Biologia, Bratislava, 62/4: 491—497, 2007 Section Cellular and Molecular Biology DOI: 10.2478/s11756-007-0098-0
Detection of early stages of apoptosis in experimental intestinal ischemia-reperfusion injury Štefan Tóth1, Mikuláš Pomfy1, Peter Wohlfahrt1, Stanislava Pingorová1, Ján Kišš1, Peter Baláž2, Slavomír Rokošný2, Roman Beňačka3 & Jarmila Veselá1 1
Department of Histology and Embryology, Faculty of Medicine, P. J. Šafárik University, Šrobárova 2, SK-04001 Košice, Slovakia; e-mail: s
[email protected] 2 Department of Transplant Surgery, IKEM, Praha, Czech Republic 3 Department of Pathophysiology, Faculty of Medicine, P. J. Šafárik University, Trieda SNP 1, SK-04001 Košice, Slovakia
Abstract: Apoptosis is a form of programmed cell death that plays an important role in small intestine ischemia-reperfusion (IR) injury. The aim of this study was to determine the total proportion of apoptotic cell death (apoptotic index) following injury induced by ischemia and during various subsequent reperfusion periods, total histopathological status and the intestine regeneration dynamics after the IR injury. Experimental animals, Wistar rats (n = 45) were divided into three experimental and one control groups. In the experimental groups 1 h ischemia was followed by 1, 4 and 24 h reperfusion. Intestinal ischemia was induced by superior mesenteric artery (SMA) occlusion. Segments of jejunum were stained with hematoxylin and eosin and studied immunohistochemically using M30 CytoDEATH and in situ TUNEL methods for apoptosis detection. Our experimental data showed that: (i) apoptosis is an important form of cell death in the small intestine after IR injury induced by SMA occlusion; (ii) maximum levels of histopathological damage and apoptotic index of mucosa occurred after 1 h ischemia and 1 h of reperfusion; and (iii) mucosa possesses great regeneration ability. The lowest levels of histopathological damage and apoptotic index were observed in the group with 1 h ischemia and 24 h reperfusion where, however, the highest mitotic index was present. Key words: apoptosis; ischemia; reperfusion; injury; regeneration. Abbreviations: CK, cytokeratin; H&E, hematoxylin and eosin; IR, ischemia-reperfusion; MI, mitotic index; PCD, programmed cell death; SMA, superior mesenteric artery; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate in situ nick end-labelling.
Introduction Programmed cell death (PCD) may be more accurately defined as cell death that is dependent on genetically encoded signals or activities within the dying cell which lead irreversibly to cell death (Fink & Cookson 2005). Apoptosis as the first type of PCD is an energy-dependent form of cell death. Apoptosis is induced by death signals in changes between the varieties of pro- and anti-inflammatory mediators. Basic morphologic and molecular characteristics of apoptosis include cell membrane blebbing, cell shrinkage, chromatin condensation, cytoskeletal reorganization, nuclear envelope injury and systemic internucleosomal DNA fragmentation (Kerr et al. 1972). Finally, apoptotic cells disintegrate into membrane-bound apoptotic bodies, which are phagocytosed by neighbouring cells and macrophages (Soini et al. 1998) without inflammatory reactions (Raff 1998). Apoptosis plays a very important role in intestine epithelium homeostasis (Fouquet et al. 2004). Apoptosis is present in the crypts of Lieberk¨ uhn and regu-
lates the total amount of progenitor stem cells (Potten 1997). It also influences the desquamation or exfoliation process of epithelial cells on the villous tips by anoikis as a phenomenon of apoptosis (Thomson et al. 2001). Anoikis is the subset of apoptosis triggered by inadequate or inappropriate cell-matrix contacts; and it maintains the correct cell number in high-turnover epithelial tissues. Under physiological conditions in the small intestine it is present mainly in the crypts of Lieberk¨ uhn (less than 1%) as spontaneous apoptosis (Potten et al. 1997). The process of apoptosis is significant under physiological and pathological conditions. Ischemia-reperfusion (IR) injury is an important event involving damage to small intestine cells and tissues and various distant vital organs (liver, lungs and kidney). Ischemia and subsequent reperfusion induces a cascade of processes which leads to cell dysfunction. Large numbers of cells lost through apoptosis may lead to changing in the vitally-important organ functions and potentially to their failure. The aim of the present study was to determine the early stages of apoptosis in jejunal epithelium in
c 2007 Institute of Molecular Biology, Slovak Academy of Sciences
