Archive | 2021
Insights into the effects of inducible and neuronal nitric oxide synthase isoenzymes in experimental intestinal heterophyiasis
Abstract
Background: Heterophyiasis is one of the food-borne trematode infections, caused by the intestinal fluke Heterophyes heterophyes. The exact role of nitric oxide (NO) in the immune response against the majority of parasites remains controversial. It proved protective against a wide range of protozoan and helminthic parasites. Nevertheless, its role in intestinal heterophyiasis is yet to be explored. Objectives: The study aims to explore the possible roles of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) in experimental intestinal heterophyiasis. Material and Methods: The experimental study design included infection of male puppies with H. heterophyes encysted metacercariae (EMC), followed by treatment with aminoguanidine (AG) and 7-nitroindazole (7-NI) drugs, as selective inhibitors of iNOS and nNOS, respectively. Controls included non-infected and infected untreated puppies. Intestinal tissue sections from all puppies were stained for histopathological and immunohistochemical (IHC) assessments. Results: Different intensities of iNOS and nNOS isoenzymes were observed in intestinal sections. The study showed the highest concentration of iNOS isoenzyme in the infected-7-NI treated group. The control noninfected puppies exhibited the highest levels of nNOS expression, with statistical significance (P<0.05). The study also showed that AG significantly reduced the degree of inflammatory cellular infiltrations. Additionally, the over-production of NO worsened the degree of intestinal apoptotic changes. Conclusion: Results obtained in the study suggested that inhibition of iNOS, to some extent, improved intestinal architecture, while inhibition of nNOS failed to eliminate experimental intestinal heterophyiasis. Abbreviations: 7-NI: 7-Nitroindazole; AG: Aminoguanidine; ECM: Encysted metacercaria; IHC: Immunohistochemical; iNOS: Inducible nitric oxide synthase; NO: Nitric oxide; NOS: Nitric oxide synthase; nNOS: Neuronal nitric oxide synthase. Nitric oxide in heterophyiasis Abdel Aziz and Elsayed 87 eNOS was first identified in endothelial cells but is also present in smooth muscle cells, epithelial cells and T cells[13]. Both nNOS and eNOS are constitutively present in many cells and tissues where they participate in normal physiological responses. On the other hand, iNOS is an immune-inflammatory factor with critical signaling transduction events during infections[14]. It is worth mentioning that the expression of NO contributes to regulating various processes in the gastrointestinal tract. The constitutive NOS enzymes have critical physiological functions such as maintaining motility and blood flow[15]. Additionally, iNOS acts as an anti-pathogen with tumoricidal activities. It is mainly involved in the defense mechanisms against infections and mediation of the inflammatory responses[16]. However, its over-production may cause detrimental consequences because of its cytotoxic activities[17]. Despite the wide evidence of using iNOS due to its anti-parasitic effects in different experimental infections of trichinellosis[18], fascioliasis[19] and strongyloidiasis[20], reports investigating the impact of nNOS in intestinal parasitic infections is lacking. Accordingly, we assessed, for the first time, the roles of iNOS and nNOS in intestinal heterophyiasis. After selective inhibition of both isoenzymes using 7-NI and AG, we examined the possible consequences of such inhibition on experimentally infected puppies through parasitological, histopathological and IHC approaches. MATERIAL AND METHODS This experimental study was conducted at the Medical Parasitology Department (Post-Graduate Research Laboratory), Faculty of Medicine, Zagazig University, Zagazig, Egypt, during the period from July 2020 to December 2020. Study design: EMC were collected to infect forty-four male puppies equally divided into four groups (11 puppies each). Control groups included non-infected puppies (G1) and infected untreated puppies (G2). Groups 3 and 4 included infected puppies treated with 7-NI and AG, respectively. Both drugs were administered after confirmation of the infection by detection of eggs in fecal samples. Drug treatment continued for 14 days post-infection. Three weeks post-infection, all puppies were sacrificed, and the whole intestines were resected, for further parasitological assessment, as well as histopathological and IHC evaluations. Experimental animals: The puppies were of matched age (5 to 7 weeks old) and weight (3-3.5 kilograms). They were housed at the Parasitology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt, in well-ventilated cages and fed bread and milk. Three consecutive fecal samples were examined to exclude any parasitic infections. Collection of EMC and infection of puppies: One hundred Mugil cephalus and 200 Tilapia nilotica fish species were collected from local fish markets in Suez and Sharkia Governorates. They were placed in iceboxes, transferred to the Laboratory, and examined for the presence of EMC according to Mahdy et al.