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Nitric Oxide Synthase in Experimental Chronic Toxoplasmosis: History
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Subjects: Parasitology
Contributor: Marwa Omar

he coccidian protozoan Toxoplasma gondii (T. gondii) is among the most prevalent zoonotic parasites worldwide. Nitric oxide (NO) production by macrophages is considered a critical microbicidal mechanism against various intracellular pathogens, including T. gondii. While the role of the inducible nitric oxide synthase isoenzyme (iNOS) has been widely investigated in both acute and chronic T. gondii infections, the specific functions of the neuronal (nNOS) isotype in the antiparasitic immune response, particularly during chronic toxoplasmosis, remain largely uncovered. Hence, this report seeks to bridge the gap regarding the potential participation of nNOS in experimental chronic T. gondii infection. Methods: The study included 56 Swiss albino mice, equally allocated into four experimental groups: (G1) negative control, (G2) infected control, (G3) infected-L-arginine-treated, and (G4) infected-7-Nitroindazole-treated. All groups except (G1) were orally infected with the avirulent (ME49) T. gondii strain. Nine weeks post-infection, all mice were euthanized for parasitological, histopathological, immunohistochemical, and biochemical analyses. Results: The NO donor, L-arginine, induced a significant reduction in the number of T. gondii cysts, together with strong nNOS immunoreactivity in the brain sections of the treated mice. Conversely, the highest parasitic burden was observed following selective nNOS inhibition with 7-Nitroindazole, exacerbating parasite-induced pathology. Conclusions: The neuronal isotype serves as a critical source of NO production during the chronic stage of T. gondii infection, thereby enhancing parasite elimination and contributing to host tissue protection.

  • Toxoplasma gondii
  • brain tissue
  • nNOS
  • 7-Nitroindazole
  • L-arginine
  • chronic toxoplasmosis
  • nitric oxide

1. Introduction

Toxoplasma gondii (T. gondii) is a coccidian parasite belonging to the phylum Apicomplexa. This obligatory intracellular protozoan is regarded as one of the most successful pathogens, as it is capable of infecting and propagating in all nucleated cell types, with a special predilection for reticuloendothelial cells [1]. Toxoplasmosis, caused by T. gondii, is a widespread zoonotic disease of significant morbidity and mortality, affecting approximately one-third of the world’s human population [2]. Despite its impact, toxoplasmosis remains a neglected parasitic infection and represents the third leading cause of the health burden from foodborne illnesses [3].
Toxoplasma gondii exhibits a broad host range, with felids serving as the only definitive hosts and all warm-blooded animals, including humans, acting as intermediate hosts. Human infection typically occurs through ingestion of tissue cysts in undercooked meat or consumption of food and water contaminated with oocysts. Additional transmission routes include congenital transfer, organ transplantation, and blood transfusion.
The life cycle of T. