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Necrotizing Enterocolitis and the Role of Anemia of Prematurity
Necrotizing Enterocolitis and the Role of Anemia of Prematurity Rachana Singh, MD, MS,* Bhavesh L. Shah, MD,* and Ivan D. Frantz, III, MD† Necrotizing enterocolitis (NEC) is one of the most common surgical diseases of preterm infants, with significant short- and long-term morbidity and mortality. Although the etiology of NEC remains elusive, multiple factors adversely affecting the intestinal mucosal integrity of preterm infants are known to be associated with NEC. Anemia and red blood cell (RBC) transfusion-related gut injury have been shown to have strong correlation with NEC. Anemia potentially compromises mucosal integrity with subsequent poor healing, and this injury may be augmented by yet unknown factors associated with RBC transfusions. Although convincing evidence is lacking, there is a need for guidelines to keep the hematocrit within clinically and physiologically relevant limits by appropriate interventions. Further investigations need to focus on assessing the interplay between anemia, chronically hypoxemic/hypoperfused intestines, and early iron therapy or other pharmacologic approaches for prevention/treatment of anemia and RBC transfusions. Semin Perinatol 36:277-282 © 2012 Elsevier Inc. All rights reserved. KEYWORDS hematocrit, hypoxemia, mucosal integrity
N
ecrotizing enterocolitis (NEC) is the most common surgical disease in very low-birth-weight (VLBW) infants, with an incidence of 7%-13%, and is associated with significant short- and long-term morbidity and mortality.1-3 NEC is thought to have a multifactorial etiology, with most of the associated factors ultimately affecting mesenteric blood flow and oxygen delivery, thereby potentially leading to hypoxemic/ischemic mucosal injury—the first step in the cascade of events leading to the onset of NEC.4,5 Recent publications suggest a consistent and significant temporal association between NEC, anemia, and red blood cell (RBC) transfusions.6-11 The objective of this review is to present our current understanding of the role of anemia of prematurity (AOP), whether causative or associative, in the onset of NEC by presenting the supporting evidence and strategies to minimize its impact.
*Division of Newborn Medicine, Department of Pediatrics, Baystate Children’s Hospital, The Western Campus of Tufts University School of Medicine, Springfield, MA. †Division of Newborn Medicine, Children’s Hospital, Boston, MA. Address reprint requests to Rachana Singh, MD, MS, Division of Newborn Medicine, Department of Pediatrics, Baystate Children’s Hospital, The Western Campus of Tufts University School of Medicine, 759 Chestnut Street, Springfield, MA 01199. E-mail: [email protected]
0146-0005/12/$-see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.semperi.2012.04.008
NEC Pathophysiology There has been extensive clinical and experimental research on understanding the pathophysiology of NEC, and significant attempts have been made to discover the one factor that initiates the cascade of events leading to the clinical manifestation of NEC.12 However, this has met with limited or minimal success in altering the incidence of NEC. The prevailing hypothesis is that NEC is a multifactorial disease process with an inverse relationship to gestational age at birth. All other factors at present are believed to be associative; therefore, the prediction of which infant will ultimately suffer from NEC is not possible. The pre- and postnatal factors associated with NEC are listed in Table 1. The current understanding of the pathophysiology of NEC is postulated to start with the preterm infant as a vulnerable host with an immature gut barrier, impaired defense mechanisms, goblet cells with scant mucus production, and an immune system that is compromised. The infant is then exposed to a relatively hostile environment of neonatal intensive care unit (NICU) where broad-spectrum antibiotic therapy diminishes the protective commensal bacteria in the gut flora and increases colonization with pathogenic bacteria. The infant may be fed nonhuman milk or hyperosmolar formulations and experience intermittent hypoxemic/ischemic events resulting in a breach in mucosal integrity. Once this initial insult occurs, inflammatory reaction with release of 277
278 Table 1 Factors Associated With Necrotizing Enterocolitis Prenatal Maternal preeclampsia Maternal cocaine use Maternal indocin for tocolysis Intrauterine growth restriction Perinatal asphyxia Postnatal RDS Hypothermia Hypotension/shock Hypoxia PDA Rapid onset and hyperosmolar enteral feedings Formula feedings Infections Hospitalization during an epidemic Colonization with necrogenic bacteria Anemia Polycythemia Thrombocytosis Packed red blood cell transfusions Exchange transfusions Congenital heart disease Congenital GI anomalies
cytokines such as platelet-activating factor and tumor necrosis factor causes further mucosal damage, which progressively leads to full-thickness necrosis and may ultimately result in intestinal perforation. Because mucosal integrity is paramount for host defense against intestinal pathogenic bacteria, it makes sense to focus on factors that can promote mucosal membrane integrity, growth, and repair as potentially preventative of NEC.