492 injury induced by ischemia and during various subsequent periods of reperfusion, and to characterize the total histopathological status and the dynamics of intestine restitution after the IR injury. Material and methods Experimental animals and grouping The experiment was performed on adult, female Wistar rats weighing 250–350 g. The animals were housed in standard conditions and had free access to commercial chow and water ad libitum. All animals (n = 45) were randomly divided into four groups. Three groups were subjected to IR injury and one group was sham-operated animals. In the first experimental group – IR1 (n = 10) – 1 h ischemia was followed by 1 h reperfusion. In the second group – IR4 (n = 10) – 1 h ischemia was followed by 4 h reperfusion. In the third group – IR24 (n = 10) – 1 h ischemia was followed by 24 h reperfusion. The sham-operated control group (S) animals were divided according to survival time into 2 h, 5 h and 25 h survival groups (each n = 5). This experiment was approved by the Committee for Ethics on Animal Experiments at the Faculty of Medicine, P. J. Šafárik University, Košice, Slovakia. Rat ischemia-reperfusion experimental model Animals were fasted for 12 h before surgery but given free access to water. Anaesthesia was performed by the intraperitoneal injection of ketamine (60–80 mg/kg) and xylazine (8– 10 mg/kg). The skin was aseptically prepped and a 6–cm midline laparotomy was performed. The superior mesenteric artery (SMA) was isolated and ischemia was induced by totally occluding it with an atraumatic microvascular clamp for 1 h. After ischemia, the clamp was carefully removed and this was followed by 1, 4 and 24 h reperfusion. In the sham-operated animals only midline laparotomy was performed omitting the SMA occlusion. The abdominal incision was closed in two layers with non-absorbable suture. All procedures were performed using an aseptic technique with animals breathing spontaneously. Body temperature was maintained by a heating pad set to 37 ◦C until the animals fully recovered from anaesthesia. Histopathological assessment All animals in the experimental and sham-operated groups were killed after reperfusion. Ten cm from Trietzi ligament 4 cm small intestine samples were harvested. The SMA is the major artery for supplying this segment of small intestine (jejunum). Harvested intestinal tissue was rinsed promptly in cold saline and immediately fixed 24–48 h in p-formaldehyde. The tissue was then embedded in paraffin wax, sectioned in 4–5 µm tissue slices and stained with hematoxylin and eosin (H&E) and studied to determine the severity of intestinal injury (histopathological injury score), calculate the mitotic index and detect the presence of apoptotic bodies. To detect apoptotic cells, two selective immunohistochemical methods (both Roche Molecular Biochemicals, Germany) were used: M30 CytoDEATH and terminal deoxyuridine in situ nick end-labelling (TUNEL). Histopathological injury index The total histopathological injury index was calculated by totalling all the scores for each sham-operated and experimental animal. This index is based on the extent and severity of histopathological abnormality or lesion in the section. H&E-stained sections of intestinal tissue were scored using a
Š. Tóth et al. semi-quantitative Park/Chiu grading system adapted from Quaedacker et al. (2000) to quantify the extent of mucosal damage. Briefly, mucosal damage was graded from 0 to 8, normal to severe, according to the following histopathological criteria: grade 0 – normal mucosa; grade 1 – subepithelial Gruenhagen’s space at the tip of the villus, often with capillary congestion; grade 2 – extension of the space with moderate epithelial lifting; grade 3 – massive epithelial lifting with a few denuded villi; grade 4 – denuded villi with exposed lamina propria and dilated capillaries, possibly with increased cellularity of lamina propria; grade 5 – disintegration of the lamina propria; grade 6 – the crypts of Lieberk¨ uhn are also affected; grade 7 – transmucosal hemorrhage and infarction is present; grade 8 – transmural infarctions and ulceration. Mitotic index Mitotic index (MI) is a measure for the proliferation status of a cell population. It is defined as the ratio between the number of cells in mitosis and the total number of cells. Conventional light microscopy of H&E-stained sections was used to quantify mitosis in the intestinal epithelium. Briefly, 10 crypts were assessed per animal for characteristic findings of mitotic cells. Detection of apoptotic bodies Apoptosis was quantified by scoring the number of apoptotic bodies identified in the crypts of H&E-stained sections, using criteria that included the presence of pyknotic nuclei, condensed chromatin, and nuclear fragmentation (karyorrhexis). Apoptotic cells can readily be identified by means of routine histological staining methods, such as H&E. Detection of apoptotic cells was based on characteristic morphological features. The cytoplasm of apoptotic cells was condensed and