Trichinosis is a food-borne zoonotic disease with worldwide distribution. It is caused by a parasitic nematode of the genus Trichinella [1]. The disease holds significant public health implications. It affects approximately ten thousand individuals annually, with a mortality rate of around 0.2% [2]. Trichinella spiralis (T. spiralis) is the most virulent pathogenic species affecting humans. It exhibits a wide global distribution and is mainly transmitted through the consumption of undercooked pork infected with the encysted larvae of the parasite [3].

The Trichinella parasite has a unique life cycle that is accomplished in a single host. The adult worms lodge in the small intestinal enterocytes [4]. There, they release newborn larvae (NBL), which migrate throughout the body until they settle in the skeletal muscles, where they become infective larvae, transforming the host muscle cells into a permanent niche. Although this parasitic occupation is efficient, it does not remain without counter-resistance from the host [5].

During the intestinal phase of infection, the immune response to trichinosis involves both Th1 and Th2 responses. Initially, Th1 responses are induced, followed by a Th2 dominance. In the small intestine, adult T. spiralis worms create an intra-multicellular residence, constituting numerous epithelial cells. As a result, the intestinal mucosa serves as the first natural barrier against the parasite. Consequently, infected epithelial cells initiate the mucosal production of proinflammatory cytokines and mediators, including nitric oxide (NO) ^[6][7][6,7].

Nitric oxide (NO) is a unique signaling molecule with diverse physiological functions. It is a product of L-arginine deamination to L-citrulline by the nitric oxide synthase (NOS) enzyme, a reaction that requires nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor [8]. Three NOS isotypes exist: neuronal nNOS (NOS I), inducible ‘i’NOS (NOS II), and endothelial eNOS (NOS III). Each of these differ in their anatomical distribution, genetic origin, and pathophysiological roles [9]. The isoforms (nNOS) and (eNOS) are constitutively present in many cells and tissues, where they engage in normal physiological responses. The inducible isotype (iNOS) is absent in resting cells. Yet, it is rapidly expressed in response to stimuli like infections or inflammation [10].

Among the calcium-dependent constitutive isoforms, (nNOS) and (eNOS), the neuronal NO synthase is the dominant isoenzyme in the small intestine of rodents and large animals. It plays critical physiological functions in regulating gut motility ^[11][12][11,12]. Because (eNOS) accounts for only a very small part of intestinal (NOS) activity, nNOS’s role may extend beyond motility regulation. Thus, it is possible that (nNOS) in the intestine also functions as the protective (eNOS). As a result, the lack of this key enzyme is associated with impaired nitric oxide production and defective gastrointestinal transit [13].

Many studies have analyzed the protective role of (NO) during Th1-inducing infections. However, fewer have examined its effects in Th2-polarizing infections, such as those involving helminth parasites. To date, the role of (NO) in the immune response against T. spiralis infection remains debatable [14]. Most studies have focused on the effects of the inducible isotype (iNOS) during both the intestinal and muscular phases of trichinosis ^[7][15][16][7,15,16]. In contrast, literature addressing the impacts of the neuronal isoform (nNOS) on different intestinal parasitic infections remains scarce. Building on this gap, this research is the first to investigate the possible implications of not only (iNOS) but also (nNOS) during the intestinal stage of experimental trichinosis.

2. Materials and Methods

2.1. The Parasite

The Trichinella spiralis (T. spiralis) strain was provided by Theodor Bilharz Research Institute (TBRI), Giza, Egypt. The larvae were originally obtained from infected pig muscles at Cairo abattoir in Elbasatin, Egypt. The life cycle was initiated and maintained through successive passages in parasite-free BALB/C mice.