Mucosal Integrity The human intestinal mucosa is derived from the endoderm with a highly complex 3-layer structure: the mucous epithelium, lamina propria, and muscularis mucosa.13 Intestinal mucosa is the largest area providing surface immunity, and is therefore also the most vulnerable to injury. A breach in the mucosal layer leaves the highly vascularized submucosa susceptible to invasion by the gut bacterial flora leading to pneumatosis intestinalis, the radiologic hall mark of NEC, as well as allowing bacterial translocation to the bloodstream with the resultant clinical manifestations of inflammation and infection. The blood flow to the gastrointestinal (GI) tract is derived from 3 sources, the celiac trunk and the superior and inferior mesenteric arteries.14 The venous drainage from the GI tract is unique, as instead of returning to the heart directly, it first flows to the liver via the portal vein, allowing substances absorbed from the gut flow first to the hepatocytes where they can be detoxified if needed. GI blood flow also has a range of dynamic regulation, wherein even in the fasting state, the splanchnic circulation receives blood flow (25% of cardiac output) that is disproportionate to the mass of the
R. Singh, B.L. Shah, and I.D. Frantz, III organs perfused (5%).15 During stress, there is a major redistribution of the systemic blood circulation with a decrease in the mesenteric gut flow because of the strong activation of the sympathetic nervous system and release of norepinephine.16 During these events of hypoperfusion and resultant ischemia/ hypoxemia, which are not uncommon for preterm infants, there may be associated mucosal injury caused by reperfusion and resultant release of cytokine/inflammatory mediators.17-20 The intestinal mucosal epithelial healing is dependent on the precise balance of migration, proliferation, and differentiation of the epithelial cells adjacent to the injured area.21,22 Attard et al23 have demonstrated that systemic hypoxia directly translated into local tissue hypoxia, and intestinal anastomotic healing was impaired. Recent studies have also disclosed constitutive expression of inducible nitric oxide synthase (iNOS) by normal intestinal epithelia, and that selective inhibition of iNOS abolished increases in nitric oxide (NO) synthesis and villous reepithelialization after injury. These results demonstrate that iNOSderived NO is a key mediator of early villous reepithelialization after acute mucosal injury.24,25 Pagani et al26 have demonstrated that the lack of hepcidin is responsible for the high inflammatory response to lipopolysaccharides in the iron deficiency state. It is possible that in this proinflammatory setting hypoxemia/ischemia-induced injury may be the initial step that sets off the cascade of events leading to the onset of NEC in preterm infants who often have lower hematocrit (Hct) levels and may experience intermittent hypoxemic events.
Anemia of Prematurity Anemia is defined by World Health Organization as hemoglobin or Hct value 2 standard deviations below (⫺2 SD) the distribution mean in a normal population of same age and gender living at the same altitude.27 This, during the neonatal period (⬍28 days of life), translates to a central venous Hct ⬍39%.28 Based on the College of American Pathologists Neonatal Red Blood Cell Transfusion Guidelines, anemia maybe classified as mild if Hct is ⱖ35% but ⬍39%, moderate if Hct is ⱖ25% but ⬍35%, and severe if Hct is ⬍25%.29 AOP is a normocytic, normochromic hypoproliferative state, an exaggerated version of the normal physiological anemia seen in term infants. This physiological decline in Hct is due to reduced erythropoietin (Epo) production in the immediate postnatal phase. For the preterm infant, it results in an Hct nadir earlier than the 8-10 weeks for term infants. Additionally, because of phlebotomy losses, nutritional deficiencies, and immune-compromised status, a significantly lower Hct level is a frequent occurrence.30-32
Anemia of Prematurity and Oxygenation In term neonates, HbF decreases exponentially from birth to approximately 25 weeks postnatally by which time it is completely replaced by HbA. However, HbA shows a biphasic pattern: first, it decreases slightly from birth to 6 weeks and then increases exponentially till all hemoglobin is almost
Relationship of NEC with anemia completely made up of HbA. Two distinct phases of postnatal erythropoiesis have been identified. The first phase (from birth to 6 weeks) is characterized by a decrease in the total amount of hemoglobin produced. In the second phase, there is an alteration in the relative proportions of HbF and HbA being synthesized.33 This transition is a much slower and prolonged process for the preterm infant, ultimately affecting both oxygen-carrying capacity and oxygen delivery to the local tissues. Oxygen is a prerequisite for successful wound healing, such as seen with NEC, because of the increased demand for reparative processes such as cell proliferation, bacterial defense, angiogenesis, and collagen synthesis. Although the role of oxygen in wound healing is not yet completely understood, many experimental and clinical observations have demonstrated that wound healing is impaired during hypoxemic states.34 In addition to the role of oxygen, recent reports suggest potential mechanisms involving NO control of blood flow via hypoxic vasodilatation. These mechanisms may be important, especially in preterm infants, because immature vascular beds, such as the splanchnic system, are at risk for hypoxemic injury secondary to shunting of blood to the brain and heart.35 Lachance et al36 have shown that oxygenation is adequately maintained in symptom-free infants with AOP. However, symptoms of anemia in preterm infants may be difficult to detect given the many other issues at play. Anemia, by decreasing the oxygen-carrying capacity of the blood to levels less than the demands of growing tissues, may enhance anaerobic metabolism and the production of byproducts such as lactic acid.37 Gutierrez et al have shown that during tissue hypoxia, the deficit arising from unequal levels of cellular adenosine triphosphate (ATP) requirements and aerobic ATP production is partially satisfied by anaerobic sources of ATP, including glycolysis, the creatine kinase reaction, and the adenylate kinase reaction. However, these reactions may set in motion cellular mechanisms that ultimately may lead to cellular dysfunction and death.38 This could again be a plausible cause for the onset of NEC in the preterm infant.
