Supplemental Insulin-Like Growth Factor-1 and Necrotizing Enterocolitis in Preterm Pigs

Abstract

Background: Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP-3) is currently tested as a therapy in preterm infants but possible effects on the gut, including necrotizing enterocolitis (NEC), have not been tested. The aim of this study was to evaluate if rhIGF-1/BP-3 supplementation in the first days after birth negatively affects clinical variables like growth, physical activity, blood chemistry and hematology and gut maturation (e.g., intestinal permeability, morphology, enzyme activities, cytokine levels, enterocyte proliferation, NEC lesions), using NEC-sensitive preterm pigs as a model for preterm infants. Methods: Preterm pigs were given twice daily subcutaneous injections of rhIGF-1/BP-3 or vehicle. Blood was collected for IGF-1 measurements and gut tissue for NEC evaluation and biochemical analyses on day 5. Results: Baseline circulating IGF-1 levels were low in preterm pigs compared with near-term pigs reared by their mother (<20 vs. 70 ng/ml). Injection with rhIGF-1/BP-3 resulted in increased plasma IGF-1 levels for up to 6 h after injection (>40 ng/mL). rhIGF-1/BP-3 treatment reduced the incidence of severe NEC lesions (7/24 vs.16/24, p = 0.01) and overall NEC severity (1.8 ± 0.2 vs. 2.6 ± 0.3, p < 0.05, with most lesions occurring in colon). In the small intestine, villi length (405 ± 25 vs. 345 ± 33 μm) and activities of the brush border peptidases aminopeptidase N and dipeptidylpeptidase IV were increased in rhIGF-1/BP-3 treated pigs, relative to control pigs (+31-44%, both p < 0.05). The treatment had no effects on body weight, blood chemistry or hematology, except for an increase in blood leucocyte and neutrophil counts (p < 0.05, i.e., reduced neonatal neutropenia). Likewise, rhIGF-1/BP-3 treatment did not affect intestinal tissue cytokine levels (IL-1β, IL-6, IL-8, TNFα,), enterocyte proliferation, goblet cell density, permeability or bacterial translocation to the bone marrow. Conclusion: Supplemental rhIGF-1/BP-3 did not negatively affect any of the measured variables of clinical status or gut maturation in preterm pigs. Longer-term safety and efficacy of exogenous rhIGF-1/BP-3 to support maturation of the gut and other critical organs in preterm newborns remain to be investigated in both pigs and infants.

Keywords: IGF-1; fetus; growth restriction; gut; infant; intestine; newborn; preterm birth.

Figure 1

Experimental setup. Schematic drawing showing the experimental setup used to evaluate basal IGF-1 levels in piglets (Experiment 1), pharmacokinetics of IGF-1(Experiment 2) and the effects of rhIGF-1/BP-3 supplementation (Experiment 3). IA+ and IA-, intra-arterial administration with (+) or without (–) maternal plasma immunization; SC, subcutaneous; PO, peroral.

Figure 2

Circulating IGF-1 levels and pharmacokinetics (mean ± SEM). (A) Basal IGF-1 plasma levels in 1–19 day-old preterm pigs reared in incubators (n = 7–107), near-term pigs reared in incubators (n = 14), and near-term pigs reared with their mother (n = 12). The 19 day-old preterm pigs were subdivided into pigs with intrauterine growth restriction (IUGR, n = 28) or non-IUGR (n = 79), and extrauterine growth restriction (EUGR, n = 26) or non-EUGR (n = 80), with growth restriction defined as the pigs with body weight within the lowest 25% of the growth percentiles. Data are presented as box plots (median and 25/75 percentiles) with whiskers = min to max. The interrupted horizontal line indicates the lower limit of quantitation (20 ng/ml) and the dotted horizontal line indicates the limit of detection (10 ng/ml) of the assay. (B) Serum IGF-1 levels in rhIGF-1/BP-3 and control preterm pigs after a single subcutaneous injection of rhIGF-1/BP-3 or vehicle on day 4 (n = 18–23) (mean ± SEM). (C) Serum IGF-1 levels at euthanasia 0–10 h after the last subcutaneous injections of rhIGF-1/BP-3 or vehicle on day 5 (n = 1–2 for samples taken 1-10 h after last dosing). t = 0 represents samples taken 15 h after last dosing (n = 16–19). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Intestinal lesions and structure. (A) Incidence of severe NEC (defined as score ≥4 in at least one gut region) in rhIGF-1/BP-3 and control preterm pigs (n = 24). (B) NEC severity score across gut regions in rhIGF-1/BP-3 and control preterm pigs (n = 24). (C) Individual NEC scores in stomach, small intestine regions (highest score of the proximal, middle and distal section) and colon regions (highest score of the colon and cecum). (D) Villus length, crypt depth and Ki67 stained area relative to total nuclei area in tunica mucosa in mid small intestine (n = 23). Values are mean±SEM. (E) Immunohistochemically staining for Ki67 with hematoxylin counterstain in a representative middle small intestine section from a preterm pig treated with rhIGF-1/BP-3 and a control preterm pig. Pictures are 10x. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Intestinal functions (means ± SEM). (A) Concentration of lactulose/mannitol (Lac/Man) ratio to asses intestinal permeability in the urine on day 5 (n = 18–22), and (B) bacterial load in bone marrow (colony forming units, CFU) to assess bacterial translocation on day 5 (n = 23–24). (C) Brush border enzyme activities in mid small intestine (n = 24, ApN, aminopeptidase N; ApA, aminopeptidase A; DPPIV, dipeptidylpeptidase). Pro-inflammatory cytokines in (D) middle small intestine and (E) colon (n = 23–24). *p < 0.05, **p < 0.01, ***p < 0.001.