Histopathological Study Post Helicobacter Pylori Infection in Mice
*1Aseel I. Ibrahim, 2Zainab I. Ibrahim, 2Zainab Jamal M. Jawad Author Affiliations:
1 Clinical laboratory Science department/ college of Pharmacy/ University of Baghdad
2 department pathology and poultry disease/ college of veterinary medicine/ university of Baghdad
Abstract:
Helicobacter pyloriisthe causesulcers in gastrointestinal tract such asgastric and pepticulcer besides the malignancy of gastric carcinoma, lymphoma andnon-gastricdiseases.
The histopathologicalchanges that occurred indifferent organs of mice postH. pylori infectionwere investigated. The bacterial isolate was taken from a patient with duodenal ulcer. The result tissue section revealed polymorphonuclear cells(PMNs) and lymphocytes infiltrated the layers of kidney, liver, and heart with degeneration.
H. pylori has a significant sever role in the incidence the inflammatory reaction in stomach.
Keyword: Helicobacter pylori, Rapid urease test, infection, diagnosis.
Introduction:
Most disorders of Helicobacter pylori infection is gastric or duodenal ulcers, which occurs where their tissues lost the protection (Wang et al., 2014), mechanism to disposethe acids&pepsin enzymes. Gastriculcer occurs when the mucosa of the stomach or duodenum was very weak for the protection.
Gastric ulcer means inflammation of stomach lining (gastritis)&the perforation occurs when the ulcer untreated or treated weak(Naito& Yoshikawa, 2002).
H. pylori produce urease enzyme which cause the release of free radicals that cause damage the epitheliumneither invading the cells of surrounding tissues &nordeveloping the immunity for repeated infection (Martin,2005).H. pylorirepresented as major etiological factor of gastritis&duodenal ulcer(NIH Consensus conference,1994;Franceschi,2002). The association between H. pylori&causes of cancer was fixed as a carcogenic by the international Agency for Research on Cancer (IARC,1994).
H. pylori colonizes epithelial cells of gastric mucosa and the infectionwas limited in stomach, duodenum and esophagus (Graham,2014).
Experimental procedure:
1- Bacterial isolation& identification:
A-The special media for isolation of H. pylori is skirrow agar with the antibiotics (polymyxin B, Vancomycin, Trimethprim B, &Amphotericin) (Dent et al.1988).
B- Bacterial isolate is taken from biopsies of duodenal ulcer patient in the Educational Baghdad hospital. Tow histological biopsies were taken , one used for bacterial culture with skirrow media then transfer to microaerophilic condition with gas generating kit from oxide for 3-7 days at 37˚C ; the second biopsy used for rapid urease test by liquid urea medium prepared by Marshall (Marshall et al. 1987).
C- H. pylori diagnosed according to Bergey's classification (Holt et al.1994); as following:
i. Microorganism examination: the smear of bacterial colonies stained with gram stain&
examined by light microscope.
ii. The motility of bacteria was examined by suspension drop with light microscope.
iii. Biochemical tests: used catalase & oxidase test.
iv. Sensitivity test for cephalothin&naldixic acid.
D- H. pyloriDiagnosed in the biopsies was done by:
i. Direct smear of biopsies according to (Montgomery et al.1988).
ii. Rapid urease test: the color of media was change from yellow to red through 5-10 min due to highly activity of urease enzyme.
2- Bacterial suspension for inoculation; prepared to contain 109bacterial cells of H. pylori per 1ml of physiological phosphate buffer (Wang et al.1997).
3-histopathological examination:
i. Thirty of white male mice BALB/CMusMusculus, aged between 8-14 weeks &
weight about 250-350 gm, healthy and good management from light to temperature available for them.
ii. All animals were infected orally with 0.1ml of bacterial suspension which contains109 bacterial cells/ml for 3 times, dose for each two days, and thenthe animals were monitored along periods of 3&4 weeks of experiment.
iii. At 4th week post infection the histological samples were taken from the animals after killing, the samples are the liver, kidney & heart preserved in 10% formalin as fixative to prepare histopathological sections was done according to Guyer instructions (Guyer,1953).
Results:
H. pylori isolated from laboratory Animals match the patient isolates.
Histopathological examined of the infected organs(kidney,liver,&heart)through the first three weeks demonstrated degenerative changes in the epithelial cells with progression of inflammatory response& infiltration of lymphocytes in all the examined tissues .In fourth week, viewed lesions characterized by intercellular swelling clearly in the renal tubular epithelium cells(Figure1)&hepatocytes(figure2,3)to vacuolar degeneration of the cardiac muscles(Figure4)&perivascular mononuclear cells cuffing, also few infiltration in the parenchyma , congestion & dilatation of blood vessels(Figure5).
Discussion:
The H. pyloribacteria can causes histopathological changes in non-stomach tissues especially in the kidney, liver & heart, although their main role in infection of the stomach by causing gastric, peptic, duodenal ulcer& gastric cancer.These changes may attribute to virulence of two factors, Vaculatingcytotoxin A (VacA) and Cytotoxin-associated gene (CagA) produced by H. pylori.
Vaculatingcytotoxin A is pore-forming toxin, secreted by H. pylori caused increased in plasma membranepermeability, changes in the structureof endosome, mitochondrial membrane permeability and then cell death (Jones et al.,2010), while CagA (Cytotoxin- associated gene A) is the second protein produced by H. pylori transfer from it into host cells by type IV secretion system (Terradot and Waksman, 2011). These two proteins are determined the pathogenesis of H. pylori(Figueiredoet al.,2005).