eosinophilic, chromatin was condensed (pyknosis) and marginated at the nuclear envelope, and nuclear fragments were later seen (karyorrhexis). The apoptotic index (AIHE) was calculated as the number of apoptotic bodies on well-oriented crypts of Lieberk¨ uhn. Briefly, 10 crypts were assessed per animal for characteristic morphologic findings of apoptotic bodies. M30 CytoDEATH immunohistochemistry Cytokeratins are intermediary filaments of epithelial cells. Cytokeratin 18 (CK18) is expressed in simple nonstratified, ductular and pseudostratified epithelia (K¨ ohler et al. 2002). Caspase-mediated cleavage of CK18 leads to exposure of a neo-epitope that can be recognized by binding of specific antibodies. The M30 antibody recognizes cleaved but not uncleaved CK18. M30 antibody permits the detection of early phases of apoptosis and is not detectible in vital epithelial cells and necrotic cells. During autophagy CK18 is reported to stay uncleaved and necrotic cells are considered to release CK18 (Ueno et al. 2005). Since the exposure of the M30 neo-epitope seems to occur in the initial phase of the apoptotic cascade, it may be used in studies on the dynamics of this process. Tissues sections were prepared as described above and processed according to the protocol described by Leers et al. (1999) and according to the manufacturer’s instructions. Sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3% H2 O2 with methanol. Pretreatment was by microwave irradiation at 600 W for 15 minutes in 0.01 M citrate buffer at pH 6.0. This yielded the best results in terms of antigen retrieval. A primary M30 monoclonal antibody (Roche Molecular Biochemicals, Germany) was used which detects
Apoptosis in intestinal ischemic-reperfusion injury
TUNEL immunohistochemistry Detection of apoptosis through the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate in situ nick end-labeling (TUNEL) in paraffin-embedded 4–µmthick sections was undertaken, using the TUNEL method (In Situ Cell Death Detection Kit, Fluorescein, Roche Molecular Biochemicals, Germany) according to the manufacturer’s instructions. The TUNEL method is based on the enzymatic ability of terminal deoxynucleotidyltransferase (TdT) to catalyze a template-independent addition of deoxyribonucleotide triphosphate to the 3’-OH ends of doubleor single-stranded DNA. Tissue sections were deparaffinized and rehydrated. Protein digestion was done by applying proteinase K (20 µg/mL) to the slides for 20 minutes at room temperature, which was followed by four washes in distilled water for 2 minutes each. Following this the sections were incubated with TUNEL-labelling mixture (TdT-FITC) in a humidified chamber at 37 ◦C for 1 h. TdT was omitted from the TUNEL-labelling mixture in the TUNEL-negative controls. Positive controls were obtained by pretreating with DNase I (3000-3 U/mL, Roche Molecular Biochemicals, Germany) prior to incubation with the TUNEL-labelling mixture. The sections were then washed with phosphate-buffered saline, and an antifade preparation containing propidium iodide (Sigma-Aldrich) was applied for nuclear staining. TUNELstained tissue sections were examined with an Olympus fluorescent microscope with Olympus DP-Soft 3.1 software. First, the propidium iodide (red) was examined through a 346-nm filter at a magnification of ×100, ×200 and ×400. Propidium iodide stains all nucleated cells (alive, necrotic, and apoptotic) in the same manner. The magnification was then increased to ×200 and a colour photomicrograph was taken. The same area as viewed for the propidium iodide staining was then similarly examined for apoptotic staining (bright green), using a 550 nm filter at a magnification of ×100, ×200 and ×400. Afterwards a colour slide photograph was taken through this filter system. The ratio of apoptotic to nonapoptotic cells (apoptotic index, AIT ) in the intestine epithelium was obtained. Under 400× magnification by fluorescent microscope, the number of apoptotic (TUNELpositive) cells was characterized by a positive staining nucleus in the TUNEL-stained sections (DNA fragmentation) counted in 10 randomly selected fields in mucosa per section. The mean number of epithelial apoptotic cells (AIT )
per one field for each animal was calculated for further statistical analysis. Statistical analysis The statistical analysis was performed using the GraphPad InStat version 3.01 (GraphPad Software, San Diego, CA). The quantitative results (apoptotic and mitotic indexes) were determined by one-way ANOVA with a multiple comparison Tukey-Kramer post-hoc test. The semi-quantitative results (histopathological injury index) were determined using the non-parametric Kruskal-Wallis test and Dunn’s posthoc test for multiple comparisons. All the results were expressed as mean ± SEM. P values less than 0.05 were considered significant.
