Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa

Takahiro Noda1, Ryuichi Iwakiri1, Kazuma Fujimoto1, Shuzo Matsuo1, and Tak Yee Aw2

1 Department of Internal Medicine, Saga Medical School, Nabeshima, Saga 849, Japan; and 2 Department of Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Apoptosis after ischemia-reperfusion (I/R) was characterized in rat small intestine. Under halothane anesthesia, the superior mesenteric artery in the rat was occluded for 15 or 60 min, followed by reperfusion. Ratios of fragmented DNA to total DNA, electrophoresis, and immunohistochemical staining were examined after I/R. Some rats were pretreated with alpha -difluoromethylornithine (DFMO) to examine the relationship between intestinal apoptosis and ornithine decarboxylase (ODC) activity. The percentage of fragmented DNA significantly increased just after ischemia and peaked at 1 h after reperfusion in the jejunum and ileum. These increases were significantly higher in the 60-min ischemia group compared with the 15-min ischemia group. This increase decreased 6 h after reperfusion. The results were corroborated by histological evaluations of the intestine under the same conditions. DFMO completely abolished elevation of ODC activity 6 h after reperfusion but did not change the percentage of fragmented DNA. Apoptosis in rat small intestine was induced by ischemia of the gut, and this process was exacerbated by reperfusion. The changes in apoptosis were independent of ODC activity.

percent fragmented DNA; DNA ladder; terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labeling; ornithine decarboxylase

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AN ISCHEMIC INSULT decreases oxygen and nutrient delivery and induces tissue injury. Paradoxically, the introduction of oxygen during reperfusion of ischemic tissues exacerbates this tissue damage. Current evidence shows that several factors can mediate injury to organs exposed to ischemia followed by reperfusion, including reactive oxygen species and inflammatory leukocytes (13, 21). The small intestine is highly sensitive to ischemia-reperfusion (I/R) (13). We previously demonstrated that occlusion of the superior mesenteric artery (SMA) followed by reperfusion causes injury to the jejunum and ileum (10, 21).

In recent years, there has been a renewed interest in the study of programmed cell death (apoptosis), a new concept of cell death proposed by Kerr et al. in 1972 (19). Apoptosis occurs in many tissues, including the gastrointestinal tract (14, 17, 24). In contrast to necrosis, apoptosis is an active process of gene-directed cellular self-destruction, and it is characterized by morphological changes such as cell shrinkage with remaining intact organelles, condensation of chromatin into crescent caps at the nuclear periphery, and eventual fragmentation of the nucleus and cytoplasm to form apoptotic bodies (1, 20, 26, 30).

We previously demonstrated a repairing process of the intestinal mucosa after I/R in rats that involved induction of ornithine decarboxylase (ODC) (9, 10). In these studies, ODC activity increased markedly after I/R in the intestinal mucosa, and increased ODC activity played an important role in repair of the intestinal mucosa damaged by I/R.

Recent evidence has shown that ischemia and/or I/R induces apoptosis in several tissues, such as the brain (15), the myocardium (16), and the liver (11). Previous studies using histological evaluations have described a spontaneous level of apoptosis as well as radiation- and chemical-induced apoptosis in small intestinal cells (14, 17, 24). However, an effect of I/R on apoptosis has not been demonstrated. The objectives of this study were to determine 1) whether I/R followed by occlusion of the SMA induces apoptosis in the small intestine and 2) whether increased ODC after I/R affects apoptosis in the small intestine.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Surgery. Male Sprague-Dawley rats (280-330 g) were used in this study. Animals were housed in wire-bottomed cages placed in a room illuminated from 0800 to 2000 (12:12-h light-dark cycle) and maintained at 21 ± 1°C. Rats were allowed access to water and chow ad libitum. Under halothane anesthesia, a laparotomy was performed. We previously demonstrated that occlusion of the SMA markedly reduced blood flow to the jejunum and the ileum but not to the duodenum (10); therefore, the SMA was occluded for 15 or 60 min with a micro-bulldog clamp. At the end of the ischemic period, the clamp was released and three drops of lidocaine were applied directly on the SMA to facilitate reperfusion.

