Necrotizing By Ian T. Cohen,
Enterocolitis
Scarlet-t D. Nelson,
in a Neonatal
Piglet Model
Rodney A. Moxley, Michael P. Hirsh, Timothy C. Counihan, and Ronald F. Martin
Worcester,
Massachusetts
l The aim of this study was to develop an animal model for necrotizing enterocolitis (NEC). Twenty-five neonatal Hanford minipigs had carotid artery and external jugular vein catheters and rectal Clinical Tonomitors placed under anesthesia. Experimental animals were subjected to a hypoxic insult (50% reduction in baseline PaO, for 30 minutes) and hypothermic stress (core temperature reduced to 35°C for 30 minutes). Regular oral diet was resumed and the survivors were euthanized 3 to 4 days later. All animals underwent necropsy with gross and histopathological evaluation of the entire bowel. Of 22 experimental animals, 14 survived (84%) and 8 (38%) died of pulmonary hemorrhage. Of the 14 survivors, 8 (57%) had gross and microscopic evidence of NEC. Six of the total 25 animals (24%) sustained rectal perforations from the tonometer. Of 3 control animals, one died of pulmonary hemorrhage and the two survivors had normal intestine. This model successfully produced gross and histological evidence of NEC. The tonometer shows promise as a predictor of NEC provided technical modifications can reduce the complication rate. Copyright o 7991 by W.E. Saunders Company INDEX WORDS:
Necrotizing
enterocolitis,
piglet model.
N
ECROTIZING enterocolitis (NEC) is the most common acquired gastrointestinal (GI) emergency in the human neonate.’ In the United States, between 2,000 and 4,000 newborn infants per year have NEC.’ The incidence increases markedly at lower gestational ages.’ Various factors have been associated with the development of NEC, including infectious agents, hypertonic feedings, asphyxia, hypovolemia, hypothermia, and exaggerated shunting reflexes, resulting in irreversible ischemic GI injury.’ Although it has not yet been determined that NEC is a complication of enteral feedings, there is strong
From the Division of Pediatric Surgery, Department of Surgery, University of Massachusetts Medical Center, Worcester, MA, and the Veterinary Diagnostic Center, University of Nebraska, Lincoln, NE. Presented at the 37th Annual International Congress of the British Association of Paediatric Surgeons, Glasgow, Scotland, July 25-27, 1990. Listed in Journal Series no. 9417, Nebraska Agn’culture Experimental Station, Lincoln, NE. Supported in pan by Tonometrics, Inc (Bethesda, MD) and Maltinckrodt (Glens Falls, NY). Address reprint requests to Ian T. Cohen, MD, Division of Pediatric Surgery, University of Massachusetts Medical Center, 55 Lake Ave N, Worcester, MA 01655. Copyright o 1991 by W.B. Saunders Company 0022-3468/91/2605-0023$03.00/O
598
and Lincoln,
Nebraska
circumstantial evidence that NEC may be a consequence of feeding? NEC research awaits the definition of an animal model closely approximating human neonatal GI form and function, which would validate conclusions drawn from experimental manipulations.4 Touloukian’ has summarized the major circulatory factors in the pathogenesis of neonatal NEC. Touloukian et al’ proposed the term “ischemic gastroenterocolitis of the neonate” to give recognition to the most probably etiology of the disease. Their experimental model used piglets ranging from 7 to 20 days of age. Using newborn piglets 6 to 96 hours of age, Alward et al7 demonstrated the effects of asphyxia on cardiac output and organ blood flow and showed no immediate change in the latter following asphyxia. Karna et al8 compared the effects of asphyxia, hypoxia, and acidosis on intestinal blood flow and oxygen uptake in newborn piglets within 72 hours of birth and showed that hypoxia played a more significant role than acidosis in asphyxia-induced intestinal ischemia. Sibbons et al9 combined hypoxia and hyperviscosity, with and without splenectomy, and showed that the neonatal piglet was capable of producing the full spectrum of NEC and that low birthweight was a significant predisposing factor. Because many of the clinical studies of NEC have not been preceded by careful laboratory evaluation, we have attempted to