The impact of oral ciprofloxacin on the structure and functions of rat gastric mucosa

Ciprofloxacin (CPX), is a fluoroquinolone antibiotic used to treat a number of gram-negative and gram-positive bacterial infections. Ciprofloxacin can cause severe side effects, ranging from tendon problems, nerve damage, to serious mood or behavior changes. The purpose of this study was to investigate how ciprofloxacin affects gastric cell lines in rats with a distinctive emphasis on physiological, histopathological, and bacteriological changes. Male albino rats (n = 21) were distributed into three groups; control, CPX, and CPX-withdrawal groups. The treated rats were given CPX tablets (12.5 mg/kg) dissolved in carboxymethyl cellulose (CMC) 0.5% orally once daily via gavage for sixty consecutive days. Control rats received only the vehicle. The withdrawal group was treated for 60 days and the drug was withdrawn for another sixty days. After completion of the experiment, all rats were sacrificed and gastric tissues were treated for light, immunohistochemical, and scanning electron microscopic examination. Image J software was used to measure immune-labeled gastric epithelial cells. Blood samples were also collected for H. Pylori immunoglobulins IgM, IgA, and IgG. Results showed that treated rats acquired significantly strongly positive tumor necrosis factor (TNFα) and significant reduction of serum level of H. pylori IgM, IgA, and IgG in all the study groups. It could be concluded that prolonged oral CPX administration to albino rats changes the gastric mucosal architecture and bacteriology.


Introduction
Ciprofloxacin is a broad-spectrum antibiotic that was once used to treat a wide range of infections. It is the first-selected agent used for the management of testicular infections. However, its antibacterial action is achieved by the accumulation of the level of reactive oxygen species (Eskandari et al., 2001;Zhang et al., 2018). In addition, inhibiting a type II topoisomerase (DNA gyrase) and topoisomerase IV leads to bacterial cell death. CPX has many serious side effects including, insomnia, restlessness, seizures, decreased appe-tite, diarrhea, crystalluria, itching, and tissue damage when given intramuscularly or subcutaneously (Michalak et al., 2017). Relationships among antibiotic usage, the gut community structure, normal physiology and metabolism, individual and public health are still being under research. Alterations in the structure of bacteria, antibiotic resistance genes (ARGs), and mobile genetic components (MGEs) after antibiotic usage are not well-understood (Xu et al., 2020). Infection with H. pylori may be linked with a multiplicity of gastro-duodenal illnesses. However, H. pylori infection is a common peptic ulcer illness and gastric cancer arises in only a lesser minority of diseased people. This study was planned to relate the pathological outcomes with the serological reaction to certain H. pylori antigens in response to CPX use in albino rats (Schumann et al., 2006). Gastric ulcers are the most mutual gastrointestinal and global disorders. They occur predominantly because of the disproportion between the damaging and aggressive causes of the mucosal barrier. The damaging elements comprise stomach hydrochloric acid (HCl), mucosal hypo-perfusion, free oxygen radicals, ethanol, Helicobacter pylori, and extreme https://doi.org/10.1016/j.sjbs.2021.11.042 1319-562X/Ó 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). consumption of non-steroidal anti-inflammatory drugs (NSAIDs) that stimulate stomach mucosal damage and lead to gastric ulceration. The avoidance or management of gastric ulcers is a medicinal challenge. Gastric ulcer treatment has major difficulties, comprising restricted effectiveness of medications against the gastric disorder and severe side effects (AL-Wajeeh et al., 2016). This research aimed to explore whether CPX treatment of rats disturbs gastric epithelial cell biology with definite orientation to the destructive elements of the gastric epithelium, in precisely, H. Pylori which exist deep within the gastric mucus, and the effects of extended local drug use on rat gastric mucosa. H. Pylori contaminates the mucosal lining of the stomach epithelium in about half of the world's population. Contamination with H. pylori leads to gastritis that will progress to chronic inflammation of the mucosal lining of the stomach. This chronic inflammation will progress from chronic atrophic gastritis to metaplasia, dysplasia, and, in 1%-3% of cases to carcinoma of the stomach. This progress from normal to malignant tissues continues and can be repressed by the suppression of the bacteria with antibiotics. CPX may influence the human gut flora even at subtherapeutic levels (Carman and Woodburn, 2001;Butt and Epplein, 2019). H. Pylori infection of the human stomach causes chronic inflammation and practices the highest threat element that progresses to peptic ulcer disease and gastric cancer. Existing regular suppression treatments practice an acid-suppressing medication and two antibiotics, nowadays commonly complemented with bismuth for an additional regular suppression in asymptomatic H. pylori carriers to destroy stomach cancer (Debraekeleer & Remaut, 2018). The mucus coat cover of the gastric epithelium delivers a defensive neutral atmosphere protecting the gastric wall cells; furthermore, it provides a generous niche for H. pylori. It consists of plenty of highmolecular-weight glycoproteins, named mucins, and some lipids, small molecules, and additional proteins. Through disulfide attachment, the mucins form a viscoelastic coat that depresses transmission proportions and is supposed to inhibit the actual transfer of antibiotics (Gotoh et al., 2002).

