Many parietal cells appeared ballooned and vacuolated with eccentric pyknotic nuclei (Fig. 9b). The bases of the glands were disorganized and most of the chief cells had deeply stained pyknotic nuclei (Fig. 9c).
The lamina propria showed focal areas of mononuclear cellular infiltration (Figs 8 and 9a). The collagen content was similar to that seen in sections of the control group.
On Alcian blue–PAS stain, the surface mucous coat, pit regions, isthmus, necks, and the bases of the glands showed an apparent increase in Alcian blue-positive reaction in comparison with the control group (Fig. 10).
Immunohistochemical staining for PCNA showed an apparent decrease in PCNA-positive reaction in the isthmus region of the fundic glands as compared with that in the control group (Fig. 11).
H&E-stained sections revealed extensive affection of the gastric mucosa. The gastric pits were wide, and an apparent decrease in the thickness of the mucosa was detected in comparison with the control group (Fig. 12).
The surface columnar mucous-secreting cells were desquamated in many areas. They appeared flattened with deeply stained pyknotic nuclei (Fig. 13a). The parietal cells showed apparent decrease in their number and size with deeply stained pyknotic nuclei (Fig. 13b). Most of the chief cells in the bases of the fundic glands were disorganized and showed pyknotic nuclei (Fig. 13c).
In certain areas, there was desquamation and shedding of the epithelium. Extensive diffuse mononuclear cellular infiltration was detected in the lamina propria (Fig. 12). Congested blood vessels were also detected (Figs 12 and 13c).
The collagen fiber content in the basal parts of the mucosa was apparently increased in comparison with the control group (Fig. 14).
Most of the specimens revealed marked reduction in both Alcian blue and PAS-positive reactions (Fig. 15a). In some areas, an apparent Alcian blue-positive reaction was seen in the bases of the glands (Fig. 15b).
Immunohistochemical staining for PCNA showed an apparent depletion in PCNA-positive reaction in the isthmus region of the fundic glands in comparison with the control group (Fig. 16).
On electron microscopy, the parietal cells showed irregular nuclei with perinuclear space. Numerous cytoplasmic vacuoles were detected and mitochondria were hardly seen. The intracellular canaliculi and microvilli were unapparent (Fig. 17).
The chief cells appeared with irregular flattened nuclei. The cytoplasm showed few irregular dilated cisternae of RER (Fig. 18).
Subgroups IVa and IVb
Examination of H&E-stained sections of group IV (IVa and IVb) showed that the structure and thickness of the fundic mucosa appeared comparable with that in the control group. The mucosa appeared intact with short narrow pits of regularly arranged fundic glands (Fig. 19).
Surface columnar mucous-secreting cells appeared intact with rod-shaped nuclei and apical vacuolated cytoplasm (Fig. 20a). Parietal cells appeared with central rounded vesicular nuclei and acidophilic cytoplasm among groups of mucous neck cells having flattened basal nuclei and pale vacuolated cytoplasm (Fig. 20b). Chief cells appeared lining the bases of the glands with basal rounded nuclei. They had basal basophilic cytoplasm with apical acidophilic zymogen granules (Fig. 20c).
On Masson’s trichrome stain, few collagenous fibers appeared between the bases of the fundic glands comparable with that in the control group (Fig. 21).
On Alcian blue–PAS stain, the surface columnar mucous-secreting cells showed a PAS-positive reaction, whereas the mucous neck cells showed Alcian blue-positive reaction. A mixed coat of both types appeared covering the surface of the mucosa (Fig. 22).
Immunohistochemical staining for PCNA showed positive reaction located at the isthmus of the glands comparable with that in the control group (Fig. 23).
On electron microscopy, subgroup IVb showed that the parietal cells appeared with rounded euchromatic nuclei and numerous mitochondria. The intracellular canaliculi showed many microvilli (Fig. 24).
