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Laboratory of Veterinary Anatomy and Embryology, Department of Veterinary Medicine, University of Antwerp, 2610 Wilrijk, Belgium and * Department of Human Nutrition, Royal Veterinary and Agricultural University, Frederiksberg DK-1958, Denmark
3To whom correspondence should be addressed. E-mail: chris.vanginneken{at}ua.ac.be.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • preterm • nitric oxide • enteral nutrition • parenteral nutrition • stereology
The first intake of enteral diets after birth is associated with remarkable intestinal growth in several mammalian species. The responses include an increase in intestinal mass, DNA, and mucosa (+50–100%) within 1–2 d of birth, as indicated in studies of pigs (1–3). Conversely, total parenteral nutrition (TPN)4 to piglets, even for just a few days, is associated with marked reductions in mucosal mass and villous heights (4–7). A degree of mucosal atrophy also occurs if TPN is instituted immediately after birth, before any access to enteral food (8). Although little is known concerning humans, it is likely that the striking intestinotrophic responses of the first enteral food are present also in infants (9). Thus, the neonatal trophic response to enteral food seems to play a key role in the transition from parenteral supply of elemental nutrients before birth to enteral intake of more complex diets after birth.
If birth occurs prematurely, a period of continued parenteral nutrition may be required to avoid digestive complications, potentially leading to life-threatening inflammatory responses, such as those seen in necrotizing enterocolitis (NEC). Nevertheless, enteral food is believed to improve maturation of the intestinal structure and function, even in preterm infants. Observations in term pigs and dogs showed that a normal trophic response can be observed at <50% of the full enteral intake (10,11), thus supporting the concept of "minimal enteral nutrition" in clinical pediatric nutrition. Nevertheless, it remains unclear whether the gut response to parenteral and enteral nutrition is similar in preterm and term neonates. An earlier study suggested that preterm pigs are less sensitive to TPN-induced mucosal atrophy, although they responded to sow’s milk given just after birth with the usual large increase in mucosal mass (8). It is likely that the highly hydrolyzed diets often used for preterm infants would be less trophic; they are also inferior to intact diets in mediating nitrogen retention (12).
Nitric oxide (NO), a gaseous free radical, is a key messenger regulating gut blood flow, motility, and inflammation. Under noninflammatory conditions, NO is synthesized predominantly by constitutive NOS-isoforms: nitric oxide synthase isozyme-3 (NOS-3) and NOS-1 (13). However, a role in inflammation is supported by studies showing that exogenously increased constitutive NOS activity may protect against histopathological NEC-lesions, which are induced by formula feeding in preterm piglets (14). In addition, an adaptive response of NOS-1 expression to feed intake (transition from parenteral to enteral feeding and from liquid to solid food intake) is indicated by the momentarily decreased number of enteric neurons expressing NOS-1 in term piglets immediately after birth (inner submucous plexus) (15) and after weaning (outer submucous plexus) (16). However, it is not known how TPN and enteral feeding affect the presence of NOS-1 in the preterm intestine.
Based on the hypothesis that the preterm pig intestine has an altered sensitivity to TPN- and oral food-induced intestinal growth, the aims of the present study were to investigate the trophic response of the preterm intestine 1) to short-term TPN and oral infusion of elemental nutrients, and 2) to different oral milk diets after a short period of TPN. We sought to quantitatively describe the morphology of the small intestine and relate this to nitrergic neurons using stereological methods to assess volume densities, surface density, numerical density, and number of nitrergic neurons. In addition, possible relations between intestinal lesions, NOS-1, and intestinal wall variables were investigated to elucidate how the preterm intestine responds to enteral diets.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Expt. 1. Nineteen piglets from the 6 litters were randomly divided into 3 groups. One group of piglets was killed within 12 h of birth and received no nutrition (NB, n = 5). Two other groups of pigs were killed for tissue collection immediately after completion of a 3-d period during which pigs were fed the elemental TPN fluid either parenterally (TPN, n = 7) or enterally (TENT, n = 7).
The parenteral nutrient solution used was prepared aseptically and based on the infusion product Nutriflex Lipid plus (Braun). The composition was based on that used previously for term and preterm pigs (8). To achieve this composition, part of the glucose and lipid was withdrawn and substituted with amino acids (Vamin 18 g N/L electrolyte free; Fresenius Kabi) and sterile water. The nutrient composition and concentrations of macrominerals per liter were as follows: total energy, 3123 kJ; glucose, 72 g; lipids, 31 g; nitrogen, 6.4 g, amino acids, 45 g; sodium, 0.88 g; potassium, 1.03 g; calcium, 0.13 g; and phosphorus, 0.37 g.
