Karen D. Crissinger

Mucosal injury induced by ischemia and reperfusion in the piglet intestine: Influences of age and feeding

The pathogenesis of neonatal necrotizing enterocolitis is unknown, but enteral alimentation, infectious agents, and mesenteric ischemia have been frequently invoked as primary initiators of the disease. To define the vulnerability of the intestinal mucosa to ischemia and reperfusion in the developing piglet, we evaluated changes in mucosal permeability using plasma-to-lumen clearance of chromium 51-labeled ethylenediaminetetraacetic acid in the ileum of anesthetized 1-day-, 3-day-, 2-wk-, and 1-mo-old piglets as a function of (a) duration of intestinal ischemia (20, 40, or 60 min of total superior mesenteric artery occlusion), (b) feeding status (fasted or nursed), and (c) composition of luminal perfusate (balanced salt solution vs. predigested cow milk-based formula). Baseline chromium 51-labeled ethylenediaminetetraacetic acid clearance was not significantly altered by ischemia, irrespective of duration, or feeding in all age groups. However, clearances were significantly elevated during reperfusion after 1 h of total intestinal ischemia in all age groups, whether fasted or fed. Reperfusion-induced increases in clearance did not differ among age groups when the bowel lumen was perfused with a balanced salt solution. However, luminal perfusion with formula resulted in higher clearances in 1-day-old piglets compared with all older animals. Thus, the neonatal intestine appears to be more vulnerable to mucosal injury induced by ischemia and reperfusion in the presence of formula than the intestine of older animals.

An animal model of necrotizing enterocolitis induced by infant formula and ischemia in developing piglets

BACKGROUND/AIMS The lipid component of piglet formula (0.5% fat) causes increased mucosal permeability in 1-day-old piglets after ischemia/reperfusion. The present study examined if luminal exposure to infant formulas (3.5% fat) and ischemia/reperfusion result in an animal model of necrotizing enterocolitis and if injury is dependent on the formula fat composition. METHODS Plasma-to-lumen clearance of 51Cr-ethylenediamine-tetraacetic acid was measured, and morphology was evaluated during luminal perfusion with preterm, term, and delipidated preterm cow milk-based infant formulas before and after ischemia/reperfusion in 1-day-old and 1-month-old piglet jejunoileum. In a separate set of experiments, a 1-2-cm segment of ileum was exteriorized and opened to expose the mucosal surface, and the villi were superfused with the above formulas (no ischemia). RESULTS Before ischemia, clearances were markedly higher for intestinal loops perfused with preterm formula than for loops perfused with term and delipidated formulas in 1-day-old animals. After ischemia, clearances in loops perfused with preterm formula were significantly greater and grossly hemorrhagic and histologically necrotic compared with loops perfused with delipidated formula (minimal injury). Superfusion with preterm formula caused diffuse hyperemia and hemorrhage into intestinal villi. CONCLUSIONS Luminal perfusion of 1-day-old piglet jejunoileum with predigested and bile acid-solubilized preterm infant formula, in combination with ischemia/reperfusion, produces an animal model of necrotizing enterocolitis, but only if the lipid fraction of the formula is present.

Developmental biology of oxidant-producing enzymes and antioxidants in the piglet intestine.

ABSTRACT: The pathogenesis of neonatal necrotizing enterocolitis is unknown, but a possible role for reactive oxygen metabolites has been postulated. We evaluated whether developmental differences exist in the levels of 1) the free radical-generating enzyme xanthine oxidase, 2) granulocyte peroxidase, an index of the resident granulocyte population, 3) free radical-scavenging enzymes (superoxide dismutase, catalase, and glutathione peroxidase), and 4) reduced glutathione, an endogenous antioxidant, in the ileal and colonic mucosa of 1-d-old, 3-d-old, 2-wk-old, and 1-mo-old piglets. We found no xanthine dehydrogenase/ oxidase activity in 1-d to 1-mo-old piglets. Mucosal granulocyte peroxidase activity was higher in older animals, indicating that there was an age-dependent infiltration of granulocytes (eosinophils, neutrophils) in the distal bowel. The peroxidase activity per circulating granulocyte, however, did not vary with age. Superoxide dismutase activity was significantly higher in 1-d-old piglets than in all older age groups; glutathione peroxidase activity was significantly lower in 1-d-old animals than that of older age groups. There was no detectable catalase activity in the mucosa when tissue was corrected for catalase activity of blood. Finally, ileal GSH levels were significantly lower in 1-d-old than in 2-wk-old and 1-mo-old animals, whereas colonic reduced glutathione activity did not differ among age groups. In conclusion, the distal bowel of the neonatal piglet appears to have a limited capacity to generate oxidants via xanthine oxidase and resident granulocytes. However, the neonatal piglet intestine has a lower capacity to detoxify hydrogen peroxide than that of older animals.

Characterization of Intestinal Collateral Blood Flow in the Developing Piglet

ABSTRACT: Interest in the pathogenesis of neonatal necrotizing enterocolitis has prompted study of the intestinal circulation in developing animals. It is conceivable that poorly developed collateral channels may predispose the neonatal intestine to ischemic insults. We therefore characterized intestinal collateral blood flow in anesthetized and ventilated 1-day and 1-month-old piglets. Intestinal blood flow was measured with radioactive microspheres (15 μm diameter) before and after either 1) total occlusion of the superior mesenteric artery (SMA) or 2) occlusion of a distal (jejunoileal) branch of the SNIA. After total SMA occlusion in 1-day and 1-month-old piglets, perfusion of the intestine via collaterals from the celiac and inferior mesenteric arteries was not evident. Jejunal, ileal, and colonic (except rectal) blood flows fell to zero 30 min after ligation of the SMA. Ligation of a distal branch of the SMA in 1-month-old animals significantly reduced total wall (by 25%) and mucosal/submucosal (by 25%) blood flows in the occluded segment. Similar experiments in 1-day-old piglets produced significantly greater reduction in total (70%) and mucosa/subumeosa (70%) blood flows. Muscle/serosa blood flows in both groups were not significantly different from control values. In conclusion, collateral perfusion of the intestine via the celiac and inferior mesenteric arteries is insignificant during acute SMA occlusion in the developing piglet. Although there is significant collateral blood flow within the SMA vascular network, perfusion between adjacent gut segments is less effective in preventing intestinal ischemia after occlusion of a branch of the SMA in neonates than in 1-month-old piglets.

The role of lipids in ischemia/reperfusion-induced changes in mucosal permeability in developing piglets

This study determined which nutrient component of formula may be responsible for changes in ischemia/reperfusion-induced mucosal permeability, as quantitated by the plasma-to-lumen clearance of 51Cr-ethylenediaminetetraacetic acid, in newborn piglets. Loops of jejunoileum in 1-day-old and 1-month-old piglets were perfused with predigested and bile acid-solubilized solutions of formula, lipid, protein, carbohydrate, delipidated formula, or fatty acid during 1 hour each of control, ischemia, and reperfusion. Luminal perfusion with formula or lipid led to significantly greater increases in mucosal permeability during reperfusion in newborn intestine than did carbohydrate or protein, whereas mucosal permeability in older animals was not different among solutions. Removal of all lipids from the formula abolished the increased mucosal permeability associated with reperfusion in newborn animals. Perfusion with oleate, a monounsaturated dietary fatty acid, led to still greater increases in reperfusion-associated permeability in newborn but not older intestine. The oleate and lipid perfusions also caused significantly increased mucosal permeability in the absence of ischemia. Thus, it appears that a lipid component of formula, probably a fatty acid, is responsible for the increase in mucosal permeability induced by ischemia/reperfusion in newborn intestine and also leads to increased mucosal permeability in the absence of ischemia/reperfusion. Investigation of the mechanism of these lipid-associated changes in mucosal permeability may provide a rationale for dietary modifications that may decrease the risk of mucosal injury during feeding and ischemic stress in immature intestine.

Fatty Acid-Induced Injury in Developing Piglet Intestine: Effect of Degree of Saturation and Carbon Chain Length

ABSTRACT: Luminal perfusion with the long-chain fatty acid (LCFA) oleate in concentrations similar to that found in premature infant formula produces a dose- and age-dependent mucosal injury in developing intestine. To investigate whether this lipid-induced phenomenon is a function of the degree of saturation and/or chain length of the fatty acid, 51Cr-EDTA plasma-to-lumen clearance was measured in jejunum and ileum of 1-d-, 3-d-, 2-wk-, and 1-mo-old piglets after perfusion with 5-mM solutions of different medium-chain saturated fatty acids and saturated and un-saturated LCFA. Mono- and polyunsaturated LCFA produced significant increases in jejunal permeability. In general, this effect was greater in piglets ≤2 wk old compared with 1-mo-old animals, but no differences were observed among the unsaturated LCFA within an age group. In contrast, the alterations in mucosal permeability induced by medium-chain fatty acids were overall more attenuated than those induced by LCFA. Our results suggest that developing intestine is vulnerable to the injurious effect of dietary fatty acids and that the lipid-induced changes in mucosal permeability appear to be a function of the fatty acid chain length. The degree of saturation of the fatty acid does not alter its cytotoxic effects.