The chief cells showed basally located euchromatic nuclei, apical moderately electron-dense secretory granules, and abundant RER (Fig. 25).
Statistical and morphometric results
The mean thickness of the gastric mucosa and the mean number of parietal cells showed significant reduction in subgroup IIIb as compared with that in the control group. The other groups showed a nonsignificant change compared with the control group. There was also a significant increase in subgroup IVb as compared with subgroup IIIb in these two parameters (Table 1).
The mean number of PCNA-positive cells showed a significant reduction in subgroups IIIa and IIIb as compared with that in the control group, whereas the other groups showed a nonsignificant difference. In contrast, there was a significant increase in subgroup IVa as compared with subgroup IIIa and also in subgroup IVb as compared with subgroup IIIb (Table 1).
The present study was carried out to evaluate the protective effects of ALA on fundic mucosal damage induced by ASA (aspirin).
In this study, administration of aspirin for 2 weeks induced focal damage to the fundic mucosa in the form of widening of the gastric pits and desquamation and sloughing of mucosal cells into the lumen. Moreover, the prolonged period of aspirin administration for 4 weeks resulted in more extensive lesions, including thinning of the mucosa, sloughing, and congestion of blood vessels.
These findings were in accordance with those of previous researchers who attributed these alterations to decreased antioxidant enzymes such as glutathione, glutathione peroxidase, catalase, and superoxide dismutase with aspirin 15. In contrast, aspirin administration was found to increase the volume of gastric juice secretion and total acidity leading to sloughing ulceration of the mucosa 12.
There was a significant reduction in the thickness of the gastric mucosa after 4 weeks of aspirin administration as compared with that in the control group. Similarly, in a clinical study, 38% of NSAIDs-treated patients showed chronic gastritis with thinning out of the gastric mucosa 16. Furthermore, in mice, aspirin resulted in gastric inflammation and decreased mucosal thickness 17.
In this study, parietal and chief cells of aspirin-treated rats appeared with pyknotic nuclei, and a significant reduction in parietal cell count was detected as compared with the control group. These changes were attributed to the generation of ROS resulting in oxidative damage in the cellular membrane and cell lysis 18 or to increased mitochondrial permeability, mitochondrial failure, and translocation of the intramitochondrial protein apoptosis-inducing factor 19.
In addition, the current study revealed congestion of the blood vessels in the lamina propria of the fundus of aspirin-treated rats. This was in accordance with a previous study in which a single oral dose of 200 mg/kg of aspirin produced hemorrhagic linear erosions with dilatation and congestion of the nearby blood vessels 20.
The cellular infiltration detected in the present study could be explained by the loss of integrity of the intercellular junctions caused by aspirin 16. This exposes the mucosa to the effect of acid and enzymes, with subsequent bacterial invasion. Bacteria will then secrete chemotactic factors for neutrophils, lymphocytes, and macrophages 16.
Consequently, many growth factors become elaborated by the inflammatory mononuclear cells attracted to the site of injury. Among these growth factors was the basic fibroblastic growth factor, which induced fibroblast proliferation and collagen deposition at the site of inflammation 21. This could explain the increased collagen content detected in this study after 4 weeks of aspirin administration.
The findings of the present study revealed that, after 2 weeks of aspirin administration, the Alcian blue binding capacity was much increased at the surface mucous coat, pit regions as well as in the mucous neck cells. These results were in agreement with those of previous researchers who observed that, in low-dose aspirin takers, the collected gastric juice showed a significant increase in the gastric mucous secretion as compared with the control group, suggesting a functional adaptive response 22.
In contrast, after 4 weeks of aspirin intake, depletion in both PAS and Alcian blue reaction was observed. These findings were in accordance with previous reports, which reported that, during long-term administration of aspirin, there was inhibition of glycoprotein biosynthesis by the epithelial cells, indicating failure of gastric adaptation against the injurious effects of aspirin 23–25.