The TPN solution was infused continuously for 3 d via a catheter inserted into an artery of the transected umbilical cord (TPN group), or via an esophageal catheter (TENT group), using automatic infusion pumps (Infusomat Secura, Braun). The nutritional goal was to provide the pigs with sufficient energy and protein to allow for a slightly positive energy balance. The infusion rate was 4 mL/(kg · h) during the first 24 h postpartum, gradually increasing to 6–8 mL/(kg · h) over the next 2 d. In this way, the pigs received an average of 450 kJ/(kg · d), 6.5 g amino acids/(kg · d), and a fluid intake of 160 mL/(kg · d) during the 3-d period.
Expt. 2. Another 3 groups of pigs were administered TPN for 3 d (similar to the group in Expt. 1) followed by the oral feeding of different milk diets (15 mL/(kg · 3 h) for 32–42 h. The 3 diets were sow’s colostrum (TPN-SOW, n = 9), cow’s colostrum (TPN-COW, n = 8), and an infant milk formula (TPN-FORM, n = 11) collected and prepared as described previously (3).
Tissue collection. For all pigs, the gastrointestinal tract was evaluated macroscopically for pathological changes, indicative of inflammation or necrosis, and digital pictures were taken of the entire gastrointestinal tract from all pigs. The small intestine from the pyloric sphincter to the ileocecal junction was rapidly excised by cutting along the mesenteric border, and its length was measured in a relaxed state. The small intestine was carefully emptied of its contents and then placed on an ice-cold metal plate and divided into 3 segments of equal length, designated proximal, middle, and distal small intestine. The 3 segments were weighed; tissue samples of 2–3 cm from the middle of each segment were removed and fixed (see below). A 10-cm segment was also taken from each intestinal region to measure intestinal dimensions. For this segment, the proportion of mucosa was determined on a dry matter basis after drying both the mucosa and the muscularis layers at 50°C for 72 h.
Tissue preparation. After rinsing, the small intestinal segments were fixed for 2 h in 4% freshly prepared paraformaldehyde (0.1 mol/L), pH 7.4. Then the fixative was washed out with 0.01 mol/L PBS solution for 24 h and stored in PBS containing 0.01% NaN3 at 4°C.
Whole-mount preparation and NADPH-diaphorase enzyme histochemistry. From each small intestinal segment, two 2–3 cm tissue slabs were randomly selected. These slabs were processed as whole-mount preparations and subjected to NADPH-diaphorase histochemistry as described in detail previously (17). Whole-mount preparations were mounted on microscope slides and scanned to count the NOS-1–containing neurons (see below).
Paraffin sections.
At random positions, 1 biopsy (3–5 mm diameter) was taken from each small intestinal segment and processed routinely for paraffin embedding. From these tissue blocks, 5-µm paraffin vertical sections were made. The first section was collected randomly from a set of 3 paraffin sections. Subsequent sections were collected a systematic distance of 3 sections apart. This resulted in 
30 sections per small intestinal segment per pig. The sections were stained with hematoxylin-eosin (HE) and were used for microscopically scoring the gastrointestinal lesions and estimating the various morphometric variables (volume densities, surface density, villous height and width, crypt depth). Mucosal sections were scored according to their appearance and the presence of atrophic or inflammatory morphological lesions (18): 0: normal morphology; 1: slight tunica mucosal and lamina propria separation; 2: increased tela submucosa and lamina propria separation with edema in the tela submucosa; 3: severe separation of the tela submucosa and lamina propria, blood congestion, and regional loss of villi; 4: blood congestion, transmural necrosis, and generalized loss of villi.
Quantitative morphological analyses. An Olympus BX50 microscope, equipped with a Sony CCD camera connected to a computer system running the software program Cast-grid (Olympus), was used for the quantitative analyses. The optimal density of the stereological grid (number of points, cycloids, unbiased counting frames), the number of sections, and the number of sample fields were estimated as described previously (19).
Estimation of the volume densities (Vv) of the various layers of the intestinal wall was done as described previously (20). In short, the grid points hitting the reference space [P(ref)] (the intestinal wall) and the number of points hitting the space of interest [P(Y)] (tunica mucosa, tela submucosa, tunica muscularis) were counted. The following equation yielded the volume densities of the various constituents of the intestinal wall: Vv(Y,ref) = 
P(Y)/P(ref).