2.2. Animals and T. spiralis Infection

In this experimental study, the sample size was calculated to be 48 mice using OpenEpi at a confidence level of 95% and 80% power of test. The laboratory-bred male Swiss albino mice were of matched age (6–8 weeks) and weight (20–25 g). The mice were selected from the animal facilities of TBRI. They were housed in well-ventilated plastic cages and maintained under controlled lighting (12 h light/12 h dark cycle) and temperature (25 ± 2 °C) with standard pelleted diets and water supplies. To exclude any parasitic infections, routine fecal examination was conducted for all mice, using direct smear analysis and concentration measures for 3 consecutive days [17]. All animal handling, breeding, and experimental procedures were conducted at the Medical Parasitology Department, Faculty of Medicine, Zagazig University, in accordance with national and institutional guidelines for the use and care of laboratory animals. T. spiralis muscle larvae were recovered from infected mice. The recovery followed the protocol implemented by Dunn and Wright [18]. Briefly, heavily infected muscles were minced and digested in equal volumes of 1% pepsin and 1% concentrated hydrochloric acid (HCl) in 1000 mL of distilled water. The mixture was incubated at 37 °C for 2 h under continuous agitation, using an electric stirrer. The digested product was passed through a 50-mesh/inch sieve to remove coarse particles. Larvae were then collected on a 200 mesh/inch sieve, washed twice, and suspended in 150 mL of tap water in a conical flask. After discarding the supernatant fluid, the larvae in the sediment were counted microscopically using a McMaster counting chamber. Experimental mice were orally infected with approximately 250–300 larvae per mouse. A tuberculin syringe with a blunt needle was used to introduce the infective larvae into the stomachs of mice after a 12 h starvation period.

2.3. Drug Regimen: Dosage Schedule

Albendazole (Bendazol; Sigma Pharmaceuticals Industries, Cairo, Egypt) was provided as a 20 mg/mL suspension and administered orally at a dose of 50 mg/kg/day, starting on the 3rd day post-infection for 3 successive days [19]. L-arginine stock solution (A8094; Sigma Pharmaceutical Industries, Cairo, Egypt) was dissolved in saline to prepare a concentration of 20 mg/mL. Each mouse in the L-arginine-treated group then received an oral dose of 0.1 mL/10 g BW/day (200 mg/kg/day) [20]. L-arginine was administered orally for 7 days before infection [16]. The nitric oxide synthase (NOS) inhibitors, aminoguanidine (AG) and 7-Nitroindazole (7-NI) (Sigma-Aldrich, St Louis, MO, USA), were administered from day 1 to day 5 post-infection. Aminoguanidine, in the form of aminoguanidine bicarbonate white powder, was dissolved in saline and given orally at 50 mg/kg/day [21]. 7-Nitroindazole was dissolved in dimethyl sulfoxide (DMSO) and administered intraperitoneally at a dose of 50 mg/kg/day [22].

2.4. Experimental Design

The forty-eight (48) mice included in the study were divided into 6 groups (8 mice each) as follows: (G1): negative control group (non-infected, non-treated), (G2): infected control group (infected, non-treated), (G3): infected–Albendazole-treated group, (G4): infected–L-arginine-treated group, (G5): infected–Aminoguanidine-treated group, and (G6): infected–7-Nitroindazole-treated group. Except for (G1), all mice were exposed to T. spiralis infection. On the 7th day post-infection, all mice (48 total) were sacrificed by cervical dislocation under anesthesia. The small intestine of each mouse was removed, opened longitudinally, and washed. A one-centimeter segment (at the junction of the proximal 1/3 and distal 2/3) was stored in 10% formalin for histopathological and immunohistochemical studies. The remaining portions of the small intestine were used to detect adult T. spiralis worms.

2.5. Assessment Measures

2.5.1. Parasitological Analysis

• Isolation and Counting of Adult Worms

First, the washed intestine was cut into 1 cm pieces and incubated at 37 °C in 10 mL of saline for 2 h to allow the worms to detach from the tissue. Next, repeated saline washing was performed until the fluid became clear. Afterward, all fluid was collected and centrifuged at 1500 rpm for 5 min. Finally, to count adult worms, the supernatant was decanted, and the sediment was examined under a dissecting microscope [23].

2.5.2. Histopathological Analysis

Histopathological examination was performed on small intestinal specimens from different study groups. The samples were fixed in 10% formalin, dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and embedded in paraffin blocks. Paraffin sections of 5 μm thickness were prepared, stained with hematoxylin and eosin (H&E), and examined under a light microscope for histopathological evaluation [24]. The extent of the intestinal inflammatory response was scored as follows: grade (+1) was defined as a mild inflammatory infiltrate, with less than one inflammatory cell focus per X100 field; grade (+2) indicated a moderate inflammatory infiltrate, with 1–5 foci/X100 field; grade (+3) corresponded to a large inflammatory infiltrate, with >5 foci/100X field; and grade (+4) reflected an extensive inflammatory infiltrate, characterized by a widespread inflammatory reaction observable throughout the tissue. The degree of inflammatory reaction was assessed by examining 10 fields for each tissue section using a semi-quantitative score [25].