Collection of intestinal mucosa. The animals were anesthetized and then euthanized. The entire small intestine was carefully removed and placed on ice. The oral 5 cm was taken as the duodenum, and the rest of the intestine was divided into two equal segments, representing the proximal (jejunum) and distal (ileum) segments. Each segment was rinsed thoroughly with normal saline (0.9% NaCl) and opened longitudinally to expose the intestinal epithelium. The mucosal layer was harvested by gentle scraping of the epithelium, using a glass slide.

DNA fragmentation assay. The mucosal scrapings were processed immediately after collection to minimize nonspecific DNA fragmentation. The amount of fragmented DNA was determined as previously described (2), with modification. Mucosal scrapings of the different intestinal segments were homogenized in 10 vol of a lysis buffer (pH 8.0) consisting of 5 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, 20 mM EDTA, and 0.5% (wt/vol) Triton X-100 (Sigma, St. Louis, MO). Aliquots (1 ml) of each sample were centrifuged for 20 min at 27,000 g to separate the intact chromatin (pellet) from the fragmented DNA (supernatant) (29). The supernatant was decanted and saved, and the pellet was resuspended in 1 ml of Tris buffer (pH 8.0) with 10 mM Tris · HCl and 1 mM EDTA. The pellet and supernatant fractions were assayed for DNA content, using diphenylamine reaction as previously described (4). The results are expressed as the percentage of fragmented DNA.

Purification of mucosal DNA and agarose gel electrophoresis. DNA was extracted from the 27,000 g fraction (29). The fragmented DNA from the various fractions was extracted with a phenol-chloroform-isoamyl alcohol mixture (25:24:1, vol/vol/vol) sequentially to remove protein. The protein-free DNA extracts were treated with 100% ethanol in 0.1 M sodium acetate at -20°C overnight to purify the DNA. The precipitated DNA was washed with 70% ethanol and resuspended in Tris buffer (pH 8.0) with 10 mM Tris · HCl and 10 mM EDTA. DNA samples were incubated with 100 µg/ml ribonuclease for 15 min at 37°C to remove RNA. Resolving agarose gel electrophoresis was performed with 1.5% gel strength containing 1.0 µg/ml ethidium bromide. Depending on the experiment, 20 µg DNA per well was loaded. DNA standards (0.5 µg per well) were included to identify the size of the DNA fragments. Electrophoresis was performed for 2 h at 70 V, and DNA was visualized by ultraviolet fluorescence.

ODC assay. ODC activity was assayed by a radiometric technique (27). Mucosal scrapings were placed in 2 ml of 0.1 M Tris buffer (pH 7.4) containing 1 mM EDTA, 50 µM pyridoxal 5-phosphate, and 5 mM dithiothreitol. The tissues were homogenized twice with a Polytron tissue homogenizer for 15 s and centrifuged at 30,000 g for 30 min. Protein content was determined, and a 200-µl aliquot of the supernatant was incubated in stoppered vials in the presence of 3.5 nmol of L-[1-14C]ornithine (52.3 mCi/mmol; New England Nuclear, Boston, MA) for 15 min at 37°C. The 14CO2 liberated by the decarboxylation of ornithine was trapped on filter paper impregnated with 20 µl of 2 N NaOH, which was suspended above the reaction mixture. The reaction was stopped by the addition of 0.3 ml 10% trichloroacetic acid. Radioactivity of the 14CO2 trapped in the filter paper was measured in an aqueous miscible scintillant (Opti-Fluor; Packard Instruments, Downers Grove, IL). The samples were counted for 5 min in a liquid scintillation spectrometer (460 CD; Packard Instruments). Results are expressed as picomoles CO2 per milligram protein per hour.