Anemia, Perfusion, and Wound Healing In preterm infants, the basal intestinal vascular resistance changes rapidly from the fetal to early neonatal life, as this fetal dormant organ becomes functional for enteral nutrition, and anemia may impair this normal transition.39,40 Jonsson et al have demonstrated that wound healing is primarily dependent on tissue perfusion and oxygen delivery to the tissues, and the presence of anemia does not seem to impair wound healing as long as the tissue perfusion is maintained. However, if perfusion is impaired in an anemic patient, then wound healing will be exceptionally vulnerable because of unmet oxygen needs.41 An experimental study in a porcine model has also demonstrated redistribution of jejunal blood flow and an increase in the ratio of mucosal to
279 systemic Hct as the main mechanisms maintaining mucosal oxygen supply during progressive anemia.42 Intestinal microvascular oxygen partial pressure and organ oxygen consumption may be limited by a critical decrease in oxygen delivery and Hct at the same time. Beyond these critical points, not only shunting of oxygen from the microcirculation could be demonstrated, but also a significant correlation between intestinal microvascular oxygen partial pressure and organ oxygen consumption has been noted.43 The cell renewal rate may increase in acute anemia by venesection, whereas it was remarkably decreased in chronic iron deficiency.44
Anemia of Prematurity, Mucosal Injury, and NEC Recent interest in transfusion-related acute gut injury (TRAGI) and NEC prompted us to investigate the relationship between NEC, AOP, and packed red blood cell (PRBC) transfusion. Although there are no uniform practice guidelines for RBC transfusion in the NICUs because of an increasing concern for transfusion associated-infections (HIV, hepatitis B and C, etc.) and gut injury, the recent trend has been toward restrictive transfusion practices.7-11,45 However, it is not possible to consider NEC and TRAGI without considering the role of AOP, as Hct and transfusion cannot be truly independent of each other. Our retrospective case-control study aimed to determine association of anemia and RBC transfusions with NEC in preterm infants. We identified 111 preterm infants with NEC ⱖstage 2a and compared them with 222 matched controls. Data from a comprehensive list of 28 clinical variables, including Hct and RBC transfusions, were recorded. Propensity scores and multivariate logistic regression models were then created to examine effects on the risk of NEC. While controlling for other factors, we reported that lower Hct level was associated with increased odds of NEC (OR ⫽ 1.10, P ⫽ 0.01). Additionally, RBC transfusions had a temporal relationship with NEC onset. Transfusion within 24 hours (OR ⫽ 7.60, P ⫽ 0.001) and 48 hours (OR ⫽ 5.55, P ⫽ 0.001) has a higher odds of developing NEC, but this association lost significance by 96 hours (OR ⫽ 2.13, P ⫽ 0.07) posttransfusion. Thus, we demonstrated that both Hct and RBC transfusions are associated with increased risk of NEC. Lower Hct was associated with higher predicted probability of NEC (Fig. 1).6 Possible mechanisms discussed earlier could explain the increase in risk of development of NEC with AOP in a preterm infant. Mally et al9, in their study evaluating the risk of NEC with PRBC transfusions, also noted that the group of infants with RBC-associated NEC had lower Hct. Blau et al in their cohort study sought to determine an association of NEC within ⬍48 hours of a PRBC transfusion. Of their cohort of 256 VLBW infants, there were 36 NEC cases, and 25% cases were associated with PRBC (n ⫽ 9). PRBC-associated cases had lower birth weight, Hct, and rapid onset of signs (⬍5 hours). Interestingly, in their study, current weight at the onset of NEC
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Figure 1 Relationship of hematocrit with probability of NEC. (Color version of figure is available online.)
did not differ; however, the more preterm the infant, the later the onset of NEC creating a centering of occurrence at a median of 31 weeks postconceptual age.46 Although Paul et al47 demonstrated correlation between transfusions and NEC, they did not control for the Hct in their 3 study groups, although the transfused groups were anemic with Hct ⬍30. In a survey of variation in transfusion practices among NICUs, we noted that NICUs with the lowest rate of transfusion had the lowest rate of NEC despite similar Hcts, supporting a role for transfusion in the pathogenesis of some NEC cases.45 These studies support our findings that anemia and its impact on the cellular mechanisms of tissue injury and repair potentially initiate the process for the development of NEC in preterm infants. El-Dib et al48 have speculated that although anemia by itself may not directly cause NEC, severe anemia may impair intestinal oxygen delivery, and thus physiologically justified increased demand of oxygen to intestinal mucosa at the time of feeding may not be met in anemic infants. Based on these data, we speculate that in anemic preterm infants, the ischemic injury sustained by the mucosal layer is aggravated by factors associated with RBC transfusions, and strategies to maintain a critical Hct may help decrease the incidence of NEC. This speculation gains further credence from the observation that Epo has been shown to be protective against NEC.49-51 However, there have been some disagreements about routine Epo therapy because of concerns for its efficacy in minimizing exposure to multiple donors, decreasing the number of transfusions, and increased incidence of retinopathy of prematurity.52-54 By contrast, Ohls et al in their National Institute of Child Health and Human Development study on long-term outcomes noted that early Epo and iron therapy did not affect anthropometric measurements or neurodevelopmental outcomes. Incidentally in their placebo group, there was a trend toward an increased incidence of
