Indigestible Material Attenuated Changes in Apoptosis in the Fasted Rat Jejunal Mucosa

Animals and Surgery.

Male Sprague-Dawley rats were used in this study. Rats were housed individually in wire-bottomed cages to prevent coprophagia. They were placed in a room illuminated from 08:00 a.m. to 08:00 p.m. (12:12-hr light:dark cycle). Animals were allowed free access to water and food ad libitum. At 7 weeks of age, rats were divided into three groups: (i) one group was fed ad libitum, 2) a second group was subjected to 72 hrs of fasting, and (iii) a third group was fed expanded polystyrene after 24 hrs of fasting. Expanded polystyrene was not digested in the rat gastrointestinal tract (13). Additional rats underwent truncal vagotomy one week before the experiment as previously described (14). Briefly, the esophagus was gently lifted just below the liver and diaphragm, and the mesentery and visible vagal fibers were cut within 2 cm of the esophagus. Six rats were tested in each group. In the fasting group, food was removed for 72 hrs until the start of the experiment. All animal protocols were approved by the Institutional Animal Review Board of Saga Medical School.

Collection of Intestinal Mucosa.

After fasting or eating expanded polystyrene, animals were anesthetized and then euthanized by exsanguination. The entire small intestine was carefully removed and placed on ice. The oral 10-cm part of the intestine was treated as the duodenum, and the rest of the intestine was divided into two equal segments representing proximal (jejunum) and distal (ileum) segments, and the jejunum was used in this study. Some pieces, approximately 2 cm in length, were resected from the middle portion of each segment and fixed in 10% neutral buffered formalin for measurement of mucosal height and for immunohistochemistry. Each segment was rinsed thoroughly with physiologic saline and opened longitudinally to expose the intestinal epithelium. The mucosal layer was harvested by gentle scraping of the epithelium with a glass slide as previously described (15). Half of the specimen was used for the DNA fragmentation assay, and the remaining half was used for apoptotic proteins assay with Western blot analysis.

DNA Fragmentation Assay.

Mucosal scrapings were processed immediately after collection to minimize nonspecific DNA fragmentation. Amounts of fragmented DNA were determined as previously described (15, 16) with some modifications. Briefly, mucosal scrapings from different intestinal segments were homogenized in 10 volumes of lysis buffer (5 mM Tris-HCl, 20 mM ethylenediaminetetra-acetic acid [EDTA], and 0.5 % [w/v] Triton X-100, pH 8.0). One-milliliter aliquots of each sample were centrifuged for 20 mins at 27,000 g to separate the intact chromatin (pellet) from the fragmented DNA (supernatant). The supernatant was decanted and saved, and the pellet was resuspended in 1 ml Tris buffer (pH 8.0) with 10 mM Tris-HCl and 1 mM EDTA. Pellet and supernatant fractions were assayed for DNA content by using a diphenylamine reaction as previously described (17). Results were expressed as the percentage of fragmented DNA.

Mucosal Height and Immunohistochemical Staining.

Tissue samples were removed from the jejunum and were immediately fixed in 10% neutral buffered formalin. Samples were then embedded in paraffin and sectioned. Specimens were stained with hematoxylin and eosin. Villus height, crypt height (depth), and mucosal height (villus height plus crypt depth) were measured with a light microscope using micrometer scale standards for fed ad libitum and fasted rats. Fragmented DNA was stained by using the terminal deoxynucleotidyl transferase–mediated dUDP-biotin nick end-labeling (TUNEL) method (18) with some modifications using an Apop Tag Kit (Oncor, Gaithersburg, MD). Specimens were dewaxed and immersed in phosphate-buffered saline containing 0.3% hydrogen peroxide for 10 mins at room temperature and then incubated with 20 μ g/ml proteinase K for 15 mins at room temperature. Seventy-five milliliters of equilibration buffer were applied directly onto the specimens for 10 mins at room temperature; subsequently 55 μ l of terminal deoxynucleotidyl transferase was added, and the mixture was incubated at 37° C for 1 hr. The reaction was terminated by transferring slides to prewarmed stop/wash buffer for 30 mins at 37° C. Specimens were covered with a few drops of rabbit serum, incubated for 20 mins at room temperature, covered with 55 μ l antidigoxigenin peroxidase, and incubated for 30 mins at room temperature. Specimens were then soaked in Tris buffer containing 0.02% diaminobenzidine and 0.02% hydrogen peroxide for 1 min for color development. Finally, specimens were counter-stained with hematoxylin. A minimum of 50 crypts were randomly selected for apoptotic index analysis, and the number of apoptotic cells was calculated. The apoptotic index was determined by dividing the number of cells by the total number of cells in the crypt column and multiplying by 100.