Estimation of the surface density of the mucosal layer (Sv) (surface of the intestinal epithelium divided by the volume of the intestinal wall) was performed as explained previously (20). SV is used to estimate the surface that is available to the intestinal wall for absorption and secretion processes. The following equation yielded the surface density: Sv(Y,ref) = (2 · I)/(l/p · P). In the equation l/p represented the length of the test line per grid point, P is the number of points hitting the intestinal wall and I is the number of intersections of the grid cycloids with the epithelial cell layer.
Using the unbiased counting frame and counting rule developed by Gundersen (21), the whole-mount preparations were scanned and the numerical density (QA) of both the submucous (inner and outer) and myenteric NOS-1–containing neurons was estimated as described earlier (15,16). Multiplying the numerical density for a given intestinal segment and plexus by the serosal surface area of the segment, yielded the number of nitrergic neurons (N) in that plexus and segment.
Statistical evaluation. The morphological data were analyzed by ANOVA with treatment and intestinal region (proximal, middle, distal) as fixed variables, litter as a covariate, and pig as the random variable, using SAS (SAS/STAT version 8.1, SAS Institute). Morphology values from the NB, TENT, and TPN groups were compared to evaluate the effect of 3 d of parenteral and enteral infusion with TPN fluid (Expt. 1). The values in the TPN and TENT groups were expressed relative to values in the NB (=1.0), to indicate the magnitude of the response 3 d after birth. Similarly, in Expt. 2, the values from TPN-SOW, TPN-FORM, and TPN-COW were compared and expressed relative to the values in the TPN group (=1.0) to indicate the magnitude of the response to 2 d of enteral feeding after a period of TPN.
QA and N did not meet the requirements for using parametric statistical methods. For these parameters, Wilcoxon Signed Rank tests were performed to detect an effect of region and Kruskall-Wallis tests were performed to detect an effect of treatment. Post hoc comparisons were carried out using the Mann-Whitney U test.
Unless stated otherwise, the values given are the means of the 3 intestinal regions. Results are expressed as means ± SEM or LSmeans ± SEM. P-values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The TPN and TENT groups had very similar increases in relative intestinal wet weight (23.8 ± 0.9 g/kg for the 2 groups, +33%, P < 0.01) after 3 d, relative to values at birth (NB group, 17.9 ± 1.2 g/kg). The intestinal dry matter proportion did not differ among the groups in Expt. 1 (16 ± 1%) but there was a significant increase in the proportion of mucosa in the intestine (from 51% at birth to 66% at 3 d, relative increases). Hence, the total mucosal weight increased 70–80% over the 3 d for both the TPN and TENT groups (Fig. 1A). There were further significant increases in intestinal mass (44%) during the enteral feeding period in all 3 groups (to 33.6 ± 1.2 g/kg, pooled values), relative to values before enteral feeding (TPN group: 24.6 ± 1.1 g/kg), although in Expt. 2 there was no change in the proportion of mucosal weight (Fig. 1A).
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40% in both groups (Fig. 1B). Similarly, intestinal length showed no significant increase in response to feeding in Expt. 2 (370 ± 11 cm) but the circumference increased significantly in pigs fed infant formula and sow’s colostrum and tended to increase in those fed cow’s colostrum (P = 0.08, Fig. 1B). Across the intestinal regions, crypt depth increased in the 3-d-old TENT group and tended to increase in the TPN group (Fig. 1C, P = 0.11). Similarly, the crypt depth increased in the TPN-SOW and TPN-FORM groups in Expt. 2 and tended to increase in the TPN-COW group (P = 0.10).
Villous height tended to increase in the TPN group in Expt. 1 (Fig. 1D, P < 0.08), relative to values at birth. Additionally, villous height increased 20% in the group fed sow’s colostrum in Expt. 2 (to 931 ± 44 µm, P < 0.05); it tended to increase in the group fed cow’s colostrum (839 ± 35 µm, P < 0.10), and was not affected in the TPN-FORM group (686 ± 39 µm), relative to values before feeding. Unlike the TPN and TENT groups in Expt. 1, all 3 oral feeding groups had increases in villous width (+27%, P < 0.001).
In the two 3-d groups in Expt. 1, surface density of the tunica mucosa (SV), relative to the intestinal wall, tended to increase (Fig. 1E, P = 0.2), whereas in Expt. 2, the SV decreased in response to feeding in the TPN-FORM group (Fig. 1E, 0.012 ± 0.001 mm–1 vs. 0.015 ± 0.001 mm–1, P = 0.02).