develop an animal model for NEC that produces the disease in a sufficient proportion of experimental animals and to a degree that the animal can survive for 1 week or more with minimal pain or distress. Survival of the animals is necessary to test factors that may predict, prevent, ameliorate, or lessen the severity of the disease process. MATERIALS AND METHODS A total of 2.5 neonatal Hanford minipigs was obtained from Charles River Laboratory (Wilmington, MA). The Hanford minipig was selected for its standard genetic background and consistent health profiles. The Laboratory Animal Care and Use Program at University of Massachusetts Medical Center at Worcester complies with all federal and state laws regarding the use of research animals and with the Public Health Service Policy on Humane Care and Use of Laboratory Animals (NIH Guide for Grants and Contracts, ~0114, no 8, June 25.1985). The facilities and care program are also currently accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). Piglets were 12 to 48 hours of age (mean, 33.6), of either sex, and had the lowest
JoornalofPediatric Surgery, Vol 26, No 5 (May), 1991: pp 598-601
NECROTIZING
ENTEROCOLITIS
IN NEONATAL
PIGLETS
birthweights of their litters. Piglets were allowed to nurse from their mother until shipment to this laboratoty. At the time of arrival, piglets were weighed, assigned an identification number, and health checked. Piglets were caged together on lambs wool bedding and fed SPF-Lac (substitute sow’s milk, Borden, Hampshire, IL) ad libitum. Experiments began the morning after arrival of the piglets. Twenty-two piglets were subjected to a 30-minute period of hypoxic-hypothermic stress. PaO, was reduced to 50% of normal value by decreasing the percent of oxygen being delivered. Core body temperature was allowed to decrease to 35°C. This occurred spontaneously while the piglets were anesthetized if no heat source, such as a water circulating pad, was provided. Three control piglets underwent the same preparation but were not allowed to become hypoxic or hypothermic. Piglets were given 0.1 mL/kg atropine sulfate intramuscularly as a preanesthetic. Anesthesia was induced with a mixture of 1.5% to 2.0% isoflurane, 60% oxygen, and 40% nitrous oxide to equal 1 Umin delivered via face mask through a nonrebreathing system. After intubation with a 2.5 or 3.0 cuffed endotracheal tube, isoflurane concentration was reduced to 1.0% to 1.5% to effect a surgical plane of anesthesia. An esophageal monitor (Mon-ATherm, Mallinckrodt, Inc, Glens Falls, NY) was placed for monitoring respirations and core body temperature. A noninvasive heart rate monitor (Datascope, Inc, Paramus, NJ) was attached to the piglet. Using strict aseptic surgical technique, size 4.OF Hemed (Gish Medical, Santa Ana, CA) central venous access catheters were placed into the left external jugular vein and carotid artery for fluid administration and blood gas monitoring. A Clinical Tonomitor (Tonometrics, Inc, Bethesda, MD) was placed in the rectum and samples were taken every 20 minutes. After completing the surgical preparation, the isoflurane was discontinued, the oxygen was reduced to 20%, and the nitrous oxide was increased to 80%. Physiological parameters were closely monitored. Fluids and/or drugs were administered only when necessary to assure survival of the piglet. A PaO, of 30 to 40 mm Hg (normal, 75 mm Hg) and body temperature of 35°C (normal. 38 to 39°C) were maintained for 30 minutes. Piglets were then recovered on 100% oxygen and body temperature was increased to normal. After recovery, piglets were returned to the animal room and resumed a normal oral diet. Three days poststress the piglets were euthanized with Euthanasia 6 Solution 2 (VetLabs Limited, Lenaxas, KS) 1 mL/lO lb. The GI tract was examined grossly and histologically.
Fig 1. Small intestine showing early stage NEC (original magnification x40).
uted to the tonometer. Normal pHi (calculated mucosal pH from the tonometer) for the piglet is 7.30 to 7.35 (Tonometrics Inc: Personal communication, November 30, 1989). Baseline pHi of the piglets in this experiment ranged from 6.62 to 7.46 (mean, 7.08).