Aim of the work
This study was designed to study gastric mucosal physiological, histopathological, immunohistochemical, and bacteriological changes associated with oral CPX administration in male albino rats.

Research settings
In this study, 21 adult male albino rats were used and divided into three groups: control, experimental, and withdrawal groups.
Group I (Control group): Consisted of seven animals that received carboxy-methyl cellulose (CMC) 0.5 % for sixty consecutive days.
Group II (CPX treated): Consisted of seven animals were received 12.5 mg/kg of ciprofloxacin, dissolved in carboxymethyl cellulose (CMC). 0.5 % orally once daily via gavage for sixty consecutive days. The dosages of CPX were similar to those used in human therapy (Khaki et al., 2008;Reagan-Shaw et al., 2008).
Group III (CPX withdrawal): Consisted of seven animals were received 12.5 mg/kg of ciprofloxacin, dissolved in carboxymethyl cellulose (CMC). 0.5% orally once daily via gavage for sixty consecutive days. The drug was stopped for another sixty consecutive days then animals were sacrificed.
On the sixtieth day, the animals of groups I and II were sacrificed and the third group animals were sacrificed after 60 days from drug withdraw. The regions of the fundus and the body of the stomach were specially chosen as they are the commonest site of drug-induced injuries. These areas were removed by a couple of scissors in-between esophageal and pyloric ends of the gastric body. The gastric mucosa was open by creating a cut along the greater curvature of the stomach (Wallace, 2001;Lim et al., 2019).

Histological examination
Rat gastric tissues in all groups were collected, washed with saline, and the gastric wall samples were put in 10% buffered formalin for fixation, handled in a tissue processing machine with paraffinembedded. Prepared for histological assessments, slices of the gastric sections were set at 5 lm thickness and discolored with hematoxylin and eosin (Hx and E) (Bancroft and Gamble, 2008;Tousson, 2016). Other gastric specimens of all groups were dissected, washed with saline, fixed in osmium tetraoxide, and coated with gold-palladium, and photographs were captured by Jeol-scanning electron microscope in Tanta faculty of medicine at electron microscopy unit (Zaki & Mohamed, 2014).

Immunohistochemical examination
Five lm sections of the stomach wall were immunohistochemically stained to estimate immune-expression of TNFa in the cytoplasm and gastric sections using Avidin-Biotin Complex (ABC) (El-Masry et al., 2020). Proliferating cell nuclear antigen (PCNA) expression in the nuclei of stomach epithelial cells (Calabrese et al., 2004). Concisely, the paraffin-embedded stomach slices were dewaxed and rehydrated. Then the slices were incubated with a monoclonal antibody against TNFa and PCNA (Dako, Carpinteria California, USA); Diaminobenzidine (DAB) was used to demonstrate the immune reaction. The sections were studied for the brownish coloration of DAB reaction and once visualized it was immediately washed off. Mayer's hematoxylin was used as a counterstain. Negative controls were prepared by excluding the primary antibodies. The cells were measured positive if showing a brownish coloring of DAB reaction related to the negative control (Buchwalow and Böcker, 2010;Hasan et al., 2021). The strength of the immunostaining was recorded according to the recording scheme defined by Mori et al. (2003) as severe (+++), moderate (++), mild (+), or nil (À).