Results Histopathological injury index Normal small intestine morphology was found in the sham-operated group (Figs 1, 2A). Animals undergoing 1 h of ischemia and 1 h of reperfusion (IR1) displayed severe mucosal damage (7.13 ± 0.13; p < 0.001 IR1 vs. IR24), manifestation of which ranged from massive epithelial lifting with few denuded villi to significant architectural distortion and lamina propria disintegration (Fig. 2B). In the second experimental group (IR4) with 1 h ischemia and 4 h reperfusion the damage was less serious (3.86 ± 0.29). Histopathological evaluation showed detachment of epithelial cells (anoikis) from the villous stroma with subepithelial Gruenhage’s spaces at the tip of the intestinal villi (Fig. 2C). In contrast, the animals from IR24 group had lower mucosal damage (1.25 ± 0.16). Morphological changes included moderate subepithelial lifting only at the villus tips and a significant degree of mucosa regeneration was evident (Fig. 2D). Mitotic index MI is an important morphological parameter of epithelial cell regeneration degree after mucosal injury. MI in the sham-operated group was 0.9 ± 0.23 (Fig. 3). After 1 h of ischemia and 1 h of reperfusion a significant
Histopathological injury index
caspase-cleaved CK18 and is specific to apoptotic epithelial cells. Primary antibody was applied at the appropriated titre (1:200) and sections were incubated overnight at 4 ◦C. Biotinylated secondary sheep anti-mouse antibody (Chemicon, Germany) was used, followed by labelling with streptavidin-POD (Roche Molecular Biochemicals, Germany). Sections were visualized with diaminobenzidine (Fluka) and lightly counterstained with Mayer’s hematoxylin (Merck, Germany). Omitting the primary M30 antibody was considered as the negative control. As M30 immunoreactivity is specific to apoptosis, the ratio of apoptotic to nonapoptotic (apoptotic index, AIM30 ) cells in the intestinal epithelium was obtained. Under 400× magnification by light microscope, the number of apoptotic (M30positive) cells was characterized by a positive staining cytoplasm (brown) in the M30 CytoDEATH-stained sections counted in 10 randomly selected fields in mucosa per section. The mean number of epithelial apoptotic cells (AIM30 ) per one field for each animal was calculated for further statistical analysis.
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Fig. 1. Histopathological injury index of jejunum. Histopathological grading was performed according to criteria established by Park/Chiu scoring system (M ± S.E.M.) in the sham-operated group S (n = 15) and the experimental groups IR1, IR4 and IR24 (each n = 10; ++ p < 0.001 S vs. IR1 and IR1 vs. IR24, + p < 0.01 S vs. IR4).
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Fig. 2. Histopathological lesions of the rat jejunum (H&E). (A) Sham-operated group – S; (B) 1 h after reperfusion – IR1; (C) 4 h after reperfusion – IR4; (D) 24 h after reperfusion – IR24.