The increased proportion of mucosa in both groups of 3-d-old pigs, relative to newborn pigs (Fig. 1A), was substantiated by the stereological measurements in that significant increases occurred for the volume density of the tunica mucosa (VV) (Fig. 1F, absolute values 0.73 ± 0.01 vs. 0.68 ± 0.02, P = 0.05), relative to the intestinal wall. This increase was reflected in a decreased density of the tela submucosa (0.13 ± 0.01 vs. 0.15 ± 0.01, data not shown) and muscle layer (0.12 ± 0.01 vs. 0.14 ± 0.01, data not shown) for the TPN and TENT groups. In Expt. 2, Vv increased only for the TPN-SOW group, relative to TPN (Fig. 1F, absolute values 0.77 ± 0.02 vs. 0.74 ± 0.02, P < 0.01), with no significant changes for the TPN-COW and TPN-FORM groups.
Examples of tissues having the characteristics of lesion scores 0–4 are shown (Fig. 2). The score tended to be higher in the TPN, compared with the TENT group (P = 0.06), but neither was significantly different from the score at birth. Generally, enteral feeding was associated with more dilatation of blood and lymphatic vessels, more goblet cells, and some separation of different layers of the intestinal wall, compared with newborn or TPN-fed pigs. When comparing the enterally fed pigs in Expt. 2, the TPN-SOW group had a lower lesion score than the TPN-FORM pigs (0.21 ± 0.08 vs. 0.97 ± 0.24, P = 0.01), with intermediate values for the TPN-COW group.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The present studies suggest that the small intestine in preterm newborn TPN-fed pigs grows in weight, rather than losing weight, as expected from earlier studies on term newborn pigs subjected to TPN after a short period of suckling (4,6,7). Although intestinal weight relative to body weight remained lowered (20–30%) in preterm TPN-fed pigs compared with pigs fed milk enterally (8,23), the preterm intestine grew, in both diameter and proportion of tissue taken up by mucosa, and with constant or increased mucosal volume and surface indices. Together, the results suggest that the preterm TPN-fed intestine does not suffer from notable mucosal atrophy after a short period of postnatal TPN, and that the area available for digestion and absorption may actually be greater after TPN than at birth. This postnatal growth of the small intestine in TPN-fed preterm pigs occurs in the face of a decreased density of NOS-1–containing myenteric neurons. This decrease appears secondary to an increased intestinal diameter because the number of NOS-1–expressing neurons remained constant. The immediate postnatal intestinal growth in preterm TPN-fed pigs could represent a continuation of the rapid gut growth phase taking place prenatally, where luminal diets are less important for gut growth (24). Only if the TPN period was of longer duration (e.g., > 1 wk) might the preterm intestine decrease in weight and area available for absorption of nutrients. These results do not preclude, however, that the function of the small intestine may be compromised, both in term and preterm neonates, even after short periods of TPN (8,25).
Our study also shows that an elemental nutrient solution infused enterally had no advantage over TPN infused parenterally in terms of the indices of mucosal growth. Enteral infusion of this relatively small amount of TPN fluid was well tolerated by preterm piglets despite its unphysiological nature (e.g., a degree of hyperosmolarity compared with intact diets). Thus, an elemental nutrient solution is inferior to milk diets in stimulating gut growth and maturation in preterm newborn pigs because both colostrum and milk formula produce marked intestinal growth when given just after birth, e.g., from 20 to 30–40 g/kg (8,23). Possibly, the absence of milk macromolecular nutrients in the TPN fluid and their separate actions to stimulate gut growth via the endogenous release of growth factors (26,27) is responsible for the deficient gross morphological growth. It could also be attributed to the direct presence of high concentrations of trophic factors (e.g., epidermal growth factor or insulin-like growth factor-1) in mammary secretions (28). Nevertheless, the complete absence of trophic effects in preterm pigs fed elemental nutrients is surprising because small amounts of enteral elemental nutrients have trophic effects in term pigs (10).
This study also shows that the gut growth induced by feeding oral milk diets after a short period of TPN is relatively small. This could be related in part to the growth that had already taken place during the TPN period. Hence, the observed increases in growth indices such as intestinal weight, mucosa volume, and circumference were diminished relative to those in preterm pigs fed milk diets immediately after birth (8,23). In general, the gut growth response to enteral feeding resulted in deeper crypts and higher and wider villi. The latter change in villous morphology was reflected in the indifferent surface density of the tunica mucosa in the enterally fed groups. Nevertheless, the gut growth response was partially diet dependent because it was only in the group fed sow’s colostrum that mucosal volume density increased.
