Ischemia/Reperfusion Injury in the Stomach: Role of Oxygen-Derived Free Radicals and Complement Regulatory System
Takashi Joh, Tadayuki Oshima, Satoshi Tanida, Makoto Sasaki, Hiromi Kataoka, and Makoto Itoh
Key words. Ischemia/Reperfusion, Free radical, Complement, 51Cr-EDTA clearance, Decay-accelerating factor (DAF)
It is well recognized that ischemia/reperfusion (I/R) causes gastric epithelial damage during the reperfusion period by oxygen free radicals-mediated mechanism. This issue concerning stomach was first reported in the rat hem- orrhagic shock model by Itoh and Guth in 1985 [1]. After this report, many scientists developed new experimental models to investigate further mecha- nisms for gastric I/R damage. We established a new local gastric I/R model [2], in which gastric epithelial damage was assessed by monitoring the blood- to-lumen clearance of 51Cr-labeled ethylenediamine tetraacetic acid (51Cr- EDTA). We also developed a guinea pig model to study healing process from gastric mucosal erosion caused by I/R [3]. Using these models, we have inves- tigated many biological factors which may act on path-physiology of gastric I/R injury such mucous, NO, complements, and complement regulatory protein. In this chapter, we mention a little bit about history, and discuss the role of complement and the complement regulatory system in gastric mucosal damage caused by I/R.
Role of Oxygen-Derived Free Radicals in Gastric Ischemia/Reperfusion Injury
Figure 1 shows mucosal lesions which were induced in the first model for gastric I/R injury reported by Itoh and Guth in 1985 [1]. In this study, 0.1 N HCl (1 ml) was instilled into the pylorus-ligated stomach of the anesthetized
73 Department of Internal Medicine and Bioregulation, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho, Nagoya 467-8601, Japan
rat, and then the rat was bled to reduce the blood pressure to less than 30mmHg. The blood pressure was maintained at less than 30 mmHg for 20min, and then the shed blood was retransfused. The area of gastric mucosal lesions was measured 20 min after the retransfusion (Fig. 1).
This study was designed to determine whether oxygen-derived free radi- cals play a role or not. For this purpose, allopurinol (an inhibitor of xanthine oxidase), superoxide dismutase (SOD; a scavenger of superoxide radicals), and dimethyl sulfoxide (DMSO; a scavenger of hydroxyl radicals) were used. Both allopurinol and SOD significantly protected against hemorrhagic shock- induced gastric lesions, although DMSO did not (Fig. 2). These results indi- cated that oxygen-derived free radicals produced in the xanthine/xanthine oxidase system [4] play an important role in the formation of gastric lesions produced by I/R plus HCl.
In 1994, we evaluated the damage to the gastric epithelium produced by local I/R without luminal instillation of 0.1 N HCl [2]. Local gastric ischemia was induced by clamping the left gastric artery which induced a 67%
reduction in blood flow only in the corpus. No measurable gross lesion was observed in this model, but epithelial damage was clearly detected by mea- surement of the blood-to-lumen clearance of 51Cr-EDTA. In the absence of Fig. 1. Hemorrhagic shock-induced gastric lesions in the rat
Fig. 2. Effects of oxygen radical scav- engers on gastric ischemia/reperfusion (I/R) injury. SOD, superoxide dismutase;
DMSO, dimethyl sulfoxide
exogenous acid, the histological damage was also minimum and could not be quantified. However, a significant increase in 51Cr-EDTA clearance was observed shortly after reperfusion in a manner that depended on the dura- tion of ischemia. This increase in clearance reached a maximum approxi- mately 10 min after reperfusion and returned rapidly toward control levels within 40–50 min after reperfusion in a manner consistent with the concept of restitution (Fig. 3). Allopurinol, SOD, and DMSO significantly reduced this leakage of51Cr-EDTA after gastric I/R. These results indicated that this model also supported the xanthine/xanthine oxidase hypothesis [4].
Role of Complement Regulatory System
It is known that I/R-related factors such as acidosis, platelet activating factor, and anoxia can activate the alternative pathway of complement, and that once activated, complement can cause tissue damage [5,6]. During the activation process, anaphylatoxins (C3a and C5a) are generated. These fragments of complement strongly activate neutrophils, bosophils and mast cells.