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Fig. 3. Mitotic index of jejunum. Number of mitotic figures detected by H&E (M ± S.E.M.) in the sham-operated group S (n = 15) and the experimental groups IR1, IR4 and IR24 (each n=10; ++ p < 0.001 vs. IR24).
decrease was noted (0.1 ± 0.10; p < 0.001 vs. IR24). After that MI rose to 0.9 ± 0.31 in the IR4 group, and to its maximum level 1.7 ± 0.34 in the IR24 group. Apoptotic bodies – apoptotic index I (AIHE ) In the sham-operated group the number of apoptotic bodies (AIHE ) was 0.3 ± 0.15 (Fig. 4). A statistically significant increase in apoptotic bodies was found in the IR1 experimental group (2.4 ± 0.27; p < 0.001 vs. IR4 and IR24). A statistically significant decrease in apoptotic bodies was seen in the IR4 (0.6 ± 0.22) and IR24 (0.4 ± 0.16). M30 CytoDEATH method – apoptotic index II (AIM30 ) In the sham-operated group the average number of M30-positive epithelial cells (AIM30 ) was 2.9 ± 0.38 (Fig. 4). M30-positivity in the epithelium was present mainly in the basal part of the crypts of Lieberk¨ uhn
Fig. 4. Apoptotic index in epithelium of jejunum. Number of apoptotic cell detected by H&E (HE), M30CytoDEATH (M30) and in situ TUNEL (TUNEL) methods (M ± S.E.M.) in the sham-operated group S (n = 15) and the experimental groups IR1, IR4 and IR24 (each n = 10; + p < 0.01 S vs. IR4, IR1 vs. IR4; ++ p < 0.001 S vs. IR1 & IR4, IR1 vs. IR4 & IR24; IR4 vs. IR24).
and at the tip of intestinal villus (Fig. 5A1 ). This localization of apoptotic cells is typical for physiological apoptosis. In the all experimental groups a statistically significant increase in M30 apoptotic cells was noted compared to the sham group (IR1, 4.2 ± 0.49; p < 0.001 vs. IR4), (IR4, 7.2 ± 0.99; p < 0.001 vs. IR24), (IR24, 2.9 ± 0.51). After IR injury M30–positive cells were predominantly localized in the apical part of intestinal villi (Fig. 5A2 -A4 ). In situ TUNEL method – apoptotic index III (AIT ) The average number of TUNEL-positive small intestine epithelial cells in the sham-operated group was 3 ± 0.39 (Fig. 4). TUNEL-positive cells were localized in the basal parts of the crypts and at the tip of the villus. After 1 h ischemia and 1 h reperfusion (IR1), AIT increased (5.3 ± 0.26; p < 0.001 vs. IR4) (Fig. 5B1 ). Significant increases in TUNEL-positive cells were present
Apoptosis in intestinal ischemic-reperfusion injury
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Fig. 5. Immunohistochemical analysis of apoptosis in the rat jejunum. Photomicrograph of the M30–positive (A1 , A2 , A3 , A4 ) and TUNEL-positive (B1 , B2 , B3 , B4 ) apoptotic epithelial cells. A1 and B1 – sham-operated group (S); A2 and B2 – 1 h after reperfusion (IR1); A3 and B3 – 4 h after reperfusion (IR4); A4 and B4 – 24 h after reperfusion (IR24).
after 1 h ischemia and 4 h reperfusion (Fig. 5B2 -B3 ) in comparison with the sham-operated controls (12 ± 0.8; p < 0.001 vs. IR24). After 24 h reperfusion the number of TUNEL-positive cells decreased to 6.5 ± 0.56 (Fig. 5B4 ). Discussion Small intestine IR is clinically connected with sepsis, hemorrhagic shock, vascular surgery procedures, small intestine transplantation and with multiorgan failure (Moore 1994). IR leads to small “intestine barrier” function failure, which is connected with bacteria and toxins translocation from the intestinal lumen through
damaged mucosa to the extraintestinal side and thence to systematic circulation (Sun 1998). Ischemia itself produces the intestinal damage, but the major damage is caused especially by reperfusion. Reperfusion of ischemic tissues is associated with local and systemic leukocyte activation and trafficking (especially neutrophils), endothelial barrier dysfunction in postcapillary venules and enhanced production of various inflammatory mediators (Carden & Granger 2000). A serious IR complication as a result of changed intestine permeability is the secondary multiorgan dysfunction syndrome which causes vitally important remote organ damage. The results of our study show that the small intes-