Mucosal cell proliferation was assessed by using 5-bromo-2′-deoxyuridine (5-BrdU) incorporation to identify cells in the S phase of the cell cycle (19). Rats were given 120 mg/kg 5-BrdU (40 g/l 5-BrdU and 4 g/l 5-fluorodeoxyuridine) at 90 mins before sacrifice. 5-BrdU was detected with a monoclonal antibody and a streptavidin–biotin staining system. The number of labeled cells in at least 10 well-oriented longitudinal crypts in each sample was determined under a light microscope by an examiner blinded to the study groups, and the number was reported as the number of 5-BrdU–labeled cells among the total crypt cells.

Isolation of Cytosolic Proteins.

Mucosal scrapings were immediately washed twice with ice cold phosphate-buffered saline (pH 7.4) and centrifuged at 1,000 g for 5 mins at 4° C. Pellets were then resuspended with two volumes of buffer A and lysed at 4° for 30 mins (20). Buffer A consisted of 250 mM sucrose (Sigma, St. Louis, MO), 20 mM N-[2-hydroxyethyl]piperazine-N,-[2-ethanesulfonic acid] (Sigma), 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μ g/ml aprotinin, 10 μ g/ml leuptin, and 1.8 mg/ml iodoacetamide). Homogenates were centrifuged at 1,000 g for 10 mins at 4° C to remove cell nuclei, and supernatants were again centrifuged at 10,000 g for 15 mins at 4° C to remove mitochondria. Supernatants of the 10,000 g spin were further centrifuged at 1,000,000 g for 1 hr at 4° C. The resulting supernatant represented the soluble cytosolic fraction, and pellets were the membranous fraction. Supernatant and pellet fractions of resuspended solutions in buffer were divided into multiple samples, which were frozen at − 80° C until immunoblotting.

Western Blot Analysis of Caspase-3.

Cleaved caspase-3 and procaspase-3 were analyzed in cytosolic fractions. Equal quantities (40 μ g) of lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). After blocking with phosphate-buffered saline containing 0.1% polyoxyethylene sorbitan monolaurate (Tween-20, Sigma) and 5% skim milk at 4° C overnight, the membrane was incubated with rabbit polyclonal anti–caspase-3 antibody (1:1000; Cell Signaling Technology, Danvers, MA) for 1 hr. Antigen-antibody complexes were detected with horseradish peroxidase–conjugated anti–rabbit IgG (1:500, Santa Cruz Biotech, Santa Cruz, CA). Detection of chemiluminescence was performed by using ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK). Densitometeric assessment of bands from the auto-radiogram was carried out by using Image Gauge VDS (Fujifilm, Tokyo, Japan). Band intensities were quantified by measuring the absolute integrated optical intensity, which estimates the band in the lane profile. Results were expressed as a ratio to β-actin densitometry units.

Statistical Analysis.

Results were expressed as the means ± SE. Data were evaluated by analysis of variance in which multiple comparisons were performed by the least significant-difference method. Differences were considered significant if the probability of the difference occurring by chance was less than 5 in 100 (P < 0.05).

Jejunal Mucosal Height in the Rats Fasted for 72 hrs and Fed Expanded Polystyrene.

Jejunal mucosal height in the 72-hr fasted rats (729 ± 10 μ m) was significantly shorter than that of the rats fed ad libitum (936 ± 12 μ m) (Figs. 1 and 2, P < 0.05), and this was mainly due to a decrease in villus height (ad libitum, 698 ± 12 μ m; 72-hr fasting, 510 ± 18 μ m) but was not due to crypt height (ad libitum, 246 ± 23 μ m; 72-hr fasting, 216 ± 13 μ m). The decrease in mucosal height in 72-hr fasted rats was reduced as a result of their eating expanded polystyrene (P < 0.05), but the recovery was not complete as indicated in Figure 2 (872 ± 17 μ m). The average volume of expanded polystyrene eaten by rats was 28 cm³.