Presented at the 47th Annual International Congress of the British Association of Paediatric Surgeons, Sorrento, Italy, July 18-21, 2000. Address reprint requests to Philip L. Glick, MD, FACS, FAAP, FRCS, Surgeon-in-Chief, Children’s Hospital of Buffalo, 219 Bryant St, Buffalo, NY 14222. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3602-0019$35.00/0 doi:10.1053/jpsu.2001.20709 334
Results: There was significantly more MPO activity in the CDH-ventilated lungs than controls similarly ventilated (3,203 ⫾ 665 versus 1,220 ⫾ 194, P ⫽ .001). There was no difference in MPO activity between the CDH and control lungs (318 ⫾ 57 v 348 ⫾ 61; P ⫽ .5). There was no difference between right and left lungs in any group.
INDEX WORDS: Congenital diaphragmatic hernia, myeloperoxidase, antioxidants, acute lung injury.
MATERIALS AND METHODS
Experimental Design Four groups of animals were studied (4 in each group). In group 1 (CDH), a left-sided diaphragmatic defect was created at 80 days’ Journal of Pediatric Surgery, Vol 36, No 2 (February), 2001: pp 334-337
MYELOPEROXIDASE ACTIVITY AS A LUNG MARKER IN CDH
Table 1. Body Weight and Wet Lung Weights for Control and CDH Animals Control
Body weight (Kg) Wet weight (g)
4 ⫾ 0.4 124 ⫾ 14
335
measured by the Micro-Lowry technique.9 Activity was expressed as units of MPO activity per milligram of protein.
CDH
4 ⫾ 0.5 53 ⫾ 8
gestation, and the lamb was delivered at 140 days. In group 2 (CDH VENT), a left-sided diaphragmatic defect was created at 80 days’ gestation; the lamb was delivered at 140 days and ventilated for 4 hours. Group 3 (CONTROL) consisted of nonoperated littermates that were delivered at 140 days. Group 4 (CONTROL VENT) consisted of nonoperated littermates that were delivered at 140 days and ventilated for 4 hours.
Fetal Surgical Procedures These studies were approved by the Animal Care Committee of the State University of New York at Buffalo. A left-sided diaphragmatic hernia was created surgically via an open hysterotomy at 80 days’ gestation as previously described.6 This stage of gestation in the sheep corresponds to the pseudoglandular phase of lung development. The pathophysiologic and morphometric changes seen in this model mirror those seen in human babies with CDH.7,8 At 140 days’ gestation, the fetuses were delivered by cesarean section. Animals in groups 1 and 3 were killed immediately, the chest opened, and the lungs perfused in situ with normal saline via the pulmonary artery. The heart and lungs were removed en bloc, and the lungs were perfused once again. The lung tissue was then snap frozen in liquid nitrogen and stored at ⫺80°C. Animals in groups 2 and 4 were instrumented while on the placental circulation. The fetal head and neck were delivered, and carotid and jugular lines were inserted through a right transverse neck incision. The trachea was then isolated and an endotracheal tube (4 mm), was inserted. The umbilical cord was then double tied and the lamb delivered, dried, wrapped in plastic, and placed on a warming blanket beneath a radiant warmer. The lambs were then ventilated for 4 hours (Servo ventilator 900 c; Siemens, Elema, Sweden). The initial rate was 40 breaths per minute with a peak inspiratory pressure (PIP) of 30 cm H2O, and a positive end expiratory pressure (PEEP) of 4 cm H2O. The inspired oxygen concentration was 100%. The rate was adjusted in an attempt to normalize PCO2, the PIP to obtain maximal PO2, and the PEEP was kept constant. Tris Hydroxymethyl amino methane (THAM) was given to maintain a base deficit of less than ⫺5 meq. Sedation was with ketamine, 3 mg/kg intravenously. At the end of the study period the lambs were killed and the lung tissue further handled as described previously.
Statistical Analysis Results are expressed as mean ⫾ SEM. Data analysis was performed using a Student’s t test with SigmaStat for Windows, Version 2.0 (1995, Jandel, Chicago, IL). P values less than .05 were considered significant.
RESULTS
Whole animal weights for both CDH and control lambs were comparable. The lung wet weights of the CDH lungs were significantly smaller than those of controls (Table 1). There were no significant differences in MPO activity between right and left lungs in any of the 4 groups. At birth, both CDH (group 1) and Control lungs (group 3) had minimal MPO activity (318 ⫾ 57 v 348 ⫾ 61, respectively). There was no significant difference between these 2 groups (P ⫽ .5). Four hours of ventilation resulted in a significant increase of MPO activity in both control (group 4) and CDH (group 2) lungs when compared with their nonventilated counterparts. After 4 hours of ventilation there was significantly more MPO activity in the CDH-ventilated lungs (group 2) than controls (group 4) similarly ventilated (3203 ⫾ 665 v 1220 ⫾ 194, respectively; P ⫽ .001; Fig 1). DISCUSSION
The current study investigated whether myeloperoxidase activity in lung tissue samples could be used as an indirect measure of acute lung injury in the CDH lamb
Myeloperoxidase Assay Myeloperoxidase was extracted from each homogenized lung tissue sample by suspending the material in 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co, St Louis, MO) in 50 mmol/L potassium phosphate buffer, pH 6.0, before sonication in an ice bath for 10 seconds. The samples were freeze-thawed 3 times, after which sonication was repeated. Suspensions were then centrifuged at 40,000g for 30 minutes and the resulting supernatant further assayed. Myeloperoxidase activity was assayed spectrophotometrically using the method of Bradley et al5: 0.1 mL of supernatant was mixed with 2.9 mL of 50 mmol/L phosphate buffer, pH 6.0, containing 0.167 mg/mL -dianisidine dihydrochloride (Sigma Chemical) and 0.0005% hydrogen peroxide (Sigma Chemical). The change in absorbance at 460 nm was measured using a spectrophotometer (U-2000; Hitachi Instruments, Webster, NY). MPO activity was then derived from the observed change in absorbance per minute. The MPO activity was normalized further to the total protein content of the supernatant, as
Fig 1. Lung MPO activities expressed as units per milligram of total protein. * P ⴝ .001, Control nonventilated versus control ventilated; ** P ⴝ .005, CDH nonventilated versus CDH ventilated; ‡ P ⴝ .001 CDH ventilated versus control ventilated.
336
MCCABE ET AL
model. The findings show that conventional ventilation and hyperoxia causes increased myeloperoxidase activity in both control and CDH lungs. This effect was significantly greater in the CDH lung samples. This assay, therefore, gives a useful direct measure of neutrophil sequestration in the lung tissue samples, and is an indirect indicator of acute lung injury being present after only 4 hours of ventilation and hyperoxia. The presence of neutrophils in the ventilated tissue offers further insight into the possible nature of the iatrogenic injury occurring in cases of CDH. Because the current study has used both conventional ventilation and hyperoxia, it is not possible to ascertain the degree of iatrogenic lung injury caused by each of the supportive measures. It would therefore be of interest to limit the effects of possible barotrauma by ventilating in a highfrequency oscillatory mode with and without hyperoxia. Hyperoxia, which is linked to the generation of various reactive oxygen species (ROS), is a necessary “evil” in the resuscitation and maintenance of CDH neonates. Under normal conditions, endogenous antioxidant clearance pathways handle these toxic compounds through a series of enzymatic and nonenzymatic pathways. However, in the setting of the CDH hypoplastic, surfactantdeficient lungs, it is conceivable that the clearance pathways for ROS first become depleted and then overwhelmed by the generation of these highly reactive radicals. Cellular responses that trigger organ injury then are initiated. ROS can induce several types of cellular damage directly (ie, lipid peroxidation, destruction of enzymes, and cleavage of DNA strands) as well as altering the activity of proteins involved in signal trans-
duction. Therefore, any attempts to manufacture and replace the scavenging enzyme system by the CDH lung are further hampered. The concept of antioxidant therapies have been investigated using recombinant enzymes such as superoxide dismutase and catalase10-12 as well as chemical scavengers such as N-acetylcysteine13 in various lung disorders. But these interventions have been limited by enzyme instability and inadequate intracellular delivery to the sites of ROS action. Partial liquid ventilation may offer another promising intervention, as demonstrated by Rotta and Steinhorn.14 They found that the degree of pulmonary neutrophil accumulation, as measured by MPO activity, in an experimental animal model of acute lung injury, was decreased when the animals received partial liquid ventilation. Perfluorocarbon used for liquid ventilation has been shown previously to decrease free radical release by alveolar macrophages15 and attenuates oxidative damage to lung tissue.16 Finally, gene therapy may become a fruitful focus for the development of novel treatments that aim to clear these toxic products at the earliest stage of iatrogenic injury.3 Despite the advances in antenatal diagnosis and neonatal care, the morbidity and mortality rate caused by CDH remain unacceptably high. Future investigations that focus on the potentiation of fetal lung growth and development in utero, or antioxidant therapies will require postnatal investigation. The MPO assay described now will offer a useful test for evidence of the early onset of acute lung injury in the CDH setting and will obviate the need for prolonged ventilatory studies.