496 tine wall structure changes are mainly influenced by the time of ischemia and reperfusion. Ikeda et al. (1998) in their complex morphological study found that 15 minutes ischemia followed by 45 and 60 minutes reperfusion changes small intestine structure. They described intestinal villi denudation and mucosa layer denudation. The maximum destruction effect following 30 minutes ischemia was described between 15 and 30 minutes reperfusion, when the mucosa layer was destroyed. Souza (2000) described small bowel local hyperemia, a lower grade of subepithelial layer edema, destruction and denudation of apical parts of villi following 30 minutes SMA occlusion and 30 minutes reperfusion. Kuenzler et al. (2002) reported on massive epithelium separation with denudation of apical parts of villi following 35 minutes SMA occlusion and 120 minutes reperfusion. In the study by Chen et al. (2005) 45 minutes small intestine ischemia and 2 h reperfusion caused mainly mucosa layer damage. In the reperfusion time interval of 6 and 12 h massive mucosa layer damage, numerous hemorrhages and necrosis with concomitant inflammation infiltration in the small intestine wall were noted. Zheng et al. (1997) identified only partial small intestine mucosa regeneration. In all small intestine biotical samples mucosa was regenerated with upper villi parts reepithelized after 48 h of reperfusion. Intestinal villi were morphometrically shorter and markedly less developed compared to intestinal villi in the control group. Following 48 and 96 h reperfusion a higher MI was noted compared to the control group. In our study the regeneration process was identified even sooner. Twenty-four h after reperfusion mucosa regeneration was evident with total intestinal villi reconstruction. According to our immunohistochemistry results apoptosis plays an important role in small intestine IR injury. For apoptosis dynamics description it is important to differentiate its presence in different compartments, i.e. cytoplasm and nucleus, which is provided by different apoptosis detection systems. Many intracellular proteins are fragmented during apoptosis by a specific protease group called caspases. One of the fragmentation substrates is CK18, an intermediate filament protein, that is an important part of the cell cytoskeleton. During apoptosis a neo-epitope in CK18 appears that becomes available at an early caspase cleavage event (MacFarlane et al. 2000). M30 CytoDEATH using the monoclonal antibody M30 is specific for this site and can be utilized specifically to recognize apoptotic cells (Leers et al. 1999). CK18 neo-epitope is highly specific for apoptotic cells, as it is not detected in vital or necrotic cells (Chu & Weiss 2002). M30 antibody cannot be used for vital or necrotic cells detection (Ueno et al. 2003). In our study the M30 CytoDEATH method was utilized for the early apoptosis recognition in small intestine epithelial cell cytoplasm. According to Pižem & C¨or (2003), CK18 neo-epitope detection in epithelial cell cytoplasm foresees DNA fragmentation in the nucleus detected by the TUNEL method. According to our results, imunohistochemical reactivity and apoptotic index determined by M30 CytoDEATH
Š. Tóth et al. are consistent with the apoptotic index detected by the TUNEL method, except for the IR4 group, where a difference was noted. We think this difference was caused by different detection sensitivities of these two methods. The M30 CytoDEATH method is more sensitive for early stages of apoptosis detection, while the TUNEL method is more sensitive for intermediate or late apoptosis stages. Apoptosis is present in the small intestine mucosa under physiological conditions (1.15%), but it shows higher incidence under pathological conditions. Under physiological conditions apoptosis presence is due to fast epithelial cell regeneration. During the IR the apoptosis level depends on many factors – type, intensity and duration of insult. Mucosa damage is accompanied by the epithelial cells easing from the apical parts of intestinal villi. According to Ikeda et al. (1998) it is an early marker of IR injury. More than 80% of eased epithelial cells showed apoptosis morphological signs (nucleus chromatin condensation and fragmentation). The rest of the eased cells had morphological signs of necrosis. In the other work by Ikeda et al. (2002), the authors considered apoptosis to be the main form of small intestine epithelial cell death after IR injury in rats. Interaction failure between epithelial cells and the extracelullar matrix (called as anoikis) plays an important role in apoptosis launch in desquamated epithelial cells. The importance of anoikis was also confirmed by Beaulieu et al. (1992), who on a molecular basis detected β1-integrin expression changes in small intestine villi and crypts. This expression failure produces epithelial cell loss and desquamation characteristic for