Abraham Lukpata

THE EFFECTS OF CRUDE EXTRACT ALLIUM SATIUM ON THE STOMACH OF ADULT FEMALE AND MALE ALBINO RATS FOLLOWING FORMALIN INHALATION TOXICITY A RESEARCH TOPIC BY UKPONG, ABASIAMA LINUS 20/ANA/189 SUBMITTED TO THE DEPARTMENT OF HUMAN ANATOMY AND FORENSIC ANTHROPOLOGY FACULTY OF BASIC MEDICAL SCIENCES CROSS RIVER UNIVERSITY OF TECHNOLOGY (CRUTECH), OKUKU CAMPUS IN FULFILMENT OF THE COURSE REQUIREMENTS FOR THE AWARD OF BARCHELOR OF SCIENCE DEGREE (B.Sc.) IN HUMAN ANATOMY JULY, 2025 CHAPTER ONE INTRODUCTION 1.1 Background of the Study The gastrointestinal (GI) tract, particularly the stomach, is vulnerable to various environmental and chemical toxins that may compromise its structural and functional integrity. Formaldehyde, widely used in industrial and medical settings, is a recognized toxicant that induces systemic and localized tissue damage upon exposure (Zhang et al., 2021). Studies indicate that inhalation of formaldehyde can lead to oxidative stress, inflammation, and cellular damage, potentially affecting gastric mucosa (Zhao et al., 2020). Given these risks, researchers have explored natural remedies with gastroprotective properties, including Allium sativum (garlic), which is known for its antioxidant, anti-inflammatory, and cytoprotective effects (Bayan, Koulivand, & Gorji, 2014). This study aims to evaluate the potential protective effects of Allium sativum crude extract on the gastric mucosa of albino rats following formalin inhalation toxicity. Formaldehyde is a ubiquitous environmental pollutant, commonly encountered in industrial processes, embalming, and laboratory settings. It is classified as a hazardous air pollutant and a known human carcinogen by the International Agency for Research on Cancer (IARC, 2021). Inhalation is the primary route of exposure in occupational and household settings, leading to systemic absorption and widespread toxicity (Tang et al., 2022). Chronic exposure has been linked to respiratory diseases, neurological impairment, and gastrointestinal disturbances, including gastric mucosal damage (Wang et al., 2020). The mechanism of formaldehyde-induced toxicity involves oxidative stress, inflammation, and direct cytotoxic effects. Oxidative stress results from excessive production of reactive oxygen species (ROS), which disrupt cellular homeostasis and cause lipid peroxidation, protein denaturation, and DNA damage (Xia et al., 2021). Studies have shown that oxidative stress plays a key role in gastric mucosal injury, leading to inflammation and ulcer formation (Zhao et al., 2020). In addition, formaldehyde exposure triggers the release of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), which further exacerbate gastric tissue damage (Liu et al., 2022). Natural compounds such as Allium sativum have gained attention for their protective effects against oxidative stress and inflammation. Garlic contains bioactive sulfur compounds, including allicin, diallyl sulfides, and s-allyl cysteine, which have been shown to exhibit strong antioxidant and anti-inflammatory properties (Bayan et al., 2014). Research suggests that garlic supplementation can enhance endogenous antioxidant defense mechanisms by upregulating superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, thereby reducing oxidative damage (Rahman & Lowe, 2021). Moreover, garlic has been reported to inhibit gastric acid secretion and enhance mucosal defense mechanisms, protecting against ulcer formation (Banerjee & Maulik, 2022). Despite the well-documented pharmacological benefits of garlic, there is limited research on its role in protecting against formaldehyde-induced gastric toxicity. This study seeks to bridge this knowledge gap by evaluating the histopathological and biochemical changes in the gastric mucosa of albino rats exposed to formalin inhalation, with and without Allium sativum intervention. 