Morphometric study
Image analysis software (Image J, 1.46a, NIH, USA) was used for Immunohistochemical quantification. Randomly selected 10 nonoverlapping fields from every slide were measured at the amplification of 400 for measurable assessment of mean zone percentage (calculated as the zone of positive immunohistochemical reaction 100/total area) and mean color intensity of the immunohistochemical reaction of TNFa and PCNA (measured by deducting the color strength of negative immunohistochemical control from the color strength of positively discolored specimens in successive slices) (Emam et al., 2021).

Laboratory investigations
Measurement of serum H. pylori immunoglobulin titers was useful in the conclusion of H. pylori infection and severity of gastritis in advance with a histopathological examination (Yamamoto et al., 1995). In all groups, blood samples were collected just before the time of scarification for H. Pylori IgM, IgA, and IgG assay with enzyme-linked immunosorbent assay (ELISA) in Al-Safwa medical laboratory in Tanta.

Statistical analysis
Quantitative records were assessed with mathematical set SPSS version 22, IBM, Armonk, NY, United States of America. All records were expressed as mean standards ± standard deviation (SD). The standard for statistical importance was significant at the P value < 0.05 for the collected data and highly significant at P value < 0.001. Analysis was done by to one way analysis (ANOVA) to evaluate significant differences between treatment groups (Dawson andTrapp, 2004, Hajrezaie et al., 2012;Altwaijry et al., 2021).

Hx. and E. sections
Control rat slices of the stomach (Group I) presented regularly arranged fundic glands, stomach pits, surface mucous cells, mucous neck cells, parietal cells, chief cells, and connective tissue with underlying submucosa and muscularis mucosa ( Fig. 1). CPX treated rat sections of the stomach wall (Group II) revealed widespread damage to the gastric mucosa in the fundus. Moreover, these rats were noticed to declare necrotic changes in the depth of gastric mucosa that reveals widespread leucocyte infiltration and edema of the sub-mucosal layer (Fig. 2). Other sections in the stomach fundus demonstrated honey combing of gastric mucosal glands, hydropic alterations in some gastric cells though other showing small pyknotic nuclei. Vacuolated mucus cells were aggregated giving rosette appearance with intact parietal cells scattered in-between (Fig. 3). Gastric sections of the CPX withdrawal group (Group III) exhibited stomach mucosa with limited necrotic areas. It also revealed submucosal layer with congested blood vessels. They also revealed dilated gastric mucosal glands with limited areas of necrosis. In addition, it exhibits nesting of parietal cells with vacuolated cytoplasm (Fig. 4).
3.1.1.1. TNFa immunohistochemical staining. TNFa -Immunostained sections of the stomach of control rats (Group I) showed mild positive reaction for TNFa expression in the cytoplasm of gastric mucosa (Group I; Fig. 5A). Severe positive reaction for TNFa expression was in the rats treated with CPX (Group II; Fig. 5B) and moderate positive reaction for TNFa expression in the gastric mucosa of the withdrawal group (Group III; Fig. 5C). Statistical analysis of morphometric results showed highly significant (P < 0.001) rise in the area percentage of TNFa positive immunoreaction in group II as linked with the control group. While, group III depicted highly significant (P < 0.001) decrease of both parameters as compared with group II. On the other hand, group III displayed a non-significant (P > 0.05) difference in immunoreactivity as compared to the control group (Table 1).
3.1.1.2. PCNA immunohistochemical staining. PCNA-Immunostained sections of the stomach of control rats (Group I) showed mild positive reaction for PCNA expression in the nuclei of gastric mucosa (Group I; Fig. 6A). Severe positive reaction for PCNA was detected in the gastric mucosa of the rats treated with CPX (Group II; Fig. 6B) and moderate positive reaction for PCNA expression were noticed in the gastric mucosa of the withdrawal group (Group III; Fig. 6C). Statistical analysis of morphometric results showed highly significant (P < 0.001) increase in the area percentage of PCNA positive immunoreaction in group II as related to control animals' group. Meanwhile, rats in group III demonstrated highly significant (P < 0.001) decrease of both parameters as compared with group II. On the other hand, group III revealed a non-significant (P > 0.05) variance in immunoreactivity as linked to the control group (Table 2).  reaching the muscularis mucosa (mm) which is exposed to gastric lumen (L) with edema and leucocytes infiltration (I) of the muscularis mucosa and submucosal layer (SU). Normal musculosa (MU) and serosa (SE) could be realized (A). Higher magnification (B) showing extensive epithelial sloughing (N), pyknotic nuclei (arrows). Dilated submucosal blood vessel (bv) with a surrounding cellular infiltrate (I) can be seen. The surface stomach mucosa having striking vacuolations of the gastric gland cells (G). Unbroken muscularis mucosa (mm) exposed to gastric lumen (L) is noticed.