RESULTS
Fourteen (64%) of the 22 experimental animals survived. Eight (36%) of the experimental and one of the control piglets died during or shortly after the procedure from pulmonary hemorrhage. Eight (57%) of the 14 survivors had gross and histological evidence of NEC (Figs 1,2, and 3). Sixteen (64%) of the piglets developed either respiratory or mixed respiratory and metabolic acidosis early in the procedure. Nine responded to sodium bicarbonate therapy. Although these are reports that piglets respire spontaneously during hypoxemia, all piglets in this study required manual ventilation during all or part of the stress period. The major complication observed in 6 (24%) of the surviving animals was rectal perforation attrib-
DISCUSSION
Kliegman has described the various models of the pathogenesis of NEC, stressing its multifactorial nature and suggesting one final common pathway.” In a further step, Kosloske has described a unifying hypothesis for the pathogenesis and prevention of NEC.” Of the three essential components in the development of the disease, mucosal injury, bacteria, and enteral substrate, the last, namely the presence or absence of enteral feeding, seems to be the most clinically significant and remediable.” In this study, the neonatal Hanford minipigs had a mean age of 33.6 hours and weighed 750 to 800 g to avoid the pitfalls of the marked physiological differ-
600
COHEN ET AL
Fig 2. Small intestine showing early stage NEC (original magnification x 100).
Fig 3. Small intestine showing mucosal ulceration (original magnification x40).
ences between newborn and 7-day-old piglets suggested by Rowe and Arango.13 A number of complications arose during this study that have been described previously, notably respiratory and metabolic acidosis. The Clinical Tonomitor14 is a type of GI tonometer that allows early detection of gut ischemia by means of a simple derived measurement of gut mucosal pH. The advantage of the Clinical Tonomitor is that it is inserted transanally, eliminating the need for surgical placement. Mucosal tissue pH is calculated with the Henderson-Hasselbalch equation using Tonomitor PCO, and arterial blood bicarbonate level. A mucosal tissue pH lower than normal reflects acidosis consequent to inadequate oxygenation. Poole et al” have shown tonometry to be a valuable tool for the early diagnosis of mesenteric ischemia. Schiedler et all’ have reported the beneficial use of tonometry to predict ischemic colitis in high-risk aortic surgery patients.
We believe the abnormal baseline pHi measurements obtained are a result of compression by the tonometer balloon causing mucosal ischemia in the rectum. Therefore, we do not believe the pHi measurements obtained are accurate for the colonic mucosa. The abnormal pHi measurements and the incidence of rectal perforation indicate that the Clinical Tonomiter is too large for neonatal use. Despite the promise of tonometry as a predictor of NEC, technical modifications are needed before any attempt is made to use it clinically because of the high incidence of rectal perforation. Development of a smaller tonometer for neonatal use is in progress. This animal model successfully produced pathological evidence of NEC similar to that observed in human neonates. The significance of this model is that the animals survive the hypoxic-hypothermic insult for a sufficient period of time to allow study of methods that may predict, prevent, ameliorate, or lessen the severity of the disease process.
NECROTIZING
ENTEROCOLITIS
IN NEONATAL
601
PIGLETS
REFERENCES 1. Kleigman RM, Fanaroff AA: Necrotizing enterocolitis. N Engl J Med 310:1093-1103,1984 2. Crissinger KD, Ryckman FC, Balistreri WF: Necrotizing enterocolitis and gastrointestinal hemorrhage, in Fanaroff AA, Martin RJ (eds): Neonatal-Perinatal Medicine (ed 4). St Louis, MO, Mosby, 1987, pp 928-932 3. Kleigman RM: Necrotizing enterocolitis: A consequence of enteral feeding in the micropremie, in The Next Frontier, Report of the 99th Ross Conference on Pediatric Research. New York, NY, Ross Laboratories, 1990, pp 122-133 4. Topalian SL, Ziegler MM: Necrotizing enterocolitis: A review of animal models. J Surg Res 37:320-336, 1984 5. Touloukian RJ: Etiologic role of the circulation, in Brown E, Sweet A (eds): Neonatal Necrotizing Enterocolitis. Philadelphia, PA, Grune & Stratton, 1980, pp 41-56 6. Touloukian RJ, Posch JN, Spencer R: The pathogenesis of ischemic gastroenterocolitis of the neonate: Selective gut mucosal ischemia in asphyxiated neonatal piglets. J Pediatr Surg 7:194-205, 1972 7. Alward CT, Hook JB, Helmrath TA, et al: Effects of asphyxia on cardiac output and organ blood flow in the newborn piglet. Pediatr Res 12:824-827, 1978 8. Karna P, Senagore A, Chou CC: Comparison of the effect of
asphyxia, hypoxia, and acidosis on intestinal blood flow and O2 uptake in newborn piglets. Pediatr Res l&929-932, 1986 9. Sibbons P, Spitz L, van Velzen D, et al: Relationship of birthweight to the pathogenesis of necrotizing enterocolitis in the neonatal piglet. Pediatr Path01 8:151-162, 1988 10. Kleigman RM: Models of the pathogenesis of necrotizing enterocolitis. J Pediatr 117:S2-S5,1990 (suppl) 11. Kosloske AM: A unifying hypothesis for pathogenesis and prevention of necrotizing enterocolitis. J Pediatr 117:S68-S74.1990 (suPPI) 12. Bloomfield S, Cohen IT, Kline AC: Feeding practice and the development of neonatal necrotizing enterocolitis. J Am Co11Nutr 2:283,1983 (abstr) 13. Rowe MI, Arango A: A critical appraisal of two common “neonatal” experimental subjects. J Pediatr Surg 12:409-412, 1977 14. Tonometric Inc: Product brochure. Bethesda, MD, Tonometric, Inc, 1990 15. Poole JW, Sammartano RJ, Boley SJ: The use of tonometry in the early diagnosis of mesenteric ischemia. Curr Surg 44:21-24, Jan-Feb1987 16. Schiedler MG, Cutler BS, Fiddian-Green RG: Sigmoid intramural pH for prediction of ischemic colitis during aortic surgery. Arch Surg 122:881-885, 1987
Enterocolitis
Scarlet-t D. Nelson,
in a Neonatal
Piglet Model
Rodney A. Moxley, Michael P. Hirsh, Timothy C. Counihan, and Ronald F. Martin
Worcester,
Massachusetts
l The aim of this study was to develop an animal model for necrotizing enterocolitis (NEC). Twenty-five neonatal Hanford minipigs had carotid artery and external jugular vein catheters and rectal Clinical Tonomitors placed under anesthesia. Experimental animals were subjected to a hypoxic insult (50% reduction in baseline PaO, for 30 minutes) and hypothermic stress (core temperature reduced to 35°C for 30 minutes). Regular oral diet was resumed and the survivors were euthanized 3 to 4 days later. All animals underwent necropsy with gross and histopathological evaluation of the entire bowel. Of 22 experimental animals, 14 survived (84%) and 8 (38%) died of pulmonary hemorrhage. Of the 14 survivors, 8 (57%) had gross and microscopic evidence of NEC. Six of the total 25 animals (24%) sustained rectal perforations from the tonometer. Of 3 control animals, one died of pulmonary hemorrhage and the two survivors had normal intestine. This model successfully produced gross and histological evidence of NEC. The tonometer shows promise as a predictor of NEC provided technical modifications can reduce the complication rate. Copyright o 7991 by W.E. Saunders Company INDEX WORDS:
Necrotizing
enterocolitis,
piglet model.