1.2 Statement of the Problem Formaldehyde exposure poses significant health risks, with growing evidence linking it to gastric mucosal damage through oxidative stress and inflammatory pathways (Wang et al., 2020). The absence of effective prophylactic or therapeutic interventions exacerbates these risks, particularly for individuals in occupational settings where formaldehyde exposure is common (Tang et al., 2022). While Allium sativum has been widely studied for its medicinal properties, its specific role in mitigating formaldehyde-induced gastric toxicity remains unexplored. This study seeks to assess the gastroprotective potential of Allium sativum crude extract, providing scientific evidence on its efficacy in alleviating formaldehyde-induced gastric injury. 1.3 Overall aim of the study This study aims to investigate the effects of crude extract of Allium sativum on the stomach of adult male and female albino rats exposed to formalin inhalation toxicity. 1.4 Specific Objectives of the Study The specific objectives of this study are to: i. Assess histopathological changes in the gastric mucosa of albino rats following formalin inhalation. ii. Evaluate the potential protective effects of Allium sativum crude extract on formalin-induced gastric damage. iii. Analyze oxidative stress markers, including malondialdehyde (MDA) and superoxide dismutase (SOD), in gastric tissues. iv. Compare the effects of Allium sativum extract with standard gastroprotective agents, if applicable. 1.5 Justification of the Study Formaldehyde is a widely used chemical with documented toxic effects on multiple organ systems, including the gastrointestinal tract (Zhao et al., 2020). Given the rising concerns about occupational and environmental formaldehyde exposure, there is an urgent need for effective protective strategies. While conventional gastroprotective drugs exist, they often come with adverse side effects, necessitating alternative approaches (Rahman & Lowe, 2021). Allium sativum is a natural, widely available compound with well-established antioxidant and anti-inflammatory properties, making it a promising candidate for mitigating chemical-induced gastric toxicity (Bayan et al., 2014). This study will contribute valuable data to the field of toxicology and gastroenterology by providing experimental evidence on the protective effects of Allium sativum against formaldehyde-induced gastric damage. 1.5 Scope of the Study This research is to the effects of crude extract of Allium sativum on the stomach of adult male and female albino rats exposed to formalin inhalation toxicity and associated changes in adult wistar rats, using routine H and E staining techniques and light microscope. 1.6 Research Hypothesis Null Hypothesis (H₀): Allium sativum crude extract does not exhibit a significant protective effect on the gastric mucosa of albino rats following formalin inhalation. Alternative Hypothesis (H₁): Allium sativum crude extract exhibits a significant protective effect on the gastric mucosa of albino rats following formalin inhalation. CHAPTER TWO LITERATURE REVIEW 2.1.1 The Stomach The stomach is a vital organ of the gastrointestinal system that functions as a reservoir and processing chamber for ingested food. It is situated in the upper left quadrant of the abdominal cavity, immediately inferior to the diaphragm and largely hidden under the left lobe of the liver. Structurally, the stomach is a muscular, hollow organ with a J-shape, and its internal environment is adapted to carry out both mechanical and chemical digestion. It is bounded proximally by the lower esophageal sphincter and distally by the pyloric sphincter, regulating the flow of chyme into the duodenum (Barrett et al., 2019). The stomach plays a crucial role in digestion by secreting hydrochloric acid and digestive enzymes, facilitating the breakdown of proteins and the absorption of certain substances. It also acts as a defense barrier against ingested pathogens, thanks to its highly acidic environment. Its role in endocrine signaling, through hormones such as gastrin, makes it a multifunctional organ within the digestive tract (Moore et al., 2018). 2.1.2 Gross Anatomy of the Stomach The gross anatomy of the stomach reveals a complex structure adapted for its role in digestion. It comprises four major anatomical regions: Cardia: The region surrounding the opening of the esophagus into the stomach. Fundus: The dome-shaped portion that lies superior to the level of the cardia and serves as a gas reservoir. Body (Corpus): The largest central region where most digestive processes occur. Pyloric Region: Consists of the pyloric antrum, pyloric canal, and pyloric sphincter, which controls the passage of chyme into the duodenum (Drake et al., 2020). 