[21]. Detection of H. heterophyes EMC was based on their characteristic location between the muscle fibers, thick cyst wall and their relative large size[22]. Further microscopic identification was by the compression method[23]. Detected EMC were separated from the infected fish samples using artificial digestion method[24]. Only EMC with morphological characteristics compatible with those of H. heterophyes were used for infection. The final volume of EMC suspension was adjusted by saline to contain ~300/0.5 ml (the infective dose)[25]. Infection of puppies was carried out orally using a stomach tube and mouth gage[26]. To collect worms, the mucosa was gently scraped, and the contents were transferred to Petri dishes containing physiological saline. The collected worms were stained using the acetocarmine stain[27] and were morphologically identified according to Witenberg[28]. Drugs: Both 7-NI and AG drugs were obtained from Sigma-Aldrich (St Louis, MO, USA). The first was dissolved in dimethylsulfoxide (DMS) and made up to final volume by adding 0.9% NaCl (DMS content was 15%). The first was given intraperitoneally at a dose of 30 mg/kg/day[29]. The second was administered by intraperitoneal injection once daily (100 mg/kg)[30]. Histopathological studies: The resected intestinal specimens from all study groups were fixed in 10% formalin, dehydrated through grades of ethyl alcohol, cleared in xylol, and then processed for paraffin embedding. Serial cut sections of 5 μ thickness were stained with haematoxylin and eosin (H&E)[31]. Sectioned worms were studied concerning their site and the surrounding cellular infiltration. Intensity of infection was estimated by grading the sectioned worms per puppy. It was calculated in a semiquantitative manner according to Seo et al.[32] as follows: score 0 (-): no worms; score 1 (+): less than 500; score 2 (++): 500 to less than 3000; and score 3 (+++): more than 3000. Other pathological changes such as intestinal polyps and apoptotic changes were also recorded. Apoptosis was graded as mean number of apoptotic cells/10 high power fields[33] where 1 = well-differentiated, 2 = moderately differentiated, and 3 = poorly differentiated; with corresponding mean numbers of apoptotic count/ high power fields of 0.86, 2.21, and 3.50, respectively. Immunohistochemical studies: The expressions of both iNOS and nNOS isoenzymes were evaluated using the avidin-biotin-peroxidase complex (ABC) method[34]. Sections from paraffin blocks were dewaxed and antigens were retrieved in a microwave oven for PARASITOLOGISTS UNITED JOURNAL 88 20 min. For blocking intrinsic peroxidase activity 3% hydrogen peroxide solution was used. Primary antibody (rabbit anti-rat nNOS and iNOS antibody) (Beijing Zhongshan Biotechnology Co., Ltd, Beijing, China), in 1:200 dilution, was added and left overnight at 4°C. Sections were then washed with phosphatebuffered saline and incubated with biotin-labelled goat anti-rabbit antibody for 35 min, then again incubated with ABC kit, PK-4000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunolabeling was attained by diaminobenzidine substrate which was added at room temperature. The slides were examined under light microscopy and the expression of the markers was recorded in a semiquantitative manner through optical density average by MetaMorph and computer image process software. Statistical methods: The data were checked and analyzed using Statistical Package for the Social Sciences (SPSS) version 22. In the current study, nonparametric statistical methods were used[35]. Mann– Whitney (U) test was used as an alternative to the independent sample t-test which is used for comparing two groups[36]. The Wilcoxon signed-ranks test was used as the non-parametric alternative for the paired samples ttest[37]. Significance was considered when P value was < 0.05. Ethical considerations: The dogs were reared and sacrificed according to the international guidelines approved by the Institutional Animal Care and Use of Zagazig University Committee (IACUCZU) for animal use in research and teaching. RESULTS Parasitological results: Light microscopic examination of the fish samples revealed H. heterophyes EMC. They were found scattered in-between the striated muscle fibers and under the scales of the examined fish specimens (Fig. 1a, b). The majority of H. heterophyes adult worms (Fig. 1c) were collected from the upper portions of the small intestines. Few H. heterophyes eggs were recovered from the fecal samples of the infected animals one-week post-infection (Fig. 1d). Intestinal sectioned worms: In G2, H. heterophyes adult worms were embedded in-between severely deformed and inflamed intestinal crypts and villi along with marked hyperplasia of lymphoid tissues (Fig. 2a, b). After administration of 7-NI in G3, worms were noticed between highly necrotic villi (Fig. 2c). In G4, AG signific