N
ECROTIZING enterocolitis (NEC) is the most common acquired gastrointestinal (GI) emergency in the human neonate.’ In the United States, between 2,000 and 4,000 newborn infants per year have NEC.’ The incidence increases markedly at lower gestational ages.’ Various factors have been associated with the development of NEC, including infectious agents, hypertonic feedings, asphyxia, hypovolemia, hypothermia, and exaggerated shunting reflexes, resulting in irreversible ischemic GI injury.’ Although it has not yet been determined that NEC is a complication of enteral feedings, there is strong
From the Division of Pediatric Surgery, Department of Surgery, University of Massachusetts Medical Center, Worcester, MA, and the Veterinary Diagnostic Center, University of Nebraska, Lincoln, NE. Presented at the 37th Annual International Congress of the British Association of Paediatric Surgeons, Glasgow, Scotland, July 25-27, 1990. Listed in Journal Series no. 9417, Nebraska Agn’culture Experimental Station, Lincoln, NE. Supported in pan by Tonometrics, Inc (Bethesda, MD) and Maltinckrodt (Glens Falls, NY). Address reprint requests to Ian T. Cohen, MD, Division of Pediatric Surgery, University of Massachusetts Medical Center, 55 Lake Ave N, Worcester, MA 01655. Copyright o 1991 by W.B. Saunders Company 0022-3468/91/2605-0023$03.00/O
598
and Lincoln,
Nebraska
circumstantial evidence that NEC may be a consequence of feeding? NEC research awaits the definition of an animal model closely approximating human neonatal GI form and function, which would validate conclusions drawn from experimental manipulations.4 Touloukian’ has summarized the major circulatory factors in the pathogenesis of neonatal NEC. Touloukian et al’ proposed the term “ischemic gastroenterocolitis of the neonate” to give recognition to the most probably etiology of the disease. Their experimental model used piglets ranging from 7 to 20 days of age. Using newborn piglets 6 to 96 hours of age, Alward et al7 demonstrated the effects of asphyxia on cardiac output and organ blood flow and showed no immediate change in the latter following asphyxia. Karna et al8 compared the effects of asphyxia, hypoxia, and acidosis on intestinal blood flow and oxygen uptake in newborn piglets within 72 hours of birth and showed that hypoxia played a more significant role than acidosis in asphyxia-induced intestinal ischemia. Sibbons et al9 combined hypoxia and hyperviscosity, with and without splenectomy, and showed that the neonatal piglet was capable of producing the full spectrum of NEC and that low birthweight was a significant predisposing factor. Because many of the clinical studies of NEC have not been preceded by careful laboratory evaluation, we have attempted to develop an animal model for NEC that produces the disease in a sufficient proportion of experimental animals and to a degree that the animal can survive for 1 week or more with minimal pain or distress. Survival of the animals is necessary to test factors that may predict, prevent, ameliorate, or lessen the severity of the disease process. MATERIALS AND METHODS A total of 2.5 neonatal Hanford minipigs was obtained from Charles River Laboratory (Wilmington, MA). The Hanford minipig was selected for its standard genetic background and consistent health profiles. The Laboratory Animal Care and Use Program at University of Massachusetts Medical Center at Worcester complies with all federal and state laws regarding the use of research animals and with the Public Health Service Policy on Humane Care and Use of Laboratory Animals (NIH Guide for Grants and Contracts, ~0114, no 8, June 25.1985). The facilities and care program are also currently accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). Piglets were 12 to 48 hours of age (mean, 33.6), of either sex, and had the lowest
JoornalofPediatric Surgery, Vol 26, No 5 (May), 1991: pp 598-601
NECROTIZING
ENTEROCOLITIS
IN NEONATAL
PIGLETS
birthweights of their litters. Piglets were allowed to nurse from their mother until shipment to this laboratoty. At the time of arrival, piglets were weighed, assigned an identification number, and health checked. Piglets were caged together on lambs wool bedding and fed SPF-Lac (substitute sow’s milk, Borden, Hampshire, IL) ad libitum. Experiments began the morning after arrival of the piglets. Twenty-two piglets were subjected to a 30-minute period of hypoxic-hypothermic stress. PaO, was reduced to 50% of normal value by decreasing the percent of oxygen being delivered. Core body temperature was allowed to decrease to 35°C. This occurred spontaneously while the piglets were anesthetized if no heat source, such as a water circulating pad, was provided. Three