2.1.2.1 Two curvatures define the stomach's contour: Lesser curvature: Forms the right border and is the site of attachment for the lesser omentum. Greater curvature: Forms the left border and provides attachment for the greater omentum. The stomach has an anterior surface that lies in contact with the diaphragm, liver, and anterior abdominal wall, and a posterior surface related to the pancreas, left kidney, and spleen (Drake et al., 2020). 2.1.3 Embryology of the Stomach Embryologically, the stomach originates from the foregut, an endodermal derivative, during the fourth week of gestation. The primordial stomach begins as a simple tubular structure but undergoes differential growth, leading to the formation of the greater and lesser curvatures. A 90-degree clockwise rotation along its longitudinal axis repositions the stomach; shifting the left side anteriorly and the right side posteriorly, affecting the alignment of the vagus nerves (Sadler, 2019). Furthermore, the dorsal mesogastrium, associated with the greater curvature, elongates to form the greater omentum. Simultaneously, the ventral mesogastrium forms the lesser omentum and contributes to the development of the liver and biliary apparatus. This embryological development is vital for understanding the stomach's anatomical relationships and potential sites for congenital anomalies (Carlson, 2022). 2.1.4. Histology of the Stomach The stomach wall consists of four principal layers: Mucosa: Composed of a simple columnar epithelium with numerous gastric pits and glands. These glands differ by region: Cardiac glands: Secrete mucus. Fundic glands: Contain parietal cells (secrete HCl and intrinsic factor) and chief cells (produce pepsinogen). Pyloric glands: Rich in mucus cells and G cells that secrete gastrin (Ross & Pawlina, 2021). Submucosa: Made up of connective tissue containing blood vessels, lymphatics, and Meissner's plexus. Muscularis externa: Comprises three layers, inner oblique, middle circular, and outer longitudinal muscles. This unique muscle arrangement facilitates churning and propulsion of food (Drake et al., 2020). Serosa: The outermost layer, consisting of visceral peritoneum. The presence of rugae (folds in the mucosa) allows the stomach to expand significantly after food intake. The stomach’s histological architecture is adapted to protect the organ from autodigestion while maintaining high digestive efficiency (Young et al., 2020). 2.1.5 Nerve Supply of the Stomach The stomach receives both extrinsic and intrinsic nerve supply: 2.1.5.1 Extrinsic innervation: Parasympathetic: Provided by the vagus nerve (cranial nerve X), stimulating gastric secretions and motility. The anterior vagal trunk supplies the anterior surface, and the posterior vagal trunk supplies the posterior surface (Drake et al., 2020). Sympathetic: Originates from spinal segments T6–T9 via the greater splanchnic nerves, synapsing in the celiac ganglia. It inhibits motility and reduces gastric secretions. 2.1.5.2 Intrinsic innervation: Myenteric (Auerbach’s) plexus: Located between muscular layers; regulates peristalsis. Submucosal (Meissner’s) plexus: Controls glandular secretions and blood flow. This complex innervation allows fine-tuned control over gastric functions including motility, enzyme secretion, and acid production (Barrett et al., 2019). 2.1.6 Functions of the Stomach The stomach performs several essential functions: i. Reservoir Function: Stores ingested food and releases it gradually into the small intestine. ii. Mechanical Digestion: Mixes food with gastric secretions using strong muscular contractions to produce chyme. iii. Chemical Digestion: Hydrochloric acid denatures proteins and activates pepsinogen to pepsin for protein digestion. iv. Enzymatic Activity: Secretes pepsin, gastric lipase, and intrinsic factor essential for vitamin B12 absorption. v. Endocrine Role: G cells in the pyloric antrum release gastrin, stimulating acid production and gastric motility. vi. Immune Defense: Acidic environment serves as a barrier to many ingested pathogens (Barrett et al., 2019). 