NEC with a lower Hct (30.3% vs 35%, 9.8% vs 5.6%).55 However, the number of cases was small. In our study, we also found that iron supplementation, another therapeutic intervention for treatment of anemia, was associated with lower risk for NEC.6 It is known that mild iron deficiency anemia may lead to decreased activity of cytochrome oxidase in buccal mucosa56 and the mucosal cells lining the intestinal villi.57 Further quantitative evidence of tissue iron depletion comes from studies of neutrophils and lymphocytes wherein neutrophils may have a decreased capacity to kill ingested bacteria, presumably because the oxidative burst is iron dependent.58-63 The iron-deficient state impairs GI functions, growth, suppressed immune functions with poor resistance to infection, and overall higher infant mortality.27 In our study, iron supplementation was associated with a lower risk of NEC, consistent with the hypothesis that prevention or treatment of anemia may be important in preventing or reducing the risk of NEC. In the past, there have been concerns about iron excess and oxidative injury in VLBW infant with limited antioxidant mechanisms. However, studies indicate that iron does not induce oxidative stress, as measured by isoprostanes and antioxidant status, when provided to stable growing low-birth-weight infants at doses ranging from 2 to 12 mg/kg/d or at a twicedaily dose of 9 mg/d.64-66
Future Directions Based on current evidence, there seems to be interplay between the intermittent hypoxic/ischemic events endured by a preterm infant during NICU stay, with mucosal integrity impairment potentially augmented by iron deficiency, AOP, and RBC transfusions. Future prospective studies are needed to address multiple questions regarding AOP, RBC transfusions, and therapies aimed toward them. For example, is there a critical Hct that has a negative influence on GI phys-
Relationship of NEC with anemia iology and its functioning, and can this be identified clinically? If there is a critical threshold, then should one avoid reaching it through judicious transfusion practices, use of Epo, or early enteral iron supplementation? If RBC transfusions worsen the injury incurred during the chronic anemic state, should transfusions be administered earlier before the injury has occurred? Which are the factors associated with the stored RBC themselves that may lead to TRAGI, such as age, preservatives, volume transfused, rate of administration, or unknown oxidants present in the stored blood that the preterm gut is unable to handle? Because the natural transition to adult HbA does not occur till 46 weeks’ corrected gestational age, is there harm in transfusing RBC with predominant HbA? Should feedings be held peritransfusion, and if so, when should one start withholding and for how long? Finally, at what gestational or postconceptional age is the infant no longer at risk of NEC? For example, is there a physiological or anatomical event analogous to retinal vascularization and risk for retinopathy of prematurity that marks the end point for NEC risk? These and many more questions remain unanswered and should be addressed in the future multicenter studies to identify clinical practices, which may help decrease the incidence of NEC.
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281 15. Barrett KE: Gastrointestinal Physiology. New York, NY, Lange Medical Books/McGraw-Hill, Medical Pub. Division, 2006: chap 1 16. Hasibeder W: Gastrointestinal microcirculation: Still a mystery? Br J Anaesth 105:393-396, 2010 17. Han X, Fink MP, Yang R, et al: Increased iNOS activity is essential for intestinal epithelial tight junction dysfunction in endotoxemic mice. Shock 21:261-270, 2004 18. Hart ML, Grenz A, Gorzolla IC, et al: Hypoxia-inducible factor-1␣dependent protection from intestinal ischemia/reperfusion injury involves ecto-5’-nucleotidase (CD73) and the A2B adenosine receptor. J Immunol 186:4367-4374, 2011 19. Pontell L, Sharma P, Rivera LR, et al: Damaging effects of ischemia/ reperfusion on intestinal muscle. Cell Tissue Res 343:411-419, 2011 20. Vollmar B, Menger MD: Intestinal ischemia/reperfusion: Microcirculatory pathology and functional consequences. Langenbecks Arch Surg 396:13-29, 2011 21. Sturm A, Dignass AU: Epithelial restitution and wound healing in inflammatory bowel disease. World J Gastroenterol 14:348-353, 2008 22. Iizuka M, Konno S: Wound healing of intestinal epithelial cells. World J Gastroenterol 17:2161-2171, 2011 23. Attard JA, Raval MJ, Martin GR, et al: The effects of systemic hypoxia on colon anastomotic healing: An animal model. Dis Colon Rectum 48: 1460-1470, 2005 24. Gookin JL, Rhoads JM, Argenzio RA: Inducible nitric oxide synthase mediates early epithelial repair of porcine ileum. Am J Physiol Gastrointest Liver Physiol 283:G157-G168, 2002 25. Kubes P, McCafferty DM: Nitric oxide and intestinal inflammation. Am J Med 109:150-158, 2000 26. Pagani A, Nai A, Corna G, et al: Low hepcidin accounts for the proinflammatory status associated with iron deficiency. Blood 118:736-746, 2011 27. World Health Organization. Iron Deficiency Anaemia. Assessment, Prevention, and Control. A guide for programme managers. WHO/ NHD/01.3. 2001, pp 1-114 28. Gomella TL, Cunnigham MD, Eyal FG, et al: Handbook of Neonatology. 4th ed. 1999, p 316 29. Kliegman RM, Walsh MC: Neonatal necrotizing enterocolitis: Pathogenesis, classification, and spectrum of disease. Curr Probl Pediatr 17: 213-288, 1987 30. Widness JA: Pathophysiology of anemia during the neonatal period, including anemia of prematurity. NeoReviews. 9:e520-e525, 2008 31. Salsbury DC: Anemia of prematurity. Neonatal Netw 20:13-20, 2001 32. Bishara N, Ohls RK: Current controversies in the management of the anemia of prematurity. Semin Perinatol 33:29-34, 2009 33. Terrenato L, Bertilaccio C, Spinelli P, et al: The switch from haemoglobin F to A: The time course of qualitative and quantitative variations of haemoglobins after birth. Br J Haematol 47:31-41, 1981 34. Schreml S, Szeimies RM, Prantl L, et al: Oxygen in acute and chronic wound healing. Br J Dermatol 163:257-268, 2010 35. Ohls RK: Core concepts: The biology of hemoglobin. NeoReviews. 12:e29-e38, 2011 36. Lachance C, Chessex P, Fouron JC, et al: Myocardial, erythropoietic and metabolic adaptations to anemia of prematurity. J Pediatr 1994; 125:278-282. 37. Reber KM, Nankervis CA, Nowicki PT: Newborn intestinal circulation. Physiology and pathophysiology. Clin Perinatol 29:23-29, 2002 38. Gutierrez G: Cellular energy metabolism during hypoxia. Crit Care Med 19:619-626, 1919 39. Caplan MS, Jilling T: New concepts in necrotizing enterocolitis. Curr Opin Pediatr 13:111-115, 2001 40. Caplan MS, Jilling T: The pathophysiology of necrotizing enterocolitis. NeoReviews 2:103-109, 2001 41. Jonsson K, Jensen JA, Goodson WH, et al: Tissue oxygenation, anemia and perfusion in relation to wound healing in surgical patients. Ann Surg 214:605-613, 1991 42. Haisjackl M, Luz G, Sparr H, et al: The effects of progressive anemia on jejunal mucosal and serosal tissue oxygenation in pigs. 8:538-544, 1997 43. van Bommel J, Siegemund M, Henny CP, et al: Critical hematocrit in
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54. Mainie P: Is there a role for erythropoietin in neonatal medicine? Early Hum Dev 84:525-553, 2008 55. Ohls RK, Ehrenkranz RA, Das A, et al. Neurodevelopmental outcome and growth at 18 to 22 months’ corrected age in extremely low birth weight infants treated with early erythropoietin and iron. Pediatrics, 2004 114:1287-1291 56. Jacobs A: Iron-containing enzymes in the buccal epithelium. Lancet 2:1331-1333, 1961 57. Dagg JH, Jackson JM, Curry B, et al: Cytochrome oxidase in latent iron deficiency (sideropenia). Br J Haematol 12:331-333, 1966 58. Chandra RK: Reduced bactericidal capacity of polymorphs in iron deficiency. Arch Dis Child 48:864-866, 1973 59. Macdougall LG, Anderson R, McNab GM, et al: The immune response in iron-deficient children: Impaired cellular defense mechanisms with altered humoral components. J Pediatr 86:833-843, 1975 60. Chandra RK, Saraya AK: Impaired immunocompetence associated with iron deficiency. J Pediatr 86:899-902, 1975 61. Srikantia SG, Prasad JS, Bhaskaram C, et al: Anaemia and immune response. Lancet 1:1307-1309, 1976 62. Yetgin S, Altay C, Ciliv G, et al: Myeloperoxidase activity and bactericidal function of PMN in iron deficiency. Acta Haematol 61:10-14, 1979 63. Dallman PR: Biochemical basis for the manifestations of iron deficiency. Annu Rev Nutr 6:12-40, 1986 64. Braekke K, Bechensteen AG, Halvorsen BL, et al: Oxidative stress markers and antioxidant status after oral iron supplementation to very low birth weight infants. J Pediatr 151:23-28, 2007 65. Miller SM, McPherson RJ, Juul SE: Iron sulfate supplementation decreases zinc protoporphyrin to heme ratio in premature infants. J Pediatr 148:44-48, 2006 66. Cheng C, Juu S: Iron balance in the neonate. NeoReviews. 12:e148, 2011
N
ecrotizing enterocolitis (NEC) is the most common surgical disease in very low-birth-weight (VLBW) infants, with an incidence of 7%-13%, and is associated with significant short- and long-term morbidity and mortality.1-3 NEC is thought to have a multifactorial etiology, with most of the associated factors ultimately affecting mesenteric blood flow and oxygen delivery, thereby potentially leading to hypoxemic/ischemic mucosal injury—the first step in the cascade of events leading to the onset of NEC.4,5 Recent publications suggest a consistent and significant temporal association between NEC, anemia, and red blood cell (RBC) transfusions.6-11 The objective of this review is to present our current understanding of the role of anemia of prematurity (AOP), whether causative or associative, in the onset of NEC by presenting the supporting evidence and strategies to minimize its impact.