control piglets underwent the same preparation but were not allowed to become hypoxic or hypothermic. Piglets were given 0.1 mL/kg atropine sulfate intramuscularly as a preanesthetic. Anesthesia was induced with a mixture of 1.5% to 2.0% isoflurane, 60% oxygen, and 40% nitrous oxide to equal 1 Umin delivered via face mask through a nonrebreathing system. After intubation with a 2.5 or 3.0 cuffed endotracheal tube, isoflurane concentration was reduced to 1.0% to 1.5% to effect a surgical plane of anesthesia. An esophageal monitor (Mon-ATherm, Mallinckrodt, Inc, Glens Falls, NY) was placed for monitoring respirations and core body temperature. A noninvasive heart rate monitor (Datascope, Inc, Paramus, NJ) was attached to the piglet. Using strict aseptic surgical technique, size 4.OF Hemed (Gish Medical, Santa Ana, CA) central venous access catheters were placed into the left external jugular vein and carotid artery for fluid administration and blood gas monitoring. A Clinical Tonomitor (Tonometrics, Inc, Bethesda, MD) was placed in the rectum and samples were taken every 20 minutes. After completing the surgical preparation, the isoflurane was discontinued, the oxygen was reduced to 20%, and the nitrous oxide was increased to 80%. Physiological parameters were closely monitored. Fluids and/or drugs were administered only when necessary to assure survival of the piglet. A PaO, of 30 to 40 mm Hg (normal, 75 mm Hg) and body temperature of 35°C (normal. 38 to 39°C) were maintained for 30 minutes. Piglets were then recovered on 100% oxygen and body temperature was increased to normal. After recovery, piglets were returned to the animal room and resumed a normal oral diet. Three days poststress the piglets were euthanized with Euthanasia 6 Solution 2 (VetLabs Limited, Lenaxas, KS) 1 mL/lO lb. The GI tract was examined grossly and histologically.
Fig 1. Small intestine showing early stage NEC (original magnification x40).
uted to the tonometer. Normal pHi (calculated mucosal pH from the tonometer) for the piglet is 7.30 to 7.35 (Tonometrics Inc: Personal communication, November 30, 1989). Baseline pHi of the piglets in this experiment ranged from 6.62 to 7.46 (mean, 7.08).
RESULTS
Fourteen (64%) of the 22 experimental animals survived. Eight (36%) of the experimental and one of the control piglets died during or shortly after the procedure from pulmonary hemorrhage. Eight (57%) of the 14 survivors had gross and histological evidence of NEC (Figs 1,2, and 3). Sixteen (64%) of the piglets developed either respiratory or mixed respiratory and metabolic acidosis early in the procedure. Nine responded to sodium bicarbonate therapy. Although these are reports that piglets respire spontaneously during hypoxemia, all piglets in this study required manual ventilation during all or part of the stress period. The major complication observed in 6 (24%) of the surviving animals was rectal perforation attrib-
DISCUSSION
Kliegman has described the various models of the pathogenesis of NEC, stressing its multifactorial nature and suggesting one final common pathway.” In a further step, Kosloske has described a unifying hypothesis for the pathogenesis and prevention of NEC.” Of the three essential components in the development of the disease, mucosal injury, bacteria, and enteral substrate, the last, namely the presence or absence of enteral feeding, seems to be the most clinically significant and remediable.” In this study, the neonatal Hanford minipigs had a mean age of 33.6 hours and weighed 750 to 800 g to avoid the pitfalls of the marked physiological differ-
600
COHEN ET AL
Fig 2. Small intestine showing early stage NEC (original magnification x 100).
Fig 3. Small intestine showing mucosal ulceration (original magnification x40).
ences between newborn and 7-day-old piglets suggested by Rowe and Arango.13 A number of complications arose during this study that have been described previously, notably respiratory and metabolic acidosis. The Clinical Tonomitor14 is a type of GI tonometer that allows early detection of gut ischemia by means of a simple derived measurement of gut mucosal pH. The advantage of the Clinical Tonomitor is that it is inserted transanally, eliminating the need for surgical placement. Mucosal tissue pH is calculated with the Henderson-Hasselbalch equation using Tonomitor PCO, and arterial blood bicarbonate level. A mucosal tissue pH lower than normal reflects acidosis consequent to inadequate oxygenation. Poole et al” have shown tonometry to be a valuable tool for the early diagnosis of mesenteric ischemia. Schiedler et all’ have reported the beneficial use of tonometry to predict ischemic colitis in high-risk aortic surgery patients.