The lesion score was higher in the TPN-FORM group, compared with the TPN-SOW piglets, indicating that sow’s milk is not only more trophic to the intestine but also better able than infant formula to protect the immature intestine against feeding-induced intestinal lesions. Formula feeding can be more detrimental to preterm neonates than to term neonates; it increases the risk of intestinal atrophy and inflammation, potentially leading to NEC (3,23,29–31). A period of TPN before the introduction of enteral diet also seemed to be associated with increased lesion scores, increased crypt depths, and lower surface densities of the tunica mucosa in the formula group, although all 3 feeding groups had degrees of mucosal atrophy at tissue collection. Thus, a period of TPN before the introduction of oral feeding does not protect the preterm intestine from the atrophy often induced by oral food introduction, particularly not when formula is used.
Finally, our studies on the numerical density and number of nitric oxide–containing neurons suggest that a rapid upregulation of NOS-1 expression in myenteric neurons may be involved in the intestinal growth response to oral milk diets, and that in addition to an increased NOS-2 expression, this may mediate the increased intestinal blood flow that most likely is required to support increased gut growth (7). In contrast to the gut growth response during the 3-d TPN period and apparently in contrast to the comparable constitutive NOS-activities in 3-wk-old piglets fed either enterally or via TPN (7), this study revealed that both the number and density of nitrergic myenteric neurons were higher in milk-fed piglets (colostrum or formula), compared with TPN. These results also differ from observations in term pigs in which both the density and number of submucous NOS-1–expressing neurons decrease after 1–2 d of enteral milk feeding (15). Nevertheless, the localization of NOS-1 in preterm pigs agreed with earlier reports of its presence in fetal and postnatal pig intestine (15,16) and with its expression in submucous and myenteric interneurons and inhibitory motor neurons (32–34).
The stimulation of NOS-1 expression in enteric neurons after enteral feeding of colostral or formula diets could be signaled by postprandial mucosal hyperemia (35,36) and peristalsis (32,34) to be conducted by small intestinal intrinsic and extrinsic regulating mechanisms. In addition, postprandial tissue hypoxia could be associated with the observed increase in NOS-1–containing neurons. In cerebellar neurons in vivo, hypoxia increases NOS-1 mRNA and protein expressions (37). In newborn pig intestine, oxygen extraction and mucosal blood flow appear to be near maximal during enteral feeding, and the increase in postprandial mucosal blood flow is generated at the expense of muscularis-serosa flow (38). Thus, the intestinal outer layers seem predisposed to tissue hypoxia during enteral feeding. This apparent preference to maintain blood flow and oxygenation at the level of the mucosal layer could offer a possible reason for the absence of a change in expression in the submucous enteric plexuses after enteral feeding.
Because the increased number of NOS-1–expressing myenteric neurons was not correlated with an increased lesion score, the increase appears to be a general physiological response of the preterm small intestine to enteral feeding, and not related to factors that protect against or lead to feeding-induced intestinal lesions and NEC. Thus, neuropathological changes leading to decreased NOS-1 expression did not appear to play an important or primary role in preterm piglets with intestinal lesions, despite the fact that decreased neuron densities were observed in diseased intestinal segments of infants with NEC (39).
Our quantitative description of intestinal morphology, coupled with our previous studies (8), suggests that preterm pigs are less sensitive to TPN-induced mucosal atrophy than term pigs. Moreover, the installation of enteral feeding after TPN resulted in a diminished trophic effect compared with fetal or preterm newborn pigs fed milk diets without a TPN period (8,23). In addition, NOS-1 cell number develops differently in preterm piglets fed milk after TPN and it seems related to the initiation of milk feeding, and not to the occurrence of feeding-induced intestinal lesions. Hence, differential regulatory mechanisms appear to be present for mucosal growth during TPN vs. enteral feeding, and in term vs. preterm pigs. These considerations are important in attempts to extend findings obtained in term newborn or suckling animals to a clinically relevant situation for preterm infants.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the Danish Agricultural and Veterinary Research Council, Danish Ministry for Food and Agriculture, by a Special Research Fund of the University of Antwerp and by a Research Grant of the Fund for Scientific Research-Flanders (Belgium) (1.5.096.04N).
4 Abbreviations used: HE, hematoxylin-eosin; I, intersection; N, number of nitrergic neurons; NB, pigs killed within 12 h of birth; NEC, necrotizing enterocolitis; NOS, nitric oxide synthase; P, point; QA, numerical density; SV, surface density; TENT, enterally fed TPN-fluid; TPN, total parenteral nutrition; TPN-COW, after TPN, 3 d enterally fed cow’s colostrum; TPN-FORM, after TPN, 3 d enterally fed formula; TPN-SOW, after TPN, 3 d enterally fed sow’s colostrum; VV, volume density.
Manuscript received 15 April 2005. Initial review completed 22 May 2005. Revision accepted 10 August 2005.
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