Immunohistochemical staining. Tissue samples were removed from the jejunum and ileum and immediately fixed in 10% neutral buffered Formalin. The samples were then embedded in paraffin and sectioned. Fragmented DNA was stained by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL) method (12) with modification using the Apop Tag kit (Oncor, Gaithersburg, MD). Specimens were dewaxed and immersed in phosphate-buffered saline containing 0.3% hydrogen peroxide for 10 min at room temperature and then incubated with 20 µg/ml proteinase K for 15 min at room temperature. Seventy-five microliters of equilibration buffer were applied directly onto the specimens for 10 min at room temperature, followed by 55 µl of TdT enzyme and incubation, which were then incubated at 37°C for 1 h. The reaction was terminated by transferring the slides to prewarmed stop/wash buffer for 30 min at 37°C. The specimens were covered with a few drops of rabbit serum and incubated for 20 min at room temperature and then covered with 55 µl of anti-digoxigenin peroxidase and incubated for 30 min at room temperature. Specimens were then soaked in Tris buffer containing 0.02% diaminobenzidine and 0.02% hydrogen peroxide for 1 min to achieve color development. Finally, the specimens were counterstained by immersion in hematoxylin.

Statistics. Results are expressed as means ± SE. Data were evaluated by analysis of variance in which multiple comparisons were carried out by the method of least significant difference. Differences were considered significant if P < 0.05.

Experiment 1. Effect of I/R on apoptosis in the small intestine. The animals were anesthetized with halothane and killed at 0, 1, 3, and 6 h after the SMA occlusion for 15 or 60 min. In control (sham) animals, the SMA was isolated in a similar fashion but was not occluded. Six animals were studied in each time point in both groups.

Experiment 2. Effect of DFMO on ODC activity and apoptosis after I/R. After the ischemic insult, rats were prepared by introducing a silicon infusion tube ~2 cm down the duodenum through the fundus of the stomach for infusion of DFMO.

During the reperfusion period, the animals in restraining cages were infused intraduodenally at 3 ml/h with saline (vehicle) or 2% DFMO dissolved in saline. DFMO was generously provided by Merrell Dow Research Institute (Cincinnati, OH). Rats were anesthetized with halothane and killed at 1 or 6 h after occlusion of the SMA for 15 or 60 min. Seven animals were studied in each group.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Experiment 1. Effect of I/R on apoptosis in the small intestine. Results on the percentage of fragmented DNA in the duodenal mucosa before and after I/R are shown in Fig. 1A. Neither ischemia nor reperfusion elicited duodenal DNA damage, and there was no difference between the 15-min and 60-min ischemia groups.


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Fig. 1.   Percentage of fragmented DNA in the intestinal mucosa after ischemia-reperfusion (I/R). Values are means ± SE. A: duodenum. B: jejunum. C: ileum. Superior mesenteric artery (SMA) was occluded for either 15 min (open circle ) or 60 min (bullet ) and then reperfused. a P < 0.05 compared with corresponding control value. b P < 0.05 compared with value at end of ischemic period. c P < 0.05 compared with 15-min ischemia group.

Figure 1B shows the ratio of fragmented DNA to total DNA after I/R in the jejunal mucosa. In the 15-min and 60-min ischemia groups, the percentage of fragmented DNA increased just after ischemia (15 min, 6.9 ± 3.4%; 60 min, 8.8 ± 2.9%) compared with the values before ischemia (15 min, 1.4 ± 0.2%; 60 min, 1.7 ± 0.4%; P < 0.05 in each group), indicating that the percentage of fragmented DNA increased during ischemia. In the 60-min ischemia group, the percentage of fragmented DNA peaked at 1 h after reperfusion (20.7 ± 2.1%; P < 0.05, compared with the value just after 60 min ischemia), and by 6 h after reperfusion, the percentage of fragmented DNA returned to baseline levels (2.0 ± 0.3%). In comparison, 15 min ischemia also resulted in enhanced DNA fragmentation, which peaked between 0 and 1 h (7.8 ± 2.1%) during reperfusion (P < 0.05 vs. the value before ischemia). However, the magnitude of DNA damage at these times was less than that in the 60-min ischemia group (P < 0.05). As it was for the 60-min ischemia group, DNA fragmentation in the 15-min ischemia group returned to baseline levels at 6 h reperfusion (1.7 ± 0.3%).