2.1.7 Clinical Relevance of the Stomach Various disorders and diseases affect the stomach: i. Gastritis: Inflammation due to H. pylori infection, alcohol, or NSAID use. ii. Peptic Ulcer Disease: Mucosal erosion often associated with H. pylori or NSAID use. iii. Gastric Carcinoma: Malignancy commonly arising from chronic gastritis or metaplasia. iv. Pernicious Anemia: Autoimmune destruction of parietal cells results in intrinsic factor deficiency and impaired B12 absorption. v. Pyloric Stenosis: Congenital hypertrophy of the pyloric muscle in infants causing projectile vomiting. vi. Gastroparesis: Delayed gastric emptying often due to autonomic neuropathy (Carlson, 2022). 2.2 Review of Related work Garlic (Allium sativum) has been extensively studied for its medicinal properties. A review by Patel and Singh (2023) highlighted the antimicrobial, anti-inflammatory, and gastroprotective effects of garlic extracts. The study noted that the sulfur-containing compounds in garlic, particularly allicin, exhibit strong antioxidant activity, neutralizing free radicals and reducing oxidative stress-induced tissue damage. Oxidative stress plays a crucial role in the pathogenesis of gastric mucosal damage. According to Ahmed et al. (2023), exposure to environmental toxins like formaldehyde induces excessive ROS production, leading to lipid peroxidation and mitochondrial dysfunction. The study further highlights that oxidative stress disrupts the gastric epithelial barrier, making the stomach more susceptible to ulcer formation and inflammatory conditions. Formaldehyde is widely recognized as a toxic compound with systemic effects. In a study by Zhang et al. (2022), chronic inhalation of formaldehyde in laboratory animals was linked to oxidative stress, apoptosis, and inflammatory responses in gastric tissues. The findings suggest that formaldehyde exposure leads to significant histopathological alterations, including mucosal erosion, inflammatory cell infiltration, and epithelial degeneration. These effects were attributed to increased levels of reactive oxygen species (ROS) and inflammatory cytokines. Research by Huang et al. (2022) demonstrated that Allium sativum extract protects against ethanol-induced gastric injury in rats. The study found that garlic supplementation significantly reduced gastric acid secretion, increased mucus production, and prevented ulcer formation. This suggests that Allium sativum could have a similar protective effect against formaldehyde-induced gastric toxicity. Recent studies also suggest that natural antioxidants can mitigate oxidative damage in gastric tissues. Kim et al. (2021) in their research examined the effects of dietary antioxidants such as flavonoids, polyphenols, and organosulfur compounds on gastric ulcer models. The study found that these compounds enhance enzymatic antioxidant defenses, reduce pro-inflammatory cytokines, and promote tissue repair, making them potential therapeutic agents against chemically induced gastric injuries. Zhang et al. (2018) conducted an experimental study on the protective effects of Allium sativum against ethanol-induced gastric injury. The findings indicated that garlic extract reduced gastric acid secretion, enhanced mucus production, and protected against ulcer formation through antioxidant and anti-inflammatory mechanisms. 2.3 ALLIUM SATIVUM Garlic (Allium sativum) is a perennial herbaceous plant widely known for its culinary and medicinal uses. Belonging to the Alliaceae family, garlic has been utilized since ancient times across various cultures not only as a food additive but also for its therapeutic properties (Nicastro et al., 2015). Recent scientific investigations have reaffirmed the pharmacological significance of garlic, attributing its effects to bioactive sulfur compounds such as allicin, diallyl disulfide, and S-allyl cysteine (Amagase, 2006). This literature review presents a comprehensive examination of the botany, phytochemistry, pharmacology, therapeutic applications, and safety profile of garlic. Figure2: Image of Garlic (Allium sativum), sources: (Nicastro et al., 2015). 