Intestinal Blood Flow and Oxygen Consumption: Responses to Hemorrhage in the Developing Piglet

ABSTRACT.: Age-related differences in the intestinal hemodynamic and oxygenation responses to arterial hemorrhage were studied in anesthetized and ventilated 1-d, 3-d, 1-wk, and 2-wk-old piglets. Steady-state values of superior mesenteric blood flow, venous pressure, and arteriovenous oxygen difference were obtained before and after 5 and 10 mL/kg arterial hemorrhage. With 5 mL/kg hemorrhage, intestinal blood flow fell significantly below baseline values, but oxygen extraction increased to maintain oxygen uptake at control levels in all age groups. In contrast to 2-wk-old piglets, the intestine of 1-d, 3-d, and 1-wk-old animals could not compensate for the greater reduction in blood flow produced by 10 mL/kg hemorrhage, resulting in a significant reduction in oxygen uptake. Thus, the intestine of developing piglets up to 1 wk of age appears to be at greater risk for tissue hypoxia induced by arterial hemorrhage than that of older animals.

Developing Intestine Is Injured during Absorption of Oleic Acid but Not Its Ethyl Ester

Although lipids are essential nutrients in the mammalian diet, we have shown that fatty acids are injurious to epithelial cells of developing piglet intestine during luminal perfusion. Furthermore, the intestine of young animals sustains greater injury than that of older piglets. In an effort to understand the mechanism for this developmental injury, we investigated whether changes in the chemical configuration of oleic acid would alter this damage. Mucosal permeability, as quantitated by the plasma-to-lumen clearance of 51chromium EDTA, was evaluated during luminal perfusion with oleic acid as compared with its ethyl (ethyl oleate) and glyceryl (glycerol-1-mono-oleate) esters, solubilized with taurocholic acid, in jejunum of 1-d-, 3-d-, 2-wk-, and 1-mo-old piglets. 51Chromium EDTA clearance increased significantly during oleic acid and glycerol-1-mono-oleate perfusion, but did not increase during perfusion with ethyl oleate or saline. This result was not secondary to failure of absorption of ethyl oleate, as [14C]oleic acid and ethyl [1-14C]oleate were absorbed to a similar extent. Furthermore, developing intestine was able to remove the ethyl group and then re-esterify the fatty acid to form triacyglycerol. These studies indicate that oleic acid-induced mucosal injury can be abolished when the carboxylic group of the fatty acid is esterified with an ethyl, but not a glycerol, group. Since the ethyl ester is also absorbed and metabolized similarly to the free fatty acid, this may provide a means of supplying long-chain fatty acids to developing intestine without causing mucosal damage.

Intestinal microcirculation in the neonate

The hypermetabolic neonatal intestine requires an appropriate supply of energy to meet its needs. Adequate regulatory mechanisms for intestinal blood flow must be present to cope with a high oxygen demand. In the developing intestine, intrinsic hemodynamic control matures as a function of postnatal age, and the newborn appears to balance this vascular immaturity by an enhanced ability to extract oxygen from the blood. When this mechanism is maximized and an additional stress is superimposed, the neonatal intestine may be at a higher risk of ischemia and injury than older intestine. Furthermore, sympathetic vasoconstrictor control predominates at birth, and the newborn is less capable of vasodilatation. This extrinsic control provides and additional ischemic risk during conditions involving sympathetic discharge. An understanding of circulatory hemodynamics in the developing intestine may allow elucidation of the role played by ischemia in mucosal injury in the newborn.

Intestinal Oxygenation and Mucosal Permeability with Luminal Mother's Milk in Developing Piglets

We have previously observed a developmental difference in mucosal permeability (i.e. the younger the animal, the greater the increase in permeability) after exposure to luminal nutrients derived from cows milk-based infant formulas. There has been tremendous speculation and some clinical evidence that mothers milk may be protective against mucosal injury in developing intestine. In this study, we hypothesized that instillation of sows milk into the intestinal lumen of developing piglets would causeno differences in either intestinal metabolic demand (oxygen uptake) or mucosal permeability among age groups. Intestinal blood flow (total and fractionated), arteriovenous oxygen content difference, venous pressure, and capillary pressure were measured, and vascular resistance and oxygen uptake were calculated, after 30 min of intraluminal instillation of predigested and solubilized sows milk in 1-d-old, 3-d-old, 2-wk-old, and 1-mo-old piglet jejunoileum. In a separate group of animals, plasma-to-lumen clearance of chromium-51 EDTA was evaluated during luminal perfusion with digested and solubilized sows milk in 1-d-old, 3-d-old, and 1-mo-old piglet jejunoileum. Intestinal oxygen uptake was similar among age groups of developing piglets, but EDTA clearance was significantly higher for intestinal segments perfused with sows milk in 1-d-old, compared with older, animals. Thus, luminal perfusion with predigested and bile acid-solubilized sows milk in 1-d-old piglet jejunoileum, compared with perfusion in older piglets, causes increased mucosal permeability in 1-d-old intestine, but this increased permeability is not due to increased intestinal oxygen uptake (i.e. increased metabolic demand).