In kidney tissue, show vacuolar degeneration (star shaped) with infiltration of lymphocytes within glomerulus, some studies showed correlation between chronic kidney disease and H.
pyloriprevalence (Ganji and Rafieian, 2017), there’s one study has not supported the correlationbetweenH. pyloripresence and the kidney infection(Wijarnpreechaet al.,2017), but our study agreement with Ganji and Rafieian study when showed agreement between H.
pylori and kidney infection.
In the liver tissue, show vacuolar degeneration & leukocyte cuffing, vasculardilatation, enlargement of hepatocytes,depression of the sinusoids. H. pylori inoculated orally may reached the hepatocytes and cause inflammation as etiological factor (Huang et al.,2009).
Rapid urease test is the most useful tool has used in our study for H. pylori diagnosis faster than other tools, rapidurease test had specificity and sensitivity reached 90%, expensive, andisthe only test was performedwithin few minutesdirectly after collect biopsy specimen by endoscopy(Atkinson& Braden, 2016; Al-Rubai, A., 2006).One study reportsthe lowering levels of fasting blood ammonia in H. pylori infection than without H. pylori infection in cirrhosis case of the liver organ(Kiniet al. 2001).Some studiesdepend on rapid urease test for evaluated the status ofH. pylori(Al-Rubai, A., 2006; Vasconezet al. 1999).
In the heart tissue, show thrombosis, perivascular leukocyte cuffing &edema with vacuolar degeneration of cardiac muscle fibers.
Diseasesof the heart like: ischemic heart disease, myocardial infarction, atherosclerosis, coronary artery disease,in addition to H. pylorihasrepresentsone of etiological factor of heart disease by releasingtwo main toxigenic nutrients, vaculating associated gene (Vac A) and cytotoxic-associated gene A (Cag A), Cytotoxin-associated gene A (Cag A) is the most virulence factorparticipating in formation of cholesterol patches in the arteriescauses autoimmune disorder, and started the immune response (Jamkhandeet al. 2016). H.
pylori adhesionto the host cell causedinflammation, cellular damage,and increases the release of most important virulence factors for H. pylori: Cag A andVac Aandfound that H. pylori strains positive toCag A are associated with heart disease morethan strainsnegative to Cag A. H. pyloristrains positive to Cag A increases the activities of cycloxygenase-1 and 2 in the endothelial cells of the blood vessels. Inflammation caused by Cag A stimulate release many cytokines like IL-1 to IL-12, monocytes, macrophages (like tumor necrosis factor α (TNF- α)), T and B lymphocytes, then causes heart disease or shock, and autoimmune reaction among anti-Cag A antibodies and the vascular wall antigens, this suggested that antibodies participated in activation the inflammatory cells within atherosclerosis lesions (Lee et al., 2018; Al-Quarashi&Hodhod, 2012).
Burden of H. pylori is to contain protein similar to the heat shock protein-60 that present on the surface of endothelial cells in the artery. Immune cross reaction occurs between human and bacterial heat shock protein-60 due to the immune response to H. pylori which in turns causes autoimmune reaction and local inflammation in the artery(Sulewska, 2004). Another speculation aboutH. pylori role in development of atherosclerotic plaque, has found that bacterial deoxyribonucleic acid in the arterywall forms patches of infection, which results in heart disease(Sulewska , 2004).The role of H. pylori in many diseases depends on the basis of rapid urease test.
Infection of H. pyloriplay role in progression of vascular disease (Lee et al.,2018;
Elkind&cole, 2006). Seroepidemiologicaland eradication studies demonstratethe relationship between atherosclerosis andthe infection with H. pylori(Ando et al.,2006).
Reached H. pyloriandtheir biochemical contain from the mucosa of the stomach to the circulation (Guoet al., 2007) are in agreement with the exposure ofendothelium to virulence factorssecreted byH. pylori. In atherosclerosis plaque sites, these factors reaches to high levels in the microenvironment of the artery wall (systemic circulation) and cause endothelial dysfunction & lesion development(this may attributed to Vac A,that altering intercellular vesicularcausingformation of vacuole (Reyratet al.,1999).
Conclusion:
The significant pathologic infection of isolated H. pylori in mice.Anti-H.pylori antibodies cross react with antigens of erythrocyte membrane in the human,which probablya guide for the relationship between infection with H. pyloriand vascular disorders . Successful H. pylori eradication led to a reduction in the platelet activation.
Acknowledgment:
I would like to thankfullDr.Yasserin the Endoscope unit in the Al-Educational Baghdad Hospital for his facilitatethe task of collecting biopsies samples, also I would like to thankful Assistant prof. Dr.Zainab Ismail Ibrahim in the Diseases and Poultry department/ veterinary collage/university of Baghdad for her efforts in reading histological sections and reviewing the research,I also thankful all employees in the Endoscope unite of the hospital.
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Figure(1):Kidney in mice, vacular degeneration (star shaped)( )with infiltration within glomerulus.( )(H&E) (X40).
Figure (2):Liver in mice, Vaculardegeneration( ) & perivascular leukocyte cuffing(
).(H&E) (X10).
Figure (3): Liver,diltation of the central vein ( ) ,enlargement of hepatocytes (vacuolar degeneration)( ) ,depletion of the sinusoids.
Figure(4):perivascular leukocyte cuffing(lymphocytes)( ) &vacuolar degeneration.(H&E) (X20).
Figure (5): Heart in mice, Show thrombosis, perivascular leukocyte cuffing & edema with vaculardegeneration( ) of cardiac muscle fibers.(H&E) (X40).