anoikis. Probstmeier et al. (1990) connects epithelium adhesivity lost in the apical part of the intestinal villi with J1/tenascin expression and function change. But Frisch & Francis (1994) unambiguously confirmed the importance of the extracelullar matrix and epithelia interaction in the apoptosis process. Epithelial cell death is triggered by epithelium desquamation, where anoikis plays a part. Also in our work we have noticed DNA fragmentation in nuclei of epithelial cells located over subepithelial edema, and in desquamated cells in the intestinal lumen. A significant apoptotic index using TUNEL was observed in intestinal epithelia after 1 h ischemia and 4 h reperfusion (14.71%). Wu et al. (2004) noticed an apoptotic index of 3.7 ± 0.2 in the control group, which may be connected with the above-mentioned mucosa layer regeneration. Significant apoptotic index increase (30.6 ± 1.5) was found in the group with 1 h ischemia and 1 h of reperfusion. According to Noda et al. (1998), 1 h ischemia and 1 h reperfusion causes 20.7 ± 2.1 % DNA fragmentation decreasing to 2.5 ± 0.3 % in the 6th hour after reperfusion. In our study, decrease to basal apoptotic index level was seen 24 h after reperfusion. This difference may be caused by different rat strains and different preoperational care (e.g. food cessation, premedication or anaesthesia) and with overall surgical capacity especially the maintaining of the lymphatic supply.
Apoptosis in intestinal ischemic-reperfusion injury In conclusion, the present study shows that maximum histopathological injury index of intestinal mucosa was induced by 1 h ischemia followed by 1 h of reperfusion. The epithelium and lamina propria were predominantly injured. The new information in our study includes the very rapid regenerations of intestinal mucosa after IR injury in our experimental model in comparison with other studies (Zheng et al. 1997) and the determination of early cytoplasmic irreversible changes in jejunal epithelium induced by 1 h of ischemia and 1 h of reperfusion. Apoptosis and its phenomenon anoikis played one of the key roles in the intestinal IR injury. Using the various immunohistochemistry methods for apoptosis detection, our study also traced the development of the apoptotic process in terms of time point and cell compartmentation. The M30 CytoDEATH immunohistochemistry method was used in the early stages of apoptosis detection, which was present in the epithelial cell cytoplasm (cytoskeletal reorganization and damage). The in situ TUNEL detection method was the most sensitive and specific for intermediate and late stages of the apoptotis detection, which is present in the nucleus (systemic internucleosomal DNA fragmentation). Detection of apoptotic bodies with routine H&E-staining is simple method for morphologic orientation, which rightly and fully corresponds with the immunohistochemistry methods. Intestinal apoptosis induced by IR injury and mucosa recovery is a rapid process, what was confirmed by MI increase, histopathological injury score and apoptotic index decrease. In the small intestine restitutio ad integrum was established after IR injury. The most significant degree of mucosal regeneration occurred 24 h after reperfusion. Acknowledgements We gratefully acknowledge the material and technical assistance and support of Drs. S. Czikková, D. Fabian and Š. Čikoš from the Institute of Animal Physiology, Slovak Academy of Sciences, Košice. We also thank A. Hantkeová, Ľ. Háberová and A. Horňáková for their technical assistance. This work was supported by the grant VEGA No. 1/2284/05. References Beaulieu J.F. 1992. Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine. J. Cell. Sci. 102: 427–436. Carden D.L. & Granger D.N. 2000. Pathophysiology of ischaemiareperfusion injury. J. Pathol. 190: 255–266. Chen W., Fu X.B., Ge S.L., Sun T.Z., Li W.J. & Sheng Z.Y. 2005. Acid fibroblast growth factor reduces rat intestinal mucosal damage caused by ischemia-reperfusion insult. World J. Gastroenterol 11: 6477–6482. Chu P.G. & Weiss L.M. 2002. Keratin expression in human tissues and neoplasms. Histopathology 40: 403–439. Fink S.L. & Cookson B.T. 2005. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect. Immun. 73: 1907–1916. Fouquet S., Lugo-Martinez V.H., Chambaz J., Cardot P., Pincon-Raymond M. & Thenet S. 2004. Control of the survival/apoptosis balance by E-cadherin: role in enterocyte anoikis. J. Soc. Biol. 198: 379–383.
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