We believe the abnormal baseline pHi measurements obtained are a result of compression by the tonometer balloon causing mucosal ischemia in the rectum. Therefore, we do not believe the pHi measurements obtained are accurate for the colonic mucosa. The abnormal pHi measurements and the incidence of rectal perforation indicate that the Clinical Tonomiter is too large for neonatal use. Despite the promise of tonometry as a predictor of NEC, technical modifications are needed before any attempt is made to use it clinically because of the high incidence of rectal perforation. Development of a smaller tonometer for neonatal use is in progress. This animal model successfully produced pathological evidence of NEC similar to that observed in human neonates. The significance of this model is that the animals survive the hypoxic-hypothermic insult for a sufficient period of time to allow study of methods that may predict, prevent, ameliorate, or lessen the severity of the disease process.
NECROTIZING
ENTEROCOLITIS
IN NEONATAL
601
PIGLETS
REFERENCES 1. Kleigman RM, Fanaroff AA: Necrotizing enterocolitis. N Engl J Med 310:1093-1103,1984 2. Crissinger KD, Ryckman FC, Balistreri WF: Necrotizing enterocolitis and gastrointestinal hemorrhage, in Fanaroff AA, Martin RJ (eds): Neonatal-Perinatal Medicine (ed 4). St Louis, MO, Mosby, 1987, pp 928-932 3. Kleigman RM: Necrotizing enterocolitis: A consequence of enteral feeding in the micropremie, in The Next Frontier, Report of the 99th Ross Conference on Pediatric Research. New York, NY, Ross Laboratories, 1990, pp 122-133 4. Topalian SL, Ziegler MM: Necrotizing enterocolitis: A review of animal models. J Surg Res 37:320-336, 1984 5. Touloukian RJ: Etiologic role of the circulation, in Brown E, Sweet A (eds): Neonatal Necrotizing Enterocolitis. Philadelphia, PA, Grune & Stratton, 1980, pp 41-56 6. Touloukian RJ, Posch JN, Spencer R: The pathogenesis of ischemic gastroenterocolitis of the neonate: Selective gut mucosal ischemia in asphyxiated neonatal piglets. J Pediatr Surg 7:194-205, 1972 7. Alward CT, Hook JB, Helmrath TA, et al: Effects of asphyxia on cardiac output and organ blood flow in the newborn piglet. Pediatr Res 12:824-827, 1978 8. Karna P, Senagore A, Chou CC: Comparison of the effect of
asphyxia, hypoxia, and acidosis on intestinal blood flow and O2 uptake in newborn piglets. Pediatr Res l&929-932, 1986 9. Sibbons P, Spitz L, van Velzen D, et al: Relationship of birthweight to the pathogenesis of necrotizing enterocolitis in the neonatal piglet. Pediatr Path01 8:151-162, 1988 10. Kleigman RM: Models of the pathogenesis of necrotizing enterocolitis. J Pediatr 117:S2-S5,1990 (suppl) 11. Kosloske AM: A unifying hypothesis for pathogenesis and prevention of necrotizing enterocolitis. J Pediatr 117:S68-S74.1990 (suPPI) 12. Bloomfield S, Cohen IT, Kline AC: Feeding practice and the development of neonatal necrotizing enterocolitis. J Am Co11Nutr 2:283,1983 (abstr) 13. Rowe MI, Arango A: A critical appraisal of two common “neonatal” experimental subjects. J Pediatr Surg 12:409-412, 1977 14. Tonometric Inc: Product brochure. Bethesda, MD, Tonometric, Inc, 1990 15. Poole JW, Sammartano RJ, Boley SJ: The use of tonometry in the early diagnosis of mesenteric ischemia. Curr Surg 44:21-24, Jan-Feb1987 16. Schiedler MG, Cutler BS, Fiddian-Green RG: Sigmoid intramural pH for prediction of ischemic colitis during aortic surgery. Arch Surg 122:881-885, 1987