In certain areas, the bases of some fundic glands of the mucosa were positive for Alcian blue staining. Chief cells could store mucous in stressful circumstances to increase the cytoprotective potential of the gastric mucosa 26. Accordingly, the basal cells detected with Alcian blue stain in group IIIb could be chief cells, which started to synthesize mucous in addition to their serous secretion in order to contribute to the gastric defensive mechanisms against aspirin.
In the present study, a significant decrease in the labeled nuclei for PCNA was detected after 2 and 4 weeks of aspirin intake. This denoted a decrease in the proliferating capacity of stem cells. This was similar to previous reports 27,28. In view of this, Wallace 29 explained that aspirin reduced the binding capacity of the epithelial growth factor to its receptor and inhibited the epithelial growth factor-signaling pathways. He added that there was an increase in the synthesis of antiproliferative products of arachidonic acid metabolism 29.
The different mechanisms by which aspirin induced mucosal damage included a direct cytotoxic mechanism through osmotic lysis 29, suppression of cyclo-oxygenase enzyme and inhibition of prostaglandin synthesis 30, reduced blood flow 31, and accumulation of oxygen free radicals 32.
In the present study, ALA administration before aspirin preserved the structure of the gastric mucosa from its injurious effects. Previous studies revealed a protective effect of ALA before indomethacin 33 and ethanol intake 3 as detected by reduced ulcer index and decreased congestion of blood vessels.
ALA is a dithiol antioxidant and is an important cofactor in pyruvate dehydrogenase and ketoglutarate dehydrogenase enzymes in the mitochondria 3. It preserves the membrane-bound ATPase enzyme 4. ALA exerts antioxidant effects on the biological systems through ROS quenching and also through an action on transition metal chelation 34. It was also capable of quenching reactive nitrogen species, thus preventing DNA damage and reduced lipid peroxidation 3. Furthermore, ALA markedly improved cysteine availability within the cell, resulting in accelerated glutathione synthesis compensating for its depletion during oxidative stress 35.
In the present study, ALA administration revealed preservation of the cells lining the fundic glands as detected by light and electron microscopy. In a previous study, DNA fragmentation, apoptosis, and cell death were significantly reduced when ALA was concomitantly administered with acetic acid 4.
The collagen fiber content was preserved with ALA administration in this study. According to previous reports, ALA reduced the transforming growth factors involved in fibroblast differentiation and collagen production 36.
Furthermore, in the current study, ALA prevented mononuclear cellular infiltration and the congestion of blood vessels. It was proved in a previous study that ALA suppressed neutrophil accumulation in the intestine, and consequently the release of toxic metabolites and proteases 5. Moreover, ALA showed potent anti-inflammatory effects through inhibition of vascular adhesion molecule and intercellular adhesion molecule production. It also inhibited chemoattractant protein secretion and natural killer cell activation and cytotoxicity 37.
The present study revealed preservation of mucous production by the glandular epithelium in groups IVa and IVb as compared with that in the control group. This was in agreement with the study by Gutierrez-Cabano 38 who found that ALA protection was related to an increase in both the fluid volume and the mucous volume retained in the gastric lumen and to the regeneration of exogenous and endogenous antioxidants.
In the present study, preservation of the proliferative cells was detected by PCNA technique in rats receiving ALA before aspirin. This indicated normal cellular proliferation, which maintained mucosal integrity. In view of this point, previous studies stated that ALA could have a dual action. It might act as a growth factor or stimulate other growth factors or receptors in normal cells. However, it might act as an antiproliferative agent in cancer cells through arrest of the cell cycle 39,40.
From the previous results, it could be concluded that ALA protected the structure of the fundic mucosa against the injurious effects of aspirin.
Clinical studies are recommended to detect the benefit of ALA when concomitantly used with aspirin.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2014 The Egyptian Journal of Histology
acetyl salicylic acid; electron microscopy; gastric mucosa; α-lipoic acid; proliferating cell nuclear antigen