Scanning electron microscopic results
Scanning electron microscopy of control rat gastric mucosa (Group I) exhibited healthy epithelial cells with gastric glands and pits (Fig. 7). CPX treated rat gastric mucosa (Group II) showed damaged epithelial cells (erosions) and widened gastric pits (Fig. 8A). Other areas showed a number of separate stomata in the basement membrane, through which young mucous epithelial cells sprouted out of the gastric glands' lamina propria. Necrotic gastric epithelium and polymerized mucus could be seen. Moreover, connective tissue meshwork of gastric gland without lining epithelial cells could be noticed (Fig. 8B). Honey combing appearance of gastric mucosa was also seen in some areas with flattened gastric glands widened gastric pits (Fig. 8C). Higher magnification showed nearby intestinalized regions revealing a plush microvillous with striated border occasionally disturbed by apical apertures of goblet cells. Widened gastric pits could be noticed with apparent H.pylori cocci (Fig. 8D). Rat gastric mucosa of the withdrawal group (Group III) showed interaction between H. pylori cocci and gastric epithelial cells in the form of intestinalized regions with plush microvilli and striated border periodically interjected by apical apertures of goblet cells (Fig. 9A). In some other areas, the basement membrane was noticed with the gastric gland cells migrating out of the lamina propria and bundles of microfilaments lying on its outer surface. Some deep gastric pits were noticed (Fig. 9B).
It also revealed partial recovery in the form of healthy epithelial cells, gastric glands with intervening gastric pits. However, Few H. pylori cocci could be seen in the depth of some gastric pits (Fig. 9C).

