Jerzy Stachura

Prostaglandin Protection of Carbon Tetrachloride-Induced Liver Cell Necrosis in the Rat

We studied whether 16,16--dimethyl prostaglandin E2 (dmPGE2) may prevent acute liver damage induced by carbon tetrachloride (CCl4) in the rat. One hundred thirty male rats were divided into the following groups: (1) controls, (2) rats given CCl4 6670 mg/kg body wt subcutaneously, (3) rats pretreated with 5 micrograms/kg dmPGE2 given subcutaneously 30 min before, and 8 and 24 h after CCl4 administration, and (4) animals given dmPGE2 only as in group 3. Liver damage was assessed by biochemical studies (SGPT, serum alkaline phosphatase, and bilirubin) and by histology. In rats receiving CCl4 alone, SGPT activities were significantly elevated to 1024 +/- 82 U/L, 1270 +/- 120 U/L, 386 +/- 48 U/L and 208 +/- 20 U/L at 24, 48, 96, and 120 h after CCl4 respectively. In animals pretreated with dmPGE2 before CCl4, SGPT activities were 201 +/- 24 U/L, 55 +/- 4.6 U/L, 28 +/- 4 U/L, and 24 +/- 4 U/L at 24, 48, 96, and 120 h after CCl4, respectively (p less than 0.01, versus animals receiving CCl4 only). Histologically, livers of rats treated with CCl4 alone showed severe centrilobular necrosis at 24 and 48 h. Livers of animals pretreated with dmPGE2 before CCl4 did not show necrosis. It is concluded that dmPGE2 protects the liver against cell necrosis induced by CCl4 in the rat.

Prostaglandin Protection of the Gastric Mucosa Against Alcohol Injury—A Dynamic Time-Related Process: Role of the Mucosal Proliferative Zone

The aim of the present study was first to resolve controversies regarding the extent of prostaglandin protection (cytoprotection) of the gastric mucosa against injury produced by 100% ethanol and second to determine time sequence and histologic, ultrastructural, and functional features of this protection. Fasted rats received intragastrically (A) 0.9% NaCl alone as a control, (B) 5 micrograms/kg of 16,16-dimethyl prostaglandin E2 dissolved in 0.9% NaCl, and (C) 100 micrograms/kg of 16,16-dimethyl prostaglandin E2 dissolved in 0.9% NaCl. Thirty minutes later, 2 ml of 100% ethanol was instilled. The gastric mucosa was assessed macroscopically, by quantitative histology, and by scanning and transmission electron microscopy for [3H]thymidine uptake, mitotic activity, ion fluxes, and gastric potential difference determined at several time intervals (between 10 min and 16 h) after ethanol administration. Between 10 min and 16 h after ethanol administration macroscopic necrosis involved 27% +/- 3% to 41% +/- 4% of the mucosal area in controls (group A), but necrosis was prevented in groups receiving 16,16-dimethyl prostaglandin E2 (groups B and C). In the control group, histology and electron microscopy showed extensive disruption of the surface epithelium and deep necrosis (greater than 0.2 mm) involving greater than 46% +/- 4% of the mucosa between 15 min and 16 h after ethanol administration. Deep necrotic lesions were completely prevented by either dose of 16,16-dimethyl prostaglandin E2 (groups B and C). The mucosal proliferative zone was severely damaged in controls (68% +/- 5%) within the first hour after ethanol administration, whereas 16,16-dimethyl prostaglandin E2 protected the zone from damage (less than 5% +/- 1%). Neither dose of 16,16-dimethyl prostaglandin E2 prevented the occurrence of initial (at 15-30 min) morphologic and functional disruption of the surface epithelium after ethanol administration. However, initial disruption of the surface epithelium by 16,16-dimethyl prostaglandin E2 (groups B and C) was followed by migration of cells from the mucosal proliferative zone; the result was prompt restoration of the surface epithelium and resumption of its barrier and transport functions.

Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats

Expression of epidermal growth factor receptor (EGFR) was studied immunohistochemically in rat gastric mucosa during healing of acetic acid-induced ulcers. In normal control gastric oxyntic mucosa, EGFR was expressed in proliferative zone cells and in some parietal cells. In mucosa of the ulcer margin, at 3, 7, and 16 days after ulcer induction, there was a 75-fold increase (over controls) in the number of cells expressing EGFR. Seventy percent of ulcers healed by the 16th day, and all were healed by the 25th day. The mucosal scar that replaced the ulcer was composed of dilated glands lined with poorly or aberrantly differentiated cells showing persistence of increased EGFR expression. An increased EGFR expression indicates an important role of EGF in ulcer healing and scar formation.

Cell Culture of Rat Gastric Fundic Mucosa

The purpose of this study was to develop a primary cell culture system of rat gastric fundic epithelial cells. The cells, isolated enzymatically, were cultured in Coons modified Hams F-12 medium supplemented with 10% fetal bovine serum, 15 mM HEPES buffer, fibronectin, and antibiotics. The inoculated cells started to grow rapidly on day 1 (doubling time, 26 h). The cells reached confluency on day 3. On phase contrast microscopy, over 90% of cells possessed epithelial characteristics. Histochemical studies showed (a) 90% of the epithelial cells contained PAS positive granules, (b) 5% of the cells gave a strong reaction for succinic dehydrogenase activity (presumably parietal cells), and (c) immunohistochemical localization of pepsinogen was negative. Ultrastructurally, microvilluslike structures, junctional complexes, Golgi apparatus, mitochondria, rough-surfaced endoplasmic reticulum, and mucous granules were observed. Mitotic figures were clearly observed on Giemsa staining and the mitotic index was maximum on day 2. Autoradiographic and biochemical studies showed these cells possessed the capability to synthesize deoxyribonucleic acid and this ability was maximum on day 2. These cells were able to synthesize and to secrete glycoprotein and this function was significantly increased by 16,16-dimethyl prostaglandin E2. Cyclic adenosine monophosphate produced by the cultured cells was enhanced by addition of 16,16-dimethyl prostaglandin E2 (p less than 0.01). This in vitro system provides a valuable model for studies of cellular functions of gastric mucosa.

Comparison of antacid, sucralfate, cimetidine, and ranitidine in protection of the gastric mucosa against ethanol injury

The abilities of antacid (Mylanta II), sucralfate, cimetidine, and ranitidine to protect the gastric mucosa against ethanol-induced necrosis were compared in a standardized, experimental rat model. Fasted rats received pretreatment with either saline, Mylanta II, 500 mg/kg of sucralfate, 50 mg/kg of cimetidine, or 50 mg/kg of ranitidine. This was followed one hour later by intragastric administration of 2 ml of 100 percent ethanol. Gastric mucosal injury was assessed four hours after administration of ethanol by quantitation of gross mucosal necrosis, assessment of mucosal histology, and determination of intragastric blood and protein concentrations. Pretreatment with Mylanta II or sucralfate significantly reduced ethanol-induced gastric mucosal necrosis. The protective effect of sucralfate was six to 10 times greater than that of Mylanta II. H2-receptor antagonists increased ethanol-induced gastric mucosal necrosis.

Quality of gastric ulcer healing: a new, emerging concept.

Assessment of gastric ulcer healing is usually based on a visual examination (by endoscopy in patients, or the evaluation of ulcer size in experimental studies), and not on histologic and ultrastructural assessment of subepithelial mucosal healing. This approach has led to the assumption that the mucosa of grossly “healed” gastric and/or duodenal ulcers returns to normal, either spontaneously or following treatment. However, the re-epithelialized mucosa of grossly “healed” experimental gastric ulcer has recently been found to have prominent histologic and ultrastructural abnormalities, including reduced height, marked dilation of gastric glands, poor differentiation and/or degenerative changes in glandular cells, increased connective tissue, and disorganized microvascular network. It has been postulated that these residual abnormalities might interfere with mucosal defense and may be the basis of ulcer recurrence. In the present article, the ulcer healing process and the role of luminal factors, transitional zone at the ulcer margin, and granulation tissue are discussed. The healing of an ulcer is accomplished by filling of the mucosal defect with epithelial cells and connective tissue to reconstruct mucosal architecture. Under influence of growth factors [predominantly epidermal growth factor (EGF) and transforming growth factor (TGFα)], the epithelial cells at the ulcer margin dedifferentiate and proliferate, supplying cells for re-epithelialization of the mucosal scar surface and reconstruction of glandular structures. Granulation tissue at the ulcer base supplies connective tissue cells to restore the lamina propria and endothelial cells and microvessels for mucosal microvasculature reconstruction. The final outcome of healing reflects a dynamic interaction between an “epithelial” component from the ulcer margin and a connective tissue component including microvessels originating from granulation tissue. Angiogenesis—the formation of new microvessels in granulation tissue—appears to be critical for the ulcer healing process. Indomethacin delays healing of experimental gastric ulcer and impairs the overall quality of ulcer healing by distorting restoration of mucosal architecture, blocking differentiation and maturation of glandular and surface epithelial cells, and inhibiting angiogenesis in granulation tissue. Aluminum-containing antacid (Maalox-70) accelerates healing of experimental gastric ulcer, improves the quality of mucosal structure reconstruction, and partly reverses the deleterious effect of indomethacin on the rate and quality of ulcer healing.

Does Sucralfate Affect the Normal Gastric Mucosa

Although the action of sucralfate on ulcerated mucosa has been demonstrated, its effect on the histology, ultrastructure, and function of normal gastric mucosa is unknown. We investigated the effect of acute administration of sucralfate on the gastric mucosal history, ultrastructure, mucosal potential difference, and luminal release of prostaglandin E2. At 15 min, 1 h, and 3 h after intragastric instillation of sucralfate, whitish incrustations of the drug were firmly adhering to the glandular mucosa. Mucosal histology after sucralfate administration demonstrated the following: disruption and exfoliation of some of the surface epithelial cells, mucosal hyperemia, prominent release of mucus from the surface epithelial cells, and edema of lamina propria and submucosa. These changes were most prominent in the areas where sucralfate was in contact with the mucosal surface. Scanning and transmission electron microscopy confirmed the above changes. Sucralfate produced a drop in gastric mucosal potential difference and a significant increase in luminal release of prostaglandin E2. Sucralfate produces distinct morphologic and functional changes in the normal gastric mucosa, which may account for its preventive and therapeutic efficacy.