2.3.1 Botanical Characteristics Garlic is a bulbous plant with a tall, erect flowering stem that grows up to 1 meter. Each bulb consists of multiple cloves, each enclosed in a papery membrane. Garlic is propagated vegetatively, as it rarely produces viable seeds (Block, 2010). It thrives in temperate climates and is cultivated globally, with major production hubs in China, India, and South Korea. 2.3.2 Phytochemical Constituents Garlic contains numerous biologically active compounds, predominantly sulfur-containing molecules. The most notable is allicin, produced enzymatically when garlic is crushed or chopped, converting alliin via alliinase (Iciek et al., 2009). Other compounds include ajoene, diallyl disulfide, diallyl trisulfide, and S-allyl cysteine (Nicastro et al., 2015). These constituents are believed to be responsible for garlic’s antimicrobial, antioxidant, anti-inflammatory, and cardioprotective properties (Nicastro et al., 2015). 2.3.3 Pharmacological Properties i. Antimicrobial Activity: Allicin and other sulfur compounds exhibit broad-spectrum antimicrobial activity against bacteria, fungi, and some viruses. Studies have demonstrated garlic's efficacy against pathogenic strains such as Escherichia coli, Staphylococcus aureus, and Candida albicans (Nicastro et al., 2015). ii. Cardiovascular Protection: Garlic has been shown to lower blood pressure, reduce cholesterol levels, and inhibit platelet aggregation (Ried et al., 2008). These effects are thought to be mediated through modulation of nitric oxide synthesis and antioxidant mechanisms. iii. Antioxidant and Anti-inflammatory Effects: The antioxidant activity of garlic is attributed to its capacity to scavenge reactive oxygen species and upregulate endogenous antioxidant enzymes (Amagase, 2006). Anti-inflammatory effects are mediated by downregulating pro-inflammatory cytokines and transcription factors such as NF-κB. iv. Anticancer Potential: Epidemiological and experimental studies have reported the anticancer effects of garlic, particularly in reducing the risk of gastrointestinal cancers (Nicastro et al., 2015). Mechanisms include induction of apoptosis, inhibition of cell proliferation, and suppression of angiogenesis. 2.3.4 Therapeutic Applications Garlic is used in the management of various health conditions including hypertension, hyperlipidemia, atherosclerosis, diabetes, and infections. Standardized garlic preparations such as aged garlic extract and garlic oil are commercially available and have demonstrated consistent efficacy in clinical trials (Banerjee & Maulik, 2002). 2.3.5 Safety and Toxicity Garlic is generally considered safe when consumed in dietary amounts. However, excessive intake may lead to gastrointestinal discomfort, allergic reactions, and bleeding complications, especially when combined with anticoagulant therapy (Petrovska & Cekovska, 2010). Topical application may cause skin irritation or dermatitis. 2.4 Formalin Formalin is an aqueous solution of formaldehyde, typically containing about 37–40% formaldehyde gas by weight with stabilizers such as methanol to prevent polymerization (World Health Organization, 2002). It is widely used in laboratories, medical facilities, and industries for its excellent preservative, disinfectant, and tissue-fixative properties. Despite its utility, formalin poses significant health hazards due to its volatile and toxic nature, and has been classified as a human carcinogen (International Agency for Research on Cancer, 2012). This review explores the chemical composition, mechanism of action, toxicology, occupational exposure, biomedical applications, and regulatory guidelines concerning formalin (Swenberg et al., 2013). 2.4.1 Chemical Composition and Properties Formalin consists primarily of formaldehyde (CH2O), a colorless, pungent-smelling gas that dissolves readily in water. The solution may also contain small amounts of methanol as a stabilizer (Swenberg et al., 2013). It is reactive and forms cross-links between primary amine groups in proteins and nucleic acids, which underlies its use as a fixative in histopathology. 2.4.2 Mechanism of Action Formalin preserves biological tissues by forming methylene bridges between amino groups in proteins, effectively "freezing" cellular structures and inhibiting autolysis and microbial growth (Buesa, 2008). While this mechanism is beneficial in biomedical applications, the same reactivity contributes to its cytotoxic and genotoxic effects upon exposure. 