Discussion
The current research studied the effect of CPX on the normal organization of gastric wall of adult rats and the possible detection of H. Pylori both histologically and serologically. The stomach of the control rats was lined by epithelial cell layers containing several gastric pits lined by mucous cells, and continuous with the tube-like glands occupied by numerous cell lines manufacturing mucus, acid, pepsinogen, and numerous hormones. Karam et al. (2003) reported that these cell lines originate from multiplying epithelial stem cells. Cells with basally basophilic cytoplasm and basal nuclei are the chief cells. These are accumulated in the lower areas of the glands. Cells with obvious eosinophilic cytoplasm and centrally located nuclei (fried egg appearance) are the parietal cells. These are focused in the middle to upper areas of the glands. The glands are separated by tinny threads of lamina propria containing the denser, flattened or irregular nuclei of fibroblasts, capillary endothelium, and numerous cells of the immune system.
Microstructural examination of gastric sections of CPX rats revealed that the CPX-induced zones of focal erosion (scattered damage) in the stomach lining that became eroded and ulcerated to the extent that the muscularis mucosae became open to the gastric lumen, widespread leucocyte infiltration and edema of the sub-mucosal stratum, congestion and dilatation of the gastric sub-mucosal blood vessels. These results were parallel to the outcomes of (Barnett et al., 2000) who detected considerable necrosis, epithelial sloughing, and shallow ulcers following drug management. Morini and Grandi (2010) and Lim et al. (2019) reported that Stomach mucosa has numerous defensive mechanisms that permit the mucosa to tolerate repeated exposure to possibly destructive factors. The discrepancy between protective and forceful factors is at the heart of the creation of lesions or ulcerations of the stomach mucosa. The dissimilarity between an erosion and ulceration is that the former is limited to the mucosa, while ulceration infiltrates to the muscularis mucosae. They also clarified how to measure gastric mucosal injuries by microscopic analysis of the stomach via light and by scanning electron microscopy. Anto et al. (2001) also indicated that, gastritis or inflammation of gastric mucosa is not a distinct illness, but relatively a collection of panics, frequently accountable for inflammatory alterations in gastric mucosa. Nonetheless, they fluctuate in their clinical features, histological expressions and causal mechanisms. Furthermore, kim et al. (2011) reported that the progress of the gastric induced mucosal injuries might furthermore be facilitated through generation of oxygen free radicles. These results were similar to that of Palacios-Espinosa et al. (2014) and Shalaby et al. (2016) who specified that in occasion of stomach ulceration, the neighboring blood vessels looked expanded and congested. In this study, there was inflammatory cellular infiltration in the lamina propria of the gastric mucosa, goes equivalent to the results recognized by Boushra et al. (2019) who stated that, there was dense cellular infiltration detected in the ulcer beds of the fundic lamina propria. They described the infiltration with mononuclear cells due to the effect of NSAIDs on the integrity of intercellular junctions and on suppressing the manufacture of the defensive mucus coat. Accordingly, the mucosa will be damaged by the acid and proteolytic enzymes associated with consequent H. pylori attack of the epithelium. These bacteria were chemotactic for WBCs, lymphocytes and macrophages. Besides, they explained that, polymorphonuclear leucocyte infiltration might be due to H. pylori infection which is associated with drug-induced ulcers. Wallace (2001) said that the existence of acid in the lumen of the stomach might not be a key element in the pathogenesis of drug-induced gastroenteropathy. It could create a significant support to the chronicity of such injuries and to bleeding by weakening the restoration procedure, interfering with hemostasis and deactivating numerous growth elements that are vital in mucosal defense and restoration.
In this study, Gastric slices from CPX group displayed moderate positive reaction for TNFa expression as matched to faint positive reaction in the control and CPX-withdrawal groups. Positive findings of the immunohistochemical staining are displayed in brown color by light microscope. This agrees with khoder et al. (2019) who exhibited that experimental animals developed augmented manufacture of mucus, reduced making of pepsinogen and augmented ghrelin-secreting cells. Apoptosis and necrosis are double distinguishing varieties of cell demise, which Ramseyer and Garvin (2013) also reported that TNFa is a type of proinflammatory cytokine that rises in chronic inflammatory conditions and mediates organ injury by exciting immune cell infiltration and cell death. Bimczok et al. (2013) and Gonciarz et al. (2019) also reported that H. pylori infection encourages cell apoptosis in concurrence with augmented oxidative stress. Raised apoptosis keeps against injurious inflammation and neoplasia; nevertheless, it diminishes cell integrity in addition to up-regulation of cell immigration and multiplication in response to damage. They also discovered that epithelial cells experiencing apoptosis as a reaction to live H. pylori were not eradicated by human gastric mononuclear phagocytes, however they were under the effect of TNF-a, produced by macrophages and extremely displayed in H. pylori-infected gastric mucosa compared to control cells. Ernst et al. (2006) and Amieva and El-Omar (2008) reported that in humans, epithelial cell mor- phology was modified by H. pylori infection which also produced interruption of tight connections, stimulation of cytokine manufacture, epithelial cell proliferation, and rise in cell death via apoptosis.