*Division of Newborn Medicine, Department of Pediatrics, Baystate Children’s Hospital, The Western Campus of Tufts University School of Medicine, Springfield, MA. †Division of Newborn Medicine, Children’s Hospital, Boston, MA. Address reprint requests to Rachana Singh, MD, MS, Division of Newborn Medicine, Department of Pediatrics, Baystate Children’s Hospital, The Western Campus of Tufts University School of Medicine, 759 Chestnut Street, Springfield, MA 01199. E-mail: [email protected]
0146-0005/12/$-see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.semperi.2012.04.008
NEC Pathophysiology There has been extensive clinical and experimental research on understanding the pathophysiology of NEC, and significant attempts have been made to discover the one factor that initiates the cascade of events leading to the clinical manifestation of NEC.12 However, this has met with limited or minimal success in altering the incidence of NEC. The prevailing hypothesis is that NEC is a multifactorial disease process with an inverse relationship to gestational age at birth. All other factors at present are believed to be associative; therefore, the prediction of which infant will ultimately suffer from NEC is not possible. The pre- and postnatal factors associated with NEC are listed in Table 1. The current understanding of the pathophysiology of NEC is postulated to start with the preterm infant as a vulnerable host with an immature gut barrier, impaired defense mechanisms, goblet cells with scant mucus production, and an immune system that is compromised. The infant is then exposed to a relatively hostile environment of neonatal intensive care unit (NICU) where broad-spectrum antibiotic therapy diminishes the protective commensal bacteria in the gut flora and increases colonization with pathogenic bacteria. The infant may be fed nonhuman milk or hyperosmolar formulations and experience intermittent hypoxemic/ischemic events resulting in a breach in mucosal integrity. Once this initial insult occurs, inflammatory reaction with release of 277
278 Table 1 Factors Associated With Necrotizing Enterocolitis Prenatal Maternal preeclampsia Maternal cocaine use Maternal indocin for tocolysis Intrauterine growth restriction Perinatal asphyxia Postnatal RDS Hypothermia Hypotension/shock Hypoxia PDA Rapid onset and hyperosmolar enteral feedings Formula feedings Infections Hospitalization during an epidemic Colonization with necrogenic bacteria Anemia Polycythemia Thrombocytosis Packed red blood cell transfusions Exchange transfusions Congenital heart disease Congenital GI anomalies
cytokines such as platelet-activating factor and tumor necrosis factor causes further mucosal damage, which progressively leads to full-thickness necrosis and may ultimately result in intestinal perforation. Because mucosal integrity is paramount for host defense against intestinal pathogenic bacteria, it makes sense to focus on factors that can promote mucosal membrane integrity, growth, and repair as potentially preventative of NEC.
Mucosal Integrity The human intestinal mucosa is derived from the endoderm with a highly complex 3-layer structure: the mucous epithelium, lamina propria, and muscularis mucosa.13 Intestinal mucosa is the largest area providing surface immunity, and is therefore also the most vulnerable to injury. A breach in the mucosal layer leaves the highly vascularized submucosa susceptible to invasion by the gut bacterial flora leading to pneumatosis intestinalis, the radiologic hall mark of NEC, as well as allowing bacterial translocation to the bloodstream with the resultant clinical manifestations of inflammation and infection. The blood flow to the gastrointestinal (GI) tract is derived from 3 sources, the celiac trunk and the superior and inferior mesenteric arteries.14 The venous drainage from the GI tract is unique, as instead of returning to the heart directly, it first flows to the liver via the portal vein, allowing substances absorbed from the gut flow first to the hepatocytes where they can be detoxified if needed. GI blood flow also has a range of dynamic regulation, wherein even in the fasting state, the splanchnic circulation receives blood flow (25% of cardiac output) that is disproportionate to the mass of the
R. Singh, B.L. Shah, and I.D. Frantz, III organs perfused (5%).15 During stress, there is a major redistribution of the systemic blood circulation with a decrease in the mesenteric gut flow because of the strong activation of the sympathetic nervous system and release of norepinephine.16 During these events of hypoperfusion and resultant ischemia/ hypoxemia, which are not uncommon for preterm infants, there may be associated mucosal injury caused by reperfusion and resultant release of cytokine/inflammatory mediators.17-20 The intestinal mucosal epithelial healing is dependent on the precise balance of migration, proliferation, and differentiation of the epithelial cells adjacent to the injured area.21,22 Attard et al23 have demonstrated that systemic hypoxia directly translated into local tissue hypoxia, and intestinal anastomotic healing was impaired. Recent studies have also disclosed constitutive expression of inducible nitric oxide synthase (iNOS) by normal intestinal epithelia, and that selective inhibition of iNOS abolished increases in nitric oxide (NO) synthesis and villous reepithelialization after injury. These results demonstrate that iNOSderived NO is a key mediator of early villous reepithelialization after acute mucosal injury.24,25 Pagani et al26 have demonstrated that the lack of hepcidin is responsible for the high inflammatory response to lipopolysaccharides in the iron deficiency state. It is possible that in this proinflammatory setting hypoxemia/ischemia-induced injury may be the initial step that sets off the cascade of events leading to the onset of NEC in preterm infants who often have lower hematocrit (Hct) levels and may experience intermittent hypoxemic events.