REFERENCES 1. Frank L, Bucher JR, Roberts RJ: Oxygen toxicity in neonatal and adult animals of various species. J Appl Physiol 45:669-704, 1978 2. Fridovich I, Freeman B: Antioxidant defences in the lung. Ann Rev Physiol 48:693-702, 1986 3. Engelhardt JF: Redox-mediated gene therapies for environmental injury: Approaches and concepts. Antioxidants and Redox Signaling 1:5-27, 1999 4. Kapur P, Holm BA, Irish MS, et al: Tracheal ligation and mechanical ventilation do not improve the antioxidant enzyme status in the lamb model of congenital diaphragmatic hernia. J Pediatr Surg 34:270-272, 1999 5. Bradley PP, Priebat DA, Christensen RD, et al: Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. J Invest Dermatol 78:206-209, 1982 6. Glick PL, Stannard VA, Leach CL: Pathophysiology of congenital diaphragmatic hernia II. The fetal lamb model is surfactant deficient. J Pediatr Surg 27:382-388, 1992 7. Adzick NS, Outwater KM, Harrison MR: 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 8. de Lorimier AA, Tierney DF, Parker HR: Hypoplastic lungs in fetal lambs with surgically produced diaphragmatic hernia. Surgery 62:12-17, 1967
9. Lowry DH, Rosebrough NJ, Farr AL, et al: Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193: 265-275, 1951 10. Turrens JF, Crapo JD, Freeman BA: Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase. J Clin Invest 73:87-95, 1984 11. Tang G, White JE, Gordon RJ, et al: Polyethylene glycolconjugated superoxide dismutase protects rats against oxygen toxicity. J Appl Physiol 74:1425-1431, 1993 12. Walther FJ, David-Cu R, Lopez SL: Antioxidant-surfactant liposomes mitigate hyperoxic lung injury in premature rabbits. Am J Physiol 269:L613-L617, 1995 13. Bernard GR: N-Acetylcysteine in experimental and clinical acute lung injury. Am J Med 91:54S-59S, 1991 14. Rotta AT, Steinhorn DM: Partial liquid ventilation reduces pulmonary neutrophil accumulation in an experimental model of systemic endotoxemia and acute lung injury. Crit Care Med 26:1707-1715, 1998 15. Smith TM, Steinhorn DM, Thusu K: A liquid perfluorochemical decreases in vitro production of reactive oxygen species by alveolar macrophages. Crit Care Med 23:1533-1539, 1995 16. Steinhorn DM, Papo MC, Fuhrman BP: Oxidative damage is reduced during partial liquid ventilation with Perflubron. Crit Care Med 24:A148, 1996
MYELOPEROXIDASE ACTIVITY AS A LUNG MARKER IN CDH
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Discussion C. Stolar (New York, NY): I like this paper very much because it speaks to our theme song of avoiding ventilating babies at all costs to try and minimise lung trauma. I think you have shown us some very interesting biologic information to support that the ventilator is a lethal device. I am surprised that you saw no difference between the right and left lungs because they can be such different sizes, particularly in this model because the compliance is so different. Is this because they are symmetrically deficient in surfactant. Why would you not see a difference in one side versus the other? A.J. McCabe (response): That was the reason we chose to make comparisons with the actual protein content of the lung, so we haven’t actually had any difference between compressed lung and a lung that is well airated. M. Hollwarth (Graz, Austria): You showed in earlier studies that there is a reduction of the amount of catalase and glutathione peroxidase in the tissue that is significant according to your statistics. My question is, do you think
that this reduction of enzymes in the tissue is significant in terms of not enough antioxidant power? I think the total amount of SOD and catalase is so huge in a cell that a reduction of about one third of the amount does not have any effects on the capacity of free oxygen radicals scavenging. A.J. McCabe (response): Our study was carried out over 4 hours, and we think that the capacity of the cell was used up gradually. I think it would be interesting to do the same studies under a shorter duration of ventilation. P.D. Losty (Liverpool, England): What sort of antenatal therapeutic strategies are you proposing? A.J. McCabe (response): I think any intervention that improves lung growth and in particular lung maturation has an important role to play in limiting the amount of trauma that we give these lungs. We have shown that the antioxidant status is not improved by tracheal ligation, which does improve lung growth, but I think perhaps a dynamic occlusion system might give us the maturation which we need in these lungs.