Vascular and Microvascular Changes—Key Factors in the Development of Acetic Acid-Induced Gastric Ulcers in Rats

The present study examined the time sequence and histologic and ultrastructural features of the formation and evolution of experimental, acetic acid-induced gastric ulcerations in rats. One hundred percent acetic acid was applied to the gastric serosa of 140 fasted male Sprague-Dawley rats through a polyethylene tube for 30 s. Gastric mucosal changes were evaluated at 1, 5, 15, and 30 min, 1 and 3 h, and 1, 2, 3, 5, 8, and 11 days after acetic acid application by visual inspection, by quantitative and qualitative light microscopy, and by transmission electron microscopy. Following exposure to acetic acid, the earliest morphologic changes occurred at 1 min and consisted of dilatation of large submucosal veins and arteries and mucosal collecting venules. Five to 15 minutes after injury, thrombi developed in submucosal veins and collecting venules, leading to microvascular stasis and mucosal necrosis. By 3 h, necrotic masses started to detach. By 24–48 h, necrotic changes penetrated the submucosa. By 72 h, most ulcers underwent transition into a “chronic” stage characterized histologically by the presence of granulation tissue at the bottom, and the appearance of a transitional healing zone at the margins. By 5 days, an increased amount of granulation tissue was observed and the gastric glands in transitional zones at the ulcer margin displayed cystic dilatation. Based on this study, we conclude that a key feature of acetic acid-induced ulcer formation is the early vascular and microvascular injury, which precedes glandular cell necrosis.

Healed experimental gastric ulcers remain histologically and ultrastructurally abnormal

The present study was designed to assess histologic and iltrastructural features of gastric mucosa in the areas of rossly healed ulcers (acetic acid-induced gastric ulcers) in ats. The specific question we studied was whether the tructure and cellular composition of the gastric mucosa in n area of grossly healed ulcer were fully restored. Eighty prague-Dawley rats underwent laparotomy; 100% acetic cid was applied to the lower gastric corpus serosa for 30 s nd the abdomen was closed. The stomachs were reopened ter 2 weeks or after 2, 3, or 4 months. Standardized gastric all specimens from the area of grossly healed ulcers were btained, processed, and evaluated by light microscopy and y transmission electron microscopy. The gastric mucosa f grossly healed ulcers demonstrated re-epithelialization at ach study time but the mucosa beneath the surface epitheium displayed prominent histologic and ultrastructural abormalities. Two different patterns of scar could be distinuished: (a) the mucosa in the area of healed ulcer was hinner (25–45% reduction vs. normal), with increased onnective tissue and poor differentiation and/or degenerative changes in the glandular cells; or (b) the mucosa dislayed ballooning dilatation of gastric glands, reduction in he microvascular network, and poor differentiation of landular cells. We conclude that (i) the subepithelial muosa of grossly healed gastric ulcer displays disorganized estoration of glandular and vascular structures and renains histologically and ultrastructurally abnormal; (ii) hese abnormalities may interfere with oxygenation, nurient supply, and with mucosal resistance and defense, and herefore could be the basis for ulcer recurrence.

Gastric microvascular endothelium: a major target for aspirin-induced injury and arachidonic acid protection. An ultrastructural analysis in the rat.

Abstract. Exposure of the gastric mucosa to aspirin results in exfoliation of the surface epithelium and deep mucosal necrosis. We assessed the changes in the mucosal microvessels during aspirin‐induced injury and arachidonic acid protection of the gastric mucosa using transmission electron microscopy. Male Spra‐gue‐Dawley rats received intragastric pretreatment with either solubilizer (control) or detergent solubi‐lized arachidonic acid (148 mg kg‐1). One hour later 1‐ml suspension of 200 mg kg‐1 body weight acidified aspirin was administered intragastrically. The ultra‐structure of mucosal microvasculature was assessed at 15 min and 4 h after aspirin administration both qualitatively and quantitatively by determining the number of necrotic or damaged capillaries in standardized mucosal sections. In addition, mucosal specimens were immunostained with a specific antiserum against vimentin, an endothelial marker, and fluorescence intensity was measured with a Nikon FX microscopic photometric system. In control rats, aspirin produced significant damage to both superficial and deeper microvessels consisting of: rupture of capillary walls, necrosis of endothelial cells, damage to endothelial organelles, deposition of fibrin and adherence of platelets to damaged endothelium. Vimentin fluorescence was reduced three‐fold. Microvascular injury preceded the development of deep necrotic lesions. Microvascular damage and deep mucosal necrosis were significantly reduced by arachidonic acid pretreatment. We conclude that gastric mucosal microvessels are the major target for aspirin‐induced injury and arachidonic acid protection.,