Anemia of Prematurity Anemia is defined by World Health Organization as hemoglobin or Hct value 2 standard deviations below (⫺2 SD) the distribution mean in a normal population of same age and gender living at the same altitude.27 This, during the neonatal period (⬍28 days of life), translates to a central venous Hct ⬍39%.28 Based on the College of American Pathologists Neonatal Red Blood Cell Transfusion Guidelines, anemia maybe classified as mild if Hct is ⱖ35% but ⬍39%, moderate if Hct is ⱖ25% but ⬍35%, and severe if Hct is ⬍25%.29 AOP is a normocytic, normochromic hypoproliferative state, an exaggerated version of the normal physiological anemia seen in term infants. This physiological decline in Hct is due to reduced erythropoietin (Epo) production in the immediate postnatal phase. For the preterm infant, it results in an Hct nadir earlier than the 8-10 weeks for term infants. Additionally, because of phlebotomy losses, nutritional deficiencies, and immune-compromised status, a significantly lower Hct level is a frequent occurrence.30-32
Anemia of Prematurity and Oxygenation In term neonates, HbF decreases exponentially from birth to approximately 25 weeks postnatally by which time it is completely replaced by HbA. However, HbA shows a biphasic pattern: first, it decreases slightly from birth to 6 weeks and then increases exponentially till all hemoglobin is almost
Relationship of NEC with anemia completely made up of HbA. Two distinct phases of postnatal erythropoiesis have been identified. The first phase (from birth to 6 weeks) is characterized by a decrease in the total amount of hemoglobin produced. In the second phase, there is an alteration in the relative proportions of HbF and HbA being synthesized.33 This transition is a much slower and prolonged process for the preterm infant, ultimately affecting both oxygen-carrying capacity and oxygen delivery to the local tissues. Oxygen is a prerequisite for successful wound healing, such as seen with NEC, because of the increased demand for reparative processes such as cell proliferation, bacterial defense, angiogenesis, and collagen synthesis. Although the role of oxygen in wound healing is not yet completely understood, many experimental and clinical observations have demonstrated that wound healing is impaired during hypoxemic states.34 In addition to the role of oxygen, recent reports suggest potential mechanisms involving NO control of blood flow via hypoxic vasodilatation. These mechanisms may be important, especially in preterm infants, because immature vascular beds, such as the splanchnic system, are at risk for hypoxemic injury secondary to shunting of blood to the brain and heart.35 Lachance et al36 have shown that oxygenation is adequately maintained in symptom-free infants with AOP. However, symptoms of anemia in preterm infants may be difficult to detect given the many other issues at play. Anemia, by decreasing the oxygen-carrying capacity of the blood to levels less than the demands of growing tissues, may enhance anaerobic metabolism and the production of byproducts such as lactic acid.37 Gutierrez et al have shown that during tissue hypoxia, the deficit arising from unequal levels of cellular adenosine triphosphate (ATP) requirements and aerobic ATP production is partially satisfied by anaerobic sources of ATP, including glycolysis, the creatine kinase reaction, and the adenylate kinase reaction. However, these reactions may set in motion cellular mechanisms that ultimately may lead to cellular dysfunction and death.38 This could again be a plausible cause for the onset of NEC in the preterm infant.
Anemia, Perfusion, and Wound Healing In preterm infants, the basal intestinal vascular resistance changes rapidly from the fetal to early neonatal life, as this fetal dormant organ becomes functional for enteral nutrition, and anemia may impair this normal transition.39,40 Jonsson et al have demonstrated that wound healing is primarily dependent on tissue perfusion and oxygen delivery to the tissues, and the presence of anemia does not seem to impair wound healing as long as the tissue perfusion is maintained. However, if perfusion is impaired in an anemic patient, then wound healing will be exceptionally vulnerable because of unmet oxygen needs.41 An experimental study in a porcine model has also demonstrated redistribution of jejunal blood flow and an increase in the ratio of mucosal to
279 systemic Hct as the main mechanisms maintaining mucosal oxygen supply during progressive anemia.42 Intestinal microvascular oxygen partial pressure and organ oxygen consumption may be limited by a critical decrease in oxygen delivery and Hct at the same time. Beyond these critical points, not only shunting of oxygen from the microcirculation could be demonstrated, but also a significant correlation between intestinal microvascular oxygen partial pressure and organ oxygen consumption has been noted.43 The cell renewal rate may increase in acute anemia by venesection, whereas it was remarkably decreased in chronic iron deficiency.44
Anemia of Prematurity, Mucosal Injury, and NEC Recent interest in transfusion-related acute gut injury (TRAGI) and NEC prompted us to investigate the relationship between NEC, AOP, and packed red blood cell (PRBC) transfusion. Although there are no uniform practice guidelines for RBC transfusion in the NICUs because of an increasing concern for transfusion associated-infections (HIV, hepatitis B and C, etc.) and gut injury, the recent trend has been toward restrictive transfusion practices.7-11,45 However, it is not possible to consider NEC and TRAGI without considering the role of AOP, as Hct and transfusion cannot be truly independent of each other. Our retrospective case-control study aimed to determine association of anemia and RBC transfusions with NEC in preterm infants. We identified 111 preterm infants with NEC ⱖstage 2a and compared them with 222 matched controls. Data from a comprehensive list of 28 clinical variables, including Hct and RBC transfusions, were recorded. Propensity scores and multivariate logistic regression models were then created to examine effects on the risk of NEC. While controlling for other factors, we reported that lower Hct level was associated with increased odds of NEC (OR ⫽ 1.10, P ⫽ 0.01). Additionally, RBC transfusions had a temporal relationship with NEC onset. Transfusion within 24 hours (OR ⫽ 7.60, P ⫽ 0.001) and 48 hours (OR ⫽ 5.55, P ⫽ 0.001) has a higher odds of developing NEC, but this association lost significance by 96 hours (OR ⫽ 2.13, P ⫽ 0.07) posttransfusion. Thus, we demonstrated that both Hct and RBC transfusions are associated with increased risk of NEC. Lower Hct was associated with higher predicted probability of NEC (Fig. 1).6 Possible mechanisms discussed earlier could explain the increase in risk of development of NEC with AOP in a preterm infant. Mally et al9, in their study evaluating the risk of NEC with PRBC transfusions, also noted that the group of infants with RBC-associated NEC had lower Hct. Blau et al in their cohort study sought to determine an association of NEC within ⬍48 hours of a PRBC transfusion. Of their cohort of 256 VLBW infants, there were 36 NEC cases, and 25% cases were associated with PRBC (n ⫽ 9). PRBC-associated cases had lower birth weight, Hct, and rapid onset of signs (⬍5 hours). Interestingly, in their study, current weight at the onset of NEC
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Figure 1 Relationship of hematocrit with probability of NEC. (Color version of figure is available online.)