2.5.3. Immunohistochemical Analysis

To investigate intestinal iNOS and nNOS expressions, immunostaining was performed using the avidin–biotin–peroxidase complex (ABC) technique [26]. Briefly, paraffin sections were cut to a thickness of 5 μm on poly-L-lysine-coated slides. The sections were deparaffinized, and endogenous peroxidase activity was blocked by incubating with 3% hydrogen peroxide for 5 min. They were then washed twice in phosphate-buffered saline (PBS) (5 min each time). Next, the sections were placed in a 0.01 mol/l citrate buffer (pH 6.0) and subjected to a 20 min microwave treatment for antigen retrieval. The primary antibodies (rabbit polyclonal anti-iNOS and rabbit polyclonal anti-nNOS antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), in 1:200 dilutions, were added and left overnight at 4 °C. Sections were washed with PBS. They were then incubated with biotin-labelled goat anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 35 min. The peroxidase activity was visualized using a diaminobenzidine (DAB) substrate (DAKO Corp, Fremont, CA, USA) applied for 5 min. Negative control slides were prepared by processing sections in the absence of primary antibody [19]. The immunohistochemical reactivities of iNOS and nNOS markers were evaluated in intestinal sections from different study groups in a semi-quantitative manner based on staining extent and intensity. The extent of staining was graded as follows: 0, <5%; 1, >5–25%; 2, >25–50%; 3, >50–75%; and 4, >75%. Meanwhile, the intensity signal was graded as follows: 0: no staining, 1: mild, 2: moderate, and 3: marked staining intensity. A final score was generated by multiplying intensity and extent, yielding a range of 0 to 12. Final scores were further classified into three categories: negative/low (scores 0–4), moderate (scores 5–8), and high expression (scores 9–12) [27]. The iNOS and nNOS immune stains were microscopically assessed in 10 fields for each tissue section using the Leica Qwin 500 image analyzer computer system (Cambridge, UK). For both histopathological and immunohistochemical analyses, biopsy specimens were randomized, coded, and evaluated blindly.

2.5.4. Biochemical Analysis

Serum samples were obtained from all treated and control mice on the 7th day post-infection. The samples were collected and stored at −20 °C until further biochemical analysis.

• Assessment of serum nitric oxide (NO) levels

Serum NO levels were measured using a colorimetric nitric oxide assay kit (BioVision Incorporated, CA, USA, Cat. No. K262–200) following the instructions of the manufacturer. Prepared standards, a sample blank, and 85 μL of each serum sample were added to a microtiter plate. The assay consisted of two steps. First, 5 μL of each nitrate reductase mixture and enzyme cofactor were added to the sample and standard wells. The plate was incubated for 60 min to convert nitrate to nitrite. After adding 5 μL of the enhancer, the plate was incubated for 10 min. In the second step, 50 μL of each Griess reagent (R1 and R2) was added to generate a deep purple compound from nitrite. Absorbance was measured at 450 nm.

• Assessment of the serum cytokines, IFN-γ, and TNF-α

Enzyme-linked immunosorbent assay (ELISA) was used to assess the serum concentrations of IFN-γ and TNF-α cytokines. The Mouse ELISA Kit (Quantikine, USA R&D Systems, Inc., Minneapolis, MN 55413, USA) was used for the quantitative determination of IFN-γ (Cat. No. MIF00) and TNF-α (Cat. No. MTA00B-1) concentrations in serum samples, according to the supplier’s protocol.

2.6. Statistical Analysis

The data collected were tabulated and analyzed using SPSS version 25 (IBM, Armonk, NY, USA). Quantitative data were described using mean, range, and standard deviation (SD). ANOVA F-test was performed to compare more than two groups for normally distributed quantitative variables. [28]. Post hoc analysis was performed for pairwise comparisons. The Bonferroni correction was applied to control the overall rate of false positives [29]. Significance was set at p values < 0.05. Treatment efficacy was calculated using the following equation: Efficacy (%) = 100 × (mean worm number in controls minus mean worm number recovered in treated mice)/mean worm number in controls [30].