An endogenous membrane-binding complement regulatory protein, decay- accelerating factor (DAF), protects host tissues from damage mediated by Fig. 3. Gastric epithelial damage induced by clamping left gastric artery
complement activation. Mucosal DAF is known to be upregulated in various inflammations including ulcerative colitis. However, gastric mucosal expres- sion of DAF after ischemia has not been well investigated. We therefore evaluated the role of complement/complement regulatory factors in damage to the gastric mucosa caused by I/R [3–6].
In these experiments, male Hartley guinea pigs (250–300 g) were used.
Animals were fasted overnight and anesthetized. Gastric ischemia was induced by clamping the left gastric and epiploic arteries for 30 min. Prior to sacrifice, stomachs were removed 0, 6, 12, 24 h, and 3 and 7 days after I/R.
Complement was suppressed by intraperitoneal administration of cobra venom factor (CVF). Pretreatment with CVF (50 units) prior to I/R reduced the serum complement value (CH50) to an undetectable level within 20–24 h of injection. Immunohistochemical studies of DAF, were performed with a specific monoclonal antibody to guinea pig DAF after local gastric I/R. In guinea pigs, alternative splicing of DAF mRNA generates both glycosylphos- phatidylinositol (GPI)-anchored types and transmembrane types (TM) of DAF (Fig. 4). The DAF mRNA and its isoforms in gastric tissues after I/R were examined by reverse transcription–polymerase chain reaction and Northern blotting analysis. Localization of gastric DAF mRNA expression after I/R was also evaluated by in situ hybridization.
To investigate isoforms expressed in the normal gastric epithelium by DAF, we separated epithelium from the remnant stromal tissue using a crypt isolation procedure [7]. Reverse transcription–polymerase chain reaction demonstrated that the GPI-anchored isoforms are predominant over the TM
Fig. 4. mRNAs for guinea pig decay-accelerating factor (DAF) isoforms and primer sets.
GPI, glycosylphosphatidylinositol; UT, untranslated
isoforms in all digestive epithelium. However, there was no difference between GPI-anchored type and TM type in the stromal tissue (Fig. 5).
Decay-accelerating factor expression was transiently upregulated after I/R.
The DAF mRNA level in gastric tissues was found to be highest at 6 h after I/R, returning to the baseline at 24 h (Fig. 6). The DAF protein in gastric tissues was highest at 24 h after I/R, returning to the baseline 6–7 days after I/R.
Strong DAF mRNA expression detected by in situ hybridization was observed in the cytoplasm of cells beneath the eroded tissues 6 h after I/R (Fig. 7).
Reverse transcription–polymerase chain reaction analysis revealed that mRNAs of the TM types had become significantly dominant by 6 h after I/R, while levels of the GPI-anchored types remained unchanged (Fig. 8). In guinea pigs depleted of complement by CVF treatment, the area of erosion and the upregulation of DAF expression after I/R were significantly limited (Fig. 6).
In summary, these data indicated that complement may play an important role in gastric I/R damage via their direct cytotoxic effects or their activation of white blood cells. The GPI-anchored form of DAF may play a protective role in normal gastrointestinal epithelial cells, while the TM-isoform of DAF might play an important role as an acute phase reactant at the site of inflam- mation in guinea pigs.
Fig. 5. Gastric DAF isoforms analyzed by reverse transcription–polymerase chain reaction. TM, transmembrane type; GPI, glycosylphosphatidylinositol anchored type
Fig. 6. Changes in gastric erosion and DAF expression after I/R. CVF, cobra venom factor
References
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2. Kawai T, Joh T, Iwata F, et al (1994) Gastric epithelial damage induced by local I/R with or without exogenous acid. Am J Physiol 266:G263–G270
3. Oshima T, Okada N, Joh T, et al (2000) Decay-accelerating factor in guinea pig stomachs following ischemia reperfusion stress. J Immunol 164:1078–1085
4. Granger DN, Rutili G, McCord JM (1981) Superoxide radicals in feline intestinal ischemia. Gastroenterology 81:22–29
5. Ikai M, Itoh M, Joh T, et al (1996) Complement plays an essential role in shock follow- ing intestinal ischaemia in rats. Clin Exp Immunol 106:156–159
6. Joh T, Ikai M, Oshima T, et al (2001) Complement plays an important role in gastric mucosal damage induced by ischemia/reperfusion in rats. Life Sci 70:109–117 7. Mizoshita T, Joh T, Oshima T, et al (2002) Differential expression of decay-accelerating
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Fig. 7. Changes in DAF expres- sion after I/R
Fig. 8. Decay-accelerating factor isoform analysis after ischemia. TM, transmembrane type; GPI, glyco- sylphosphatidylinositol anchored type
