Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium

Abstract

Background and aims: Injuries caused by ischaemia and ischaemia/reperfusion in the small intestine have been widely accepted as resulting in necrosis. The aim of this study was to ascertain whether apoptosis also occurs.

Methods: Intestinal epithelium from rats subjected to ischaemia (15-90 minutes) and ischaemia/reperfusion (15 minutes ischaemia followed by 15-75 minutes of reperfusion) was studied using histological, immunohistochemical, and molecular biological methods as well as FACS.

Results: Mucosal injury was induced by both ischaemia and ischaemia/reperfusion. Detachment of epithelial cells from the villous stroma was an early morphological change indicating mucosal injury. More than 80% of the detached cells exhibited characteristic morphological features of apoptosis (condensation of chromatin and nuclear fragmentation). The remainder demonstrated necrotic features. The apoptotic cells eventually underwent spontaneous degeneration with membrane rupture, a process morphologically identical to necrosis. DNA fragmentation was also confirmed by immunohistochemical methods and agarose gel electrophoresis.

Conclusion: Apoptosis is a major mode of cell death in the destruction of rat small intestinal epithelial cells induced by ischaemia and ischaemia/reperfusion injury. Disruption of epithelial cell-matrix interactions ("anoikis") may play an important part in induction of apoptosis in detached enterocytes.

Figure 1

Sections of jejunum following ischaemia for 15 minutes (A1-3), 30 minutes (B1-3), and 60 minutes (C1-3), and in a group given 75 minutes' reperfusion followed by 15 minutes of ischaemia (D1-3). In A1-3 no significant changes are apparent except for dilatation of Gruenhagen's space. Apoptotic cells are present at the tip of the villi (A2, 3). In B1-3 note the amorphous substance between the villous stroma and the base of the epithelial cells (B2). Some enterocytes are TUNEL positive (B3). In C1-3 the upper regions of the villi are destroyed. Many detached enterocytes show both condensed and fragmented nuclei which are TUNEL positive (C2, 3). Some enterocytes located in the lower region, which are detached from the villous stroma, are also TUNEL positive (C2, 3 arrow). Note the amorphous substance (*) between the epithelium and villous stroma (vs) in C2. In D1-3 notable destruction of the upper two thirds of the villi is evident but the tips are covered with enterocytes after 75 minutes of reperfusion (D2). Most of the detached cells show condensed and fragmented nuclei and also TUNEL positive staining, in contrast to the enterocytes located in the villi (D3). Serial sections A1-D1 and A2-D2 were stained with haematoxylin and eosin and sections A3-D3 were stained using the TUNEL method. Original magnification: ×40 (A1-D1), ×100 (B2, B3), ×150 (D2, D3), ×200 (A2, A3, C2, C3).

Figure 2

Electron microphotographs of jejunum from groups given (A) sham ischaemia, (B) 90 minutes of ischaemia, and (C) 45 minutes of reperfusion. (A) Enterocytes located in the middle part of the villi show no remarkable changes. (B) An enterocyte showing compaction and segregation of chromatin against the nuclear envelope (arrow) is observed between normal appearing cells. The amorphous substance is evident as blebs limited by a simple membrane (*) (arrowhead, basement membrane; V, vessel). (C) Detached cells have characteristic features of apoptosis but still preserve microvilli (arrow) and intact mitochondria. Original magnification: ×2500 (A and B), ×5000 (C).

Figure 3

Fluorescence microphotographs of detached enterocytes stained with the DNA binding fluorochromes HO342 and PI: (A) 60 minutes' ischaemia, and (B) 45 minutes' reperfusion. (C) High power magnification of fig 3B. (D) Light microscopical finding for the cells in fig 3C. (A) Condensed and fragmented nuclei are HO342 positive (arrows) (green~blue) in contrast to the PI positive oval or round nuclei (arrowheads) (red~yellow). (B) Some condensed and fragmented nuclei are HO342 (large arrows) and others are PI (small arrows) positive. On the other hand, oval or round nuclei preserving their normal morphology are PI positive (arrowheads). (C) and (D) A HO342 positive cell with a fragmented nucleus has a round shape with apoptotic features (arrow) while a PI positive cell with an oval nucleus demonstrates a relatively normal morphology (arrowhead). Original magnification: ×80 (A and B), ×160 (C and D).

Figure 4

Flow cytometric analysis of FITC-Annexin V binding and PI uptake of detached enterocytes undergoing apoptosis versus necrosis. Detached enterocytes (1 × 10⁴) were analysed after both ischaemia (A) and reperfusion (B). (C-E) Frequencies of live, necrotic, and apoptotic cells after ischaemia (30, 60, and 90 minutes) and reperfusion (15, 45, and 75 minutes). The lower left quadrant of each panel shows live cells (FITC-Annexin^-/PI^-), the lower right quadrant represents apoptotic cells (FITC-Annexin⁺/PI^-) and the upper right quadrant contains the non-viable, necrotic cells (FITC-Annexin⁺/PI⁺). These latter comprise both PI^high cells (G₁) and PI^low cells (G₂). (A) PI^high cells have oval or round nuclei and preserve their normal morphology as in fig 3B (arrowheads) and PI^low cells are round in shape with condensed and fragmented nuclei as in fig 3B (small arrows). Numbers within the dot plots represent the percentages of cells in each quadrant. One representative experiment out of five is shown. More than 80% of the detached cells show apoptotic features (C-E). Significant differences between the ischaemia and reperfusion groups were only observed for the early phase of the experiment. *p<0.01 compared with 30 minutes' ischaemia.

Figure 5

Agarose gel electrophoresis of DNA extracted from small intestine after 30 minutes (lane 1) and 60 minutes (lane 2) of ischaemia and 45 minutes of reperfusion (lane 3). Both the ischaemia (lanes 1 and 2) and ischaemia/reperfusion groups (lane 3) show ladder patterns.

Figure 6

Time course of percentage DNA fragmentation after ischaemia and ischaemia/reperfusion. The percentage of fragmented DNA in the ischaemia and ischaemia/reperfusion groups was measured at various time points. No significant differences between ischaemia and ischaemia/reperfusion injury were observed.