Figure 1C shows the results on DNA fragmentation in the ileal mucosa. In the 15-min and 60-min ischemia groups, the percentage of fragmented DNA significantly increased just after ischemia (15 min, 4.9 ± 1.0%; 60 min, 12.3 ± 2.0%) compared with the value before ischemia (15 min, 1.5 ± 0.2%; 60 min, 2.4 ± 0.4%; P < 0.05 in each group). Increases in the percentage of fragmented DNA peaked at 1 h after I/R in both groups (15 min, 10.0 ± 1.0%; 60 min, 25.3 ± 4.8%; P < 0.05 in each group compared with the value just after ischemia), and this increase returned to the value before the ischemia at 6 h after I/R (15 min, 2.8 ± 0.4%; 60 min, 3.1 ± 0.4%). The increase in the percentage of fragmented DNA in the 60-min ischemia group was larger than that in the 15-min ischemia group (P < 0.05), similar to that seen in the jejunum (Fig. 1B).

Resolving agarose gel electrophoresis was performed to evaluate the nature of the fragmented DNA in the jejunal and ileal mucosa after I/R. As shown in Fig. 2, A and B, agarose gel electrophoresis of the fragmented DNA obtained from the jejunal mucosa at 15- and 60-min ischemia, respectively, revealed distinct DNA ladders. These ladders are characteristic of apoptosis (29), although they are not useful for quantitative analysis because the same doses of fragmented DNA were loaded onto each lane. Similar DNA laddering on agarose gel electrophoresis was obtained from ileal mucosa of animals subjected to 15 and 60 min I/R (data not shown).


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Fig. 2.   Agarose gel electrophoresis of fragmented DNA from jejunal mucosa after I/R. A: jejunal mucosa of 15-min ischemia group. B: jejunal mucosa of 60-min ischemia group. Twenty micrograms of fragmented DNA were loaded. Lanes 1-4, DNA before ischemia and 0, 1, and 6 h after reperfusion, respectively. The ladder, which is characteristic of apoptosis, was clearly shown on each lane in both the 15-min and 60-min ischemia groups. Lanes 5 and 6 contained marker DNA from phi ×174 Hae III and Lambda EcoR III digest (Wako Pure Chemical), respectively.

The immunohistochemical staining (TUNEL) of jejunal segments before and after I/R is shown in Fig. 3 (15 min ischemia) and Fig. 4 (60 min ischemia). Before I/R, few apoptotic cells were observed at the villus tips (Figs. 3A and 4A). Immediately after ischemia, the number of apoptotic cells increased in both the 15-min ischemia group (Fig. 3B) and the 60-min ischemia group (Fig. 4B). The number of apoptotic cells increased at 1 h after I/R (15-min ischemia group, Fig. 3C; 60-min ischemia group, Fig. 4C). Interestingly, only a few apoptotic cells were seen in the jejunal mucosa 6 h after I/R in both groups (15-min ischemia group, Fig. 3D; 60-min ischemia group, Fig. 4D). These histological findings on I/R-induced apoptosis corroborated the data obtained using biochemical determination of DNA fragmentation.


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Fig. 3.   Light micrographs of upper jejunum stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method before and after 15 min occlusion of rat SMA. A: before ischemia. B: immediately after 15 min ischemia. C: 1 h after reperfusion. D: 6 h after reperfusion. Magnification, ×50. Stained cell bodies are apoptotic cells.


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Fig. 4.   Light micrographs of upper jejunum stained by TUNEL method before and after 60 min occlusion of rat SMA. A: before ischemia. B: immediately after 60 min ischemia. C: 1 h after reperfusion. D: 6 h after reperfusion. Magnification, ×50. Stained cell bodies are apoptotic cells.