In the current study, scanning electron microscopy of gastric mucosa of CPX group exhibited damaged epithelial cells, erosions with widened gastric pits. Zaki & Mohamed, 2014 also reported that ulcerations at higher magnification presented the connective tissue framework of stomach gland corion restituted of normal epithelial cells but showing young traveling cells emergent out from glandular pit. The fundus of the stomach presented gastric ulcer spreading up to the muscularis mucosa. The mucosal cells adjoining the round gastric ulcer are the body's challenge to heal the damage by wrapping the ulcer. Round these cells the coarse texture of healthy gastric mucosa could be seen.
A scanning electron microscope photomicrograph of other areas of the stomach wall of CPX treated rats revealed mucus, widened gastric pits with Interaction between H. pylori cocci and gastric epithelial cells. Moreover, intestinalized region with a plush microvillus, striated border occasionally disturbed by apical stom-ata of goblet cells was noticed. This agrees with Winborn and Weser (1983) who registered features consistent with those described in chronic gastritis; represented by amplification of the ''cobblestone relief" look of the luminal surface of the stomach and the existence of several little, short, bulging microvilli projecting from the free edge of the surface mucous cells to the stomach lumen.
Furthermore, the scanning electron microscopy of the gastric mucosa of rats of the CPX group recorded widened gastric pits, Fig. 8. A scanning electron photomicrograph of gastric mucosa of CPX treated rat (Group II) showing damaged epithelial cells with erosions and ulceration (N) and widened gastric pits (P) (A). Other areas show a number of separate holes in the basement membrane, over which Young mucous epithelial cells (red arrows) sprouting out of the gastric glands lamina propria (G). Necrotic gastric epithelium (N) and polymerized mucus (U) can be seen. Moreover, connective tissue meshwork of gastric gland without lining epithelial cells (ct) could be noticed (B). Honey combing appearance of gastric mucosa is also seen in some areas with flattened gastric glands (G) and widened gastric pits (P) (C). Higher magnification shows a nearby intestinalized region revealing a plush microvillous (m) with striated border occasionally interjected by apical apertures of goblet cells (red arrows) with surrounding widened gastric pits (P). Few H. pylori cocci (H) could be seen (Fig. 7D). (SEM A, B & Cx100; Dx500).   Results are expressed as mean ± SD. ** P < 0.001 means highly significant variances. Results are expressed as mean ± SD. ** P < 0.001 means highly significant variances.    (1999) and Kusters et al. (2006) indicated that H. pylori is a gram-negative bacterium, measuring 2 to 4 lm in length and 0.5-1 lm in width. Though frequently spiral-shaped, the bacterium could seem like a rod, while coccoid forms look after extended in vitro culture or antibiotic therapy which was recognized in the current study by scanning electron microscopy. These coccoids could not be cultured in vitro and are supposed to signify unviable cells, while it had been proposed that coccoid forms might elucidate a viable, non-culturable form.
It was documented in this research that CPX treated rats exhibited insignificant deviation of serum anti H. Pylori immunoglobulins compared to the control and CPX-withdrawal groups. Granberg et al. (1993) mentioned that investigation for IgG antibodies, is a respectable and dependable test for the recognition of antibodies to H. pylori and as a clue of H. pylori infection. The determination of IgA antibodies might be used as a test that supplements the IgG antibody assessment. This also agrees with Shao et al. (2003) who determined the frequency of H. pylori infection by the serum anti-H. pylori immunoglobulin G (IgG) and IgA antibody reactions, and the significance of clinical presentations in the identification of H. pylori contamination in patients with gastric atrophy, intestinal metaplasia, and dysplasia by enzymelinked immune-adsorbent assays (ELISA). Silva et al., 2001 also specified that the recognition of H. pylori in the gastric epithelium and its association with the peptic disease lead to a notable alteration in the controlling of peptic ulcers. It is a universal accord that suppression of the bacterium is the cornerstone of peptic ulcer therapy. The low rates of suppression observed in mono and dual treatments encouraged the usage of double or three antibiotics and a proton pump inhibitor. Dresner et al., 1996 reported that CPX once combined with omeprazole and bismuth is effective for the eradication of H pylori. Dore et al., 2012 also displayed that Ciprofloxacin-based therapy treated 65% of patients as an alternate to bismuth established quadruple treatment. This agrees with our serological findings of significant reduction of serum level of H. Pylori IgM, IgA, and IgG in CPX group.

Conclusion
It could be concluded that prolonged oral CPX administration to albino rats changes gastric mucosal physiology and architecture but never aggravates infection with H. Pylori. It is also recommended to accurately track patients under treatment concomitantly to avoid any possible side effects of gastric ulcerations and erosions.