did not differ; however, the more preterm the infant, the later the onset of NEC creating a centering of occurrence at a median of 31 weeks postconceptual age.46 Although Paul et al47 demonstrated correlation between transfusions and NEC, they did not control for the Hct in their 3 study groups, although the transfused groups were anemic with Hct ⬍30. In a survey of variation in transfusion practices among NICUs, we noted that NICUs with the lowest rate of transfusion had the lowest rate of NEC despite similar Hcts, supporting a role for transfusion in the pathogenesis of some NEC cases.45 These studies support our findings that anemia and its impact on the cellular mechanisms of tissue injury and repair potentially initiate the process for the development of NEC in preterm infants. El-Dib et al48 have speculated that although anemia by itself may not directly cause NEC, severe anemia may impair intestinal oxygen delivery, and thus physiologically justified increased demand of oxygen to intestinal mucosa at the time of feeding may not be met in anemic infants. Based on these data, we speculate that in anemic preterm infants, the ischemic injury sustained by the mucosal layer is aggravated by factors associated with RBC transfusions, and strategies to maintain a critical Hct may help decrease the incidence of NEC. This speculation gains further credence from the observation that Epo has been shown to be protective against NEC.49-51 However, there have been some disagreements about routine Epo therapy because of concerns for its efficacy in minimizing exposure to multiple donors, decreasing the number of transfusions, and increased incidence of retinopathy of prematurity.52-54 By contrast, Ohls et al in their National Institute of Child Health and Human Development study on long-term outcomes noted that early Epo and iron therapy did not affect anthropometric measurements or neurodevelopmental outcomes. Incidentally in their placebo group, there was a trend toward an increased incidence of
NEC with a lower Hct (30.3% vs 35%, 9.8% vs 5.6%).55 However, the number of cases was small. In our study, we also found that iron supplementation, another therapeutic intervention for treatment of anemia, was associated with lower risk for NEC.6 It is known that mild iron deficiency anemia may lead to decreased activity of cytochrome oxidase in buccal mucosa56 and the mucosal cells lining the intestinal villi.57 Further quantitative evidence of tissue iron depletion comes from studies of neutrophils and lymphocytes wherein neutrophils may have a decreased capacity to kill ingested bacteria, presumably because the oxidative burst is iron dependent.58-63 The iron-deficient state impairs GI functions, growth, suppressed immune functions with poor resistance to infection, and overall higher infant mortality.27 In our study, iron supplementation was associated with a lower risk of NEC, consistent with the hypothesis that prevention or treatment of anemia may be important in preventing or reducing the risk of NEC. In the past, there have been concerns about iron excess and oxidative injury in VLBW infant with limited antioxidant mechanisms. However, studies indicate that iron does not induce oxidative stress, as measured by isoprostanes and antioxidant status, when provided to stable growing low-birth-weight infants at doses ranging from 2 to 12 mg/kg/d or at a twicedaily dose of 9 mg/d.64-66
Future Directions Based on current evidence, there seems to be interplay between the intermittent hypoxic/ischemic events endured by a preterm infant during NICU stay, with mucosal integrity impairment potentially augmented by iron deficiency, AOP, and RBC transfusions. Future prospective studies are needed to address multiple questions regarding AOP, RBC transfusions, and therapies aimed toward them. For example, is there a critical Hct that has a negative influence on GI phys-
Relationship of NEC with anemia iology and its functioning, and can this be identified clinically? If there is a critical threshold, then should one avoid reaching it through judicious transfusion practices, use of Epo, or early enteral iron supplementation? If RBC transfusions worsen the injury incurred during the chronic anemic state, should transfusions be administered earlier before the injury has occurred? Which are the factors associated with the stored RBC themselves that may lead to TRAGI, such as age, preservatives, volume transfused, rate of administration, or unknown oxidants present in the stored blood that the preterm gut is unable to handle? Because the natural transition to adult HbA does not occur till 46 weeks’ corrected gestational age, is there harm in transfusing RBC with predominant HbA? Should feedings be held peritransfusion, and if so, when should one start withholding and for how long? Finally, at what gestational or postconceptional age is the infant no longer at risk of NEC? For example, is there a physiological or anatomical event analogous to retinal vascularization and risk for retinopathy of prematurity that marks the end point for NEC risk? These and many more questions remain unanswered and should be addressed in the future multicenter studies to identify clinical practices, which may help decrease the incidence of NEC.
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