The histological evaluations further revealed that damage to the small intestine in the 15-min ischemia group was small, with slightly edematous villus tips just after the ischemic period and throughout the period 1-6 h after I/R. In contrast, the histology of the jejunal segment of the 60-min ischemia group showed substantial destruction of the mucosal layer at 1 h postischemia. Additionally, mucosal integrity was partially restored at 6 h after the I/R insult.

Experiment 2. Effect of DFMO on ODC activity and apoptosis after I/R. Table 1 summarizes the ODC activity and the percentage of fragmented DNA in the jejunum before and 6 h after I/R. The ODC activity markedly increased at 6 h after I/R in the vehicle-treated control group, and this increase of ODC activity was completely inhibited by administration of DFMO. In contrast to differences in ODC activity, the percentage of fragmented DNA did not differ significantly between vehicle-treated controls and DFMO-treated animals, indicating that increased ODC activity was not correlated with induction of apoptosis after I/R.

                              
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Table 1.   Effect of DFMO on the percentage of fragmented DNA and ODC activity in rat jejunal mucosa after I/R

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although it is well known that the intestinal mucosa is highly sensitive to I/R (13), the relationship between I/R-induced apoptosis and tissue injury in the small intestine is not clear. Several recent studies have shown that I/R induces apoptosis in the brain (15), the myocardium (16), and the liver (11). Additionally, the severity of apoptotic cell death in the brain was shown to be proportional to the duration of the ischemic period (15). In the current study, we found that I/R, following occlusion of the SMA, induced apoptosis in the jejunum and ileum. Apoptosis did not occur in the duodenum, where the blood flow was not decreased after occlusion of the SMA (10). The percentage of fragmented DNA (quantified as the proportion of fragmented DNA to total DNA) increased during ischemia and was maximal at 1 h after reperfusion. Furthermore, the magnitude of DNA fragmentation was directly proportional to the duration of the ischemic insult, as evidenced by a significantly higher percentage of fragmented DNA after 60-min ischemia compared with 15-min ischemia. The biochemical quantification of apoptosis was corroborated with histological examination using immunohistochemical staining of apoptotic cells by the TUNEL method.

It is notable that induction of apoptosis by intestinal ischemia occurred during the ischemic phase and continued into the early reperfusion phase. The finding that maximal DNA fragmentation occurred at 1 h postreperfusion followed by a return to baseline values by 6 h suggests that I/R induction of intestinal apoptosis and mucosal recovery are rapid processes. This suggestion is consistent with previous findings that apoptotic cells can appear within a few minutes after a stimulus (30) and the whole sequence of apoptosis was often completed within 1-4 h (3, 6). Our present results are consistent with I/R initiation of apoptosis in the small intestine during the ischemic period and continued into the first few hours of reperfusion. By 6 h, DNA fragmentation was low and returned to baseline values, indicating a cessation of apoptosis and restoration of the injured mucosa. The reason for this interesting kinetics of induction of mucosal cell death and restoration is unclear. There are two possible explanations. First, it may be that there is a time-dependent increase in apoptosis-promoting factors during ischemia and early phases of reperfusion, which rapidly decreased with prolonged reperfusion. Second, corresponding to the decline in apoptosis-promoting factors, there may be simultaneous induction of inhibitors of apoptosis as well as promoters of tissue repair during the later phase of reperfusion.