2.4.3 Biomedical Applications i. Tissue Fixation: Formalin is the gold standard for preserving tissues in pathology and anatomy laboratories. It ensures structural preservation for histological examination. ii. Embalming and Preservation: Formalin is widely used in embalming fluids for the preservation of cadavers in medical training and funeral practices. iii. Disinfectant: Due to its antimicrobial properties, formalin is used in sterilization processes and as a disinfectant in healthcare settings (Swenberg et al., 2013). 2.4.4 Toxicology and Health Effects Exposure to formalin occurs primarily through inhalation, skin contact, and, less commonly, ingestion. Acute effects include irritation of the eyes, nose, and respiratory tract, while chronic exposure has been linked to nasal and nasopharyngeal cancers (WHO, 2002; Zhang et al., 2010). Carcinogenicity: The IARC (2012) classifies formaldehyde as a Group 1 human carcinogen. Epidemiological studies have shown increased risks among workers in industries using formaldehyde. Neurotoxicity and Behavioral Effects: Animal studies indicate that inhalation or chronic exposure to formalin may lead to neuroinflammation, behavioral changes, and memory impairment (Yahya et al., 2017). Reproductive and Developmental Toxicity: Evidence from animal studies suggests possible adverse effects on reproductive health and embryonic development at high exposure levels (Swenberg et al., 2013). 2.4.5 Occupational Exposure and Safety Professionals working in medical laboratories, mortuaries, and industrial settings are at increased risk of exposure. Protective measures include adequate ventilation, personal protective equipment (PPE), and adherence to permissible exposure limits (PEL) set by regulatory bodies like OSHA and NIOSH (Occupational Safety and Health Administration, 2011). 2.4.6 Regulatory Standards and Guidelines The Occupational Safety and Health Administration (OSHA) have set the permissible exposure limit (PEL) for formaldehyde at 0.75 ppm as an 8-hour time-weighted average (TWA) (OSHA, 2011). The American Conference of Governmental Industrial Hygienists (ACGIH) recommends a threshold limit value (TLV) of 0.3 ppm as a ceiling limit (ACGIH, 2020). WHO and other international agencies emphasize strict regulations and guidelines for the safe use and disposal of formalin-containing products. CHAPTER THREE MATERIALS AND METHODS 3.1 Research Design This study adopted an experimental design aimed at investigating the protective effects of crude extract of Allium sativum (garlic) against stomach toxicity induced by formalin inhalation in adult male and female albino rats. The experiment involved formalin inhalation exposure, garlic extract treatment, and assessment of body weight changes, pancreatic weight, and histological changes. 3.2 Study Area The research study was conducted in the Department of Anatomy and Forensic Anthropology Histology laboratory situated in the northern region of Cross River State at the University of Cross River Okuku campus. 3.3 Materials and Their Uses S/N Material Use 1. Adult male and female albino rats Experimental animals for toxicity induction and treatment evaluation 2. Formalin solution Induction of oxidative stress through inhalation toxicity 3. Fresh garlic bulbs (Allium sativum) Preparation of crude aqueous extract for antioxidant treatment 4. Distilled water Solvent for garlic extraction and animal hydration 5. Cage Housing and maintenance of experimental rats under hygienic conditions. 6. Weighing balance Measurement of body weight and pancreas weight 7. Syringes and oral cannula Administration of garlic extract 8. Light microscope Examination of histological pancreatic sections 9. Histology reagents (Formalin, Paraffin wax, Hematoxylin and Eosin stains) Tissue processing and staining for microscopic evaluation 10. Dissection kit Organ harvesting (pancreas). 3.4 Experimental Animals Twenty-five (25) male and female healthy adult albino rats, weighing between 111–250g, were used for the study. The rats were sourced from the human anatomy animal house of University of Cross River (UNICROSS) Okuku campus. They were kept in standard ventilated cages with free access to food and water, and allowed to acclimatize for one week under controlled conditions. 