Small-bowel transplantation is performed today in case of various diseases. However, despite the increasing numbers of small-bowel transplantations that are performed, mortality and morbidity are still higher as compared with transplantation of other organs. New anti-rejection medication has been developed, and understanding of immunological aspects and operative procedures has improved the success rate of transplantation. However, preservation of tissue during storage is another important step in the transplantation procedure, especially to delay ischemia of the donor small bowel, which is estimated to occur after 12–24 h, thus providing more time for transport of the organ to the recipient, tissue matching, preparation of the operation team, and allowing improved post-operative functions of the transplanted organ at lower cost.

Today, a major principle of organ preservation is hypothermia at 4–10°C when flushing the organ with a preservation solution via the vascular system. Tissue damage that occurs during preservation may be related to the type of the solution, the period of preservation and the nature of organ to be transplanted. During the preservation process, hypoxia and consecutive ischemia develop in the organ, leading to decreased ATP levels and pH in the cells, that result at first in reversible changes, and with prolonged ischemia in irreversible cell damage. Small intestinal mucosa is known as one of the most susceptible organs to ischemia. Ischemic damage of the mucosa is manifested by epithelial detachment from the lamina propria at the tips of villi and it extends down to the bottom of the crypts. Hypertonic Euro-Collins (EC) solution (KH₂PO₄, 2.04 g/l; K₂HPO₄, 7.4 g/l; KCl, 1.12 g/l; NaHCO₃, 0.84 g/l; and glucose, 35 g/l; osmolarity, 355 mOsml/l and pH 7.2 at 4°C) has been widely used for the preservation of organs in experimental and clinical studies. The preservation period for small bowel has experimentally been extended up to 24 h using Ringer’s lactate (RL) solution. In the present study, we compared at the light microscopical and electron microscopical level damage of the small bowel tissue at different periods of hypoxia using EC and RL solutions for preservation.

In the present study, male and female Wistar Albino rats (220–250 g) from the Animal Laboratory of the Marmara University have been used. They were kept on a 12-h night/day cycle at 22±1°C and were fed standard diet with water freely available. The study has been approved by the ethic commission of the Marmara University. Three experimental groups of 5 rats each were treated with physiological saline solution (group 1), EC solution (group 2) and RL solution (group 3). After anesthesia by ether inhalation and opening of the abdomen, small bowels were perfused via the ascending aorta with physiological saline solution, EC or RL until the solution from the superior vena cava appeared to be clear. Ileal grafts with a length of approx. 6 cm were resected at a distance of 3 cm proximal from the ileocaecal valve. The intestinal lumen was flushed with 20 ml cold saline solution. Perfusion without storage served as controls. Biopsies containing the entire intestinal wall were taken from the grafts and kept in the different preservation solutions at 4°C for 3, 6 and 12 h.

Intestinal tissue samples were prepared for routine light microscopical examination. Paraffin sections were stained with hematoxylin and eosin (H–E) and periodic acid-Schiff (PAS), and examined using a BH-2 photomicroscope (Olympus, Tokyo, Japan).

For the demonstration of permeability of intercellular tight junctions at the electron microscopical level, the lanthanum chloride method was used and 1 mm³-sized tissue samples were fixed at room temperature for 2 h in a 2.5% cacodylate-buffered glutaraldehyde solution (0.1 M; pH 7.4), containing 4% lanthanum chloride. Following dehydration in an increasing series of alcohol containing 4% lanthanum chloride, tissue samples were embedded in epon 812, then sections of 60 nm thickness were cut with an ultramicrotome (Leica, Vienna, Austria), contrasted with uranyl acetate and lead citrate, and examined with a 1200 EXII transmission electron microscope (Jeol, Tokyo, Japan). Histopathological assessment of the intestinal tissue was performed by using criteria shown in Table 1 and modified after. Data are expressed as mean±SEM. Statistically significant differences between groups were identified using the Friedman non-parametric test and the Dunn’s multiple comparisons test. Differences were considered to be significant at p<0.05.

The light microscopic analysis of biopsies of intestinal tissue that was merely perfused with the different preservation solutions revealed normal morphology of the submucosa and muscularis externa, undamaged goblet cells and a normal amount of PAS-positive mucus.

Figure 1. Photomicrographs at the LM level (a, b) and EM level (c) of small bowel of rat that was not stored. (a) Mucosa and submucosa are normal. H–E staining. (b) Normal amount of mucus and goblet cells are present. PAS staining. (c) Enterocytes are present with normal microvilli (m) and impermeable tight junctions (?). Bars: 100 ?m (a), 25 ?m (b) and 500 nm (c).

Electron microscopy of biopsies of perfused ileum that had not been stored indicated a normal ultrastructure of villus epithelial cells, goblet cells and Paneth cells, whereas there was virtually no intercellular penetration of lanthanum via tight junctions.

Preservation of tissue in physiological saline solution for 3 h caused shedding of villus epithelium from the lamina propria, and damage of the villi. Damage of crypt epithelium was found to occur locally, but the deeper parts of the crypts remained intact. PAS staining showed diminished amounts of mucus in the lumen but goblet cells in crypts of intact epithelium were not damaged. Preservation of tissue in EC solution for 3 h resulted in local separation of epithelium from the lamina propria, and damage of epithelium at the luminal side of the crypts with undamaged deeper regions, whereas goblet cell degeneration was observed in zones of damaged epithelium covering villi and crypts. The amounts of mucus were slightly decreased in those areas. Preservation in RL showed epithelial damage in the apical regions of villi, but integrity of crypts was maintained. The amounts of mucus were diminished moderately, but goblet cells showed a normal morphology.

Figure 2. Photomicrographs at the LM level (a, b) and EM level (c) of small bowel of rat that was stored for 3 h in saline solution. (a) Most villi show apical epithelial rupture (?), and degeneration whereas the structure of the bottom of the crypt () is normal. H–E staining. (b) Decreased amounts of mucus (?) and normal goblet cells are present. PAS staining. (c) Enterocytes with irregular microvilli (m) and leakage of lanthanum chloride via tight junctions (?) are apparent. Bars: 100 ?m (a), 25 ?m (b) and 1 ?m (c).

Figure 3. Photomicrographs at the LM level (a, b) and EM level (c) of small bowel of rat that was stored for 3 h in EC solution. (a) Epithelial separation from the lamina propria in the apical region of a villus (?) occurs. H–E staining. (b) Moderately decreased amounts of mucus (?) are present. PAS staining. (c) Regular microvilli (m) and leakage of lanthanum chloride into intercellular spaces are shown. Bars: 100 ?m (a), 25 ?m (b) and 500 nm (c).