In the present study we hypothesized that increased polyamine levels may be responsible for modulating apoptosis because ODC activity increased markedly after an I/R stimulus. Previous studies have documented that some growth factors inhibit apoptosis in many organs (28). The effect of polyamines or ODC on apoptosis appears to be contradictory. After fasting, apoptosis of the rat intestinal mucosa increased when ODC activity was suppressed (17). Circadian variation of apoptosis in the rat intestinal mucosa mirrored ODC activity, i.e., apoptosis was induced in the light period (17), whereas ODC activity was increased in the dark period (8). Desiderio et al. (7) showed that the ODC activity was transiently elevated and polyamine levels rapidly fell below control values in apoptotic thymocytes, whereas ODC elevation was long-lasting and increases of polyamines were persistent in proliferating thymocytes. Packham and Cleveland (23) reported that, in vitro studies using cultured cells, ODC mediated c-myc-induced apoptosis that was blocked by DFMO. In our current study, the increase in ODC activity in the small intestine after I/R was completely abolished by DFMO. However, the percentage of fragmented DNA was not affected by DFMO treatment, indicating that suppression of increased ODC activity by DFMO did not enhance apoptosis. Thus these results indicate that ODC activity did not influence apoptosis in the small intestine after I/R.

The mechanism of I/R-induced apoptosis and the factors that control the ischemic vs. the reperfusion phases are not known. Several studies have shown that free radicals can cause apoptosis (5, 25), which would be consistent with enhanced oxyradical generation during I/R. However, other reports show the lack of an association of oxygen radicals with enhanced apoptosis (22). What is clear is that the regulation of intestinal cell death and proliferation is highly complex and is likely to be controlled by a variety of factors (18). Thus further study is needed to identify and delineate these regulatory factors. In particular, the mechanism of I/R-induced apoptosis and subsequent tissue repair warrants further exploration. Regardless of the mechanism, the present study shows that I/R substantially causes intestinal cell death by the process of apoptosis and that the injured intestinal mucosa is capable of rapid and significant recovery during reperfusion.

    ACKNOWLEDGEMENTS

We thank Dr. E. H. W. Bohme of Marion Merrell Dow Research Institute for the generous supply of alpha -difluoromethylornithine.

    FOOTNOTES

This research was supported in part by Grants-in-Aid for Scientific Research 07670600 (R. Iwakiri) and 08670601 (K. Fujimoto) from the Ministry of Education of Japan, and by a grant from Takeda Science Foundation (K. Fujimoto). T. Y. Aw is the recipient of an American Heart Association Established Investigatorship Award (National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-44510).

Address for reprint requests: R. Iwakiri, Division of Gastroenterology, Dept. of Internal Medicine, Saga Medical School, 5-1-1 Nabeshima, Saga 849, Japan.

Received 28 February 1997; accepted in final form 29 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Arends, M. J., and A. H. Wyllie. Apoptosis. Mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 2: 223-254, 1991.

2.   Aw, T. Y., P. Nicotera, L. Manzo, and S. Orrenius. Tributyltin stimulates apoptosis in rat thymocytes. Arch. Biochem. Biophys. 283: 46-50, 1990[Medline].

3.   Bursch, W., S. Paffe, B. Putz, G. Barthel, and R. Schulte-Hermann. Determination of the length of the histological stages of apoptosis in normal liver and in altered hepatic foci of rats. Carcinogenesis 11: 847-853, 1990[Abstract].

4.   Burton, K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: 315-323, 1956.

5.   Buttke, T. M., and P. A. Sandstrom. Oxidative stress as a mediator of apoptosis. Immunol. Today 15: 1-4, 1994[Medline].

6.   Coles, H. S. R., J. F. Burne, and M. C. Raff. Large-scale normal cell death in the developing rat kidney and its reduction by epidermal growth factor. Development 118: 777-784, 1993[Abstract/Free Full Text].

7.   Desiderio, M. A., E. Grassilli, E. Bellesia, P. Salomoni, and C. Franceschi. Involvement of ornithine decarboxylase and polyamines in glucocorticoid-induced apoptosis of rat thymocytes. Cell Growth Differ. 6: 505-513, 1995[Abstract].

8.   Fujimoto, K., D. N. Granger, L. R. Johnson, V. H. Price, T. Sakata, and P. Tso. Circadian rhythm of ornithine decarboxylase activity in small intestine of fasted rats. Proc. Soc. Exp. Biol. Med. 100: 409-413, 1992.