3.5 Ethical Considerations Ethical approval for the study was obtained from the Ethical Committee of University of Cross River State (UNICROSS), Okuku Campus, for the study to be carried out in the department of Anatomy and Forensic Anthropology histology laboratory 3.6 Preparation of Garlic (Allium sativum) Extract Fresh garlic bulbs were peeled, washed, and dried. 100g of the bulbs were blended using a blender to obtain a paste. The paste was soaked in 50mL of distilled water and was filtered.The filtrate (aqueous garlic extract) was stored in a refrigerator. 3.7 Formalin Inhalation Exposure Formalin toxicity was induced by exposing rats to formalin vapors for 9 days. This exposure model was modified from the method described by Patil et al. (2014) to simulate occupational inhalation toxicity. 3.8 Administration and Treatment The body weight of each rat was recorded at the start of the experiment and final body weights were recorded before sacrifice. The Wistar rats were grouped into six categories based on their weights, 5 rats each in group one-four, 2 in group five and 3 in group six has showed below respectively. GROUPS NUMBER OF RATS ADMINISTRATION/TREATMENTS Group 1: (Control Group) 5 Rats with no formalin exposure and no garlic treatment Group 2: (Formalin only Female) 5 Female rats exposed to formalin vapors only without any treatment. Group 3: (Formalin + Garlic Female) 5 Female rats exposed to formalin vapors and treated with 1.73ml of garlic extract orally. Group 4: (Garlic only Female) 5 Female rats were treated with 1.5ml of garlic extract orally without formalin exposure. Group 5: (Formalin only Male) 2 Male rats were exposed to formalin vapors only without any treatment. Group 6: (Formalin + Garlic Male) 3 3 Male rats were exposed to formalin vapors and treated with 1.1ml. garlic extract without formalin exposure. 3.9 Sacrifice and Histological Analysis of the Pancreas After the 14-day exposure and treatment period, rats were sacrificed, and the stomach was harvested for histological analysis. This included detailed examination of the Stomach to evaluate the extent of protection or damage in response to the administered substances. The tissues were carefully excised and rinsed in normal saline and were fixed immediately in 10% buffered formalin for 48 hours. Tissues were dehydrated, cleared, and embedded in paraffin wax. Sections of 5 μm thickness were cut using a microtome and stained with Haematoxylin and Eosin (H&E) for light microscopic examination. The tissues were evaluated for structural changes such as: Degeneration of acinar cells, Islet cell destruction, inflammatory infiltration, Edema or fibrosis 3.10 Tissue processing The stomach is fixed in 10% formalin were rapidly processed for routine paraffin embedding. The process began with tissue dehydration, which was carried out for duration of one hour through ascending grades of ethanol to ensure complete dehydration. The ethanol grades used were as follows: 70% ethanol 80% ethanol 90% ethanol 100% ethanol Following dehydration, the tissues were cleared in xylene to remove any remaining alcohol and prepare them for paraffin infiltration. The clearing process was conducted as follows: 1. Absolute alcohol 1 and Xylene for 1 hour 2. Xylene 1 for 1 hour 3. Xylene II for 1 hour Once cleared, the tissues were infiltrated in two changes of molten paraffin wax at 55°C in an oven, each for 1 hour. The tissues were then embedded in paraffin wax using brass embedding molds smeared with glycerin. This step ensured that the paraffin-blocked tissues could be easily separated from the molds after embedding. After embedding, the paraffin-blocked tissues were embedded and mounted on wooden blocks for sectioning using a rotary microtome. Sections were obtained from a clean glass slide smeared with egg albumen to facilitate adherence. The slides were then dried on a hot plate at a temperature of 40°C overnight to enhance adherence. Once dried, the slides were stored in slide racks until ready for staining. 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