9.   Fujimoto, K., D. N. Granger, V. H. Price, and P. Tso. Ornithine decarboxylase is involved in repair of small intestine after ischemia-reperfusion in rats. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G523-G529, 1991[Abstract/Free Full Text].

10.   Fujimoto, K., V. H. Price, D. N. Granger, R. Specian, S. Bergstedt, and P. Tso. Effect of ischemia-reperfusion on lipid digestion and absorption in rat intestine. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G595-G602, 1991.

11.   Fukuda, K., M. Kojiro, and J. F. Chiu. Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 7777 tissue: apoptosis or necrosis? Am. J. Pathol. 142: 935-946, 1993[Abstract].

12.   Gavrieli, Y., Y. Sherman, and S. A. Ben-Sasson. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119: 493-450, 1992[Abstract].

13.   Granger, D. N., and R. J. Korthuis. Physiologic mechanisms of postischemic tissue injury. Annu. Rev. Physiol. 57: 311-332, 1995[Medline].

14.   Han, H., T. Iwanaga, and T. Fujita. Species differences in the process of apoptosis in epithelial cells of the small intestine: an ultrastructural and cytochemical study of luminal cell elements. Arch. Histol. Cytol. 56: 83-90, 1993[Medline].

15.   Hill, I. E., J. P. MacManus, I. Rasquinha, and U. I. Tuor. DNA fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat. Brain Res. 676: 398-403, 1995[Medline].

16.   Itoh, G., J. Tamura, M. Suzuki, Y. Suzuki, H. Ikeda, M. Koike, M. Nomura, T. Jie, and K. Ito. DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am. J. Pathol. 146: 1325-1331, 1995[Abstract].

17.   Iwakiri, R., and T. Y. Aw. Apoptosis in rat small intestine: circadian rhythm and effect of feeding and fasting (Abstract). Gastroenterology 108: A292, 1995.

18.   Johnson, L. R., and S. A. McCormack. Regulation of gastrointestinal mucosal growth. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 611-641.

19.   Kerr, J. F. R., A. H. Wyllie, and A. R. Currie. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26: 239-257, 1972[Medline].

20.   Kroemer, G., P. Petit, N. Zamzami, J. L. Vayssiére, and B. Mignotte. The biochemistry of programmed cell death. FASEB J. 9: 1277-1287, 1995[Abstract/Free Full Text].

21.   Kurtel, H., K. Fujimoto, B. J. Zimmerman, D. N. Granger, and P. Tso. Ischemia-reperfusion-induced mucosal dysfunction: role of neutrophils. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G490-G496, 1991[Abstract/Free Full Text].

22.   Muschel, R. J., E. J. Bernhard, L. Garza, W. G. McKenna, and C. J. Koch. Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling. Cancer Res. 55: 995-998, 1995[Abstract].

23.   Packham, G., and J. L. Cleveland. Ornithine decarboxylase is a mediator of c-myc-induced apoptosis. Mol. Cell. Biol. 14: 5741-5747, 1994[Abstract].

24.   Potten, C. S. The significance of spontaneous and induced apoptosis in the gastrointestinal tract of mice. Cancer Metastasis Rev. 11: 179-195, 1992[Medline].

25.   Ratan, R. R., and J. M. Baraban. Apoptotic death in an in vitro model of neuronal oxidative stress. Clin. Exp. Pharmacol. Physiol. 22: 309-310, 1995[Medline].

26.   Searle, J. J. F., R. Kerr, and C. J. Bishop. Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol. Annu. 17: 229-259, 1982[Medline].

27.   Tabata, K., and L. R. Johnson. Mechanism of induction of mucosal ornithine decarboxylase by food. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G370-G374, 1986[Medline].

28.   Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456-1462, 1995[Medline].

29.   Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284: 555-556, 1980[Medline].

30.   Wyllie, A. H., J. F. R. Kerr, and A. R. Currie. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68: 251-306, 1980[Medline].


AJP Gastroint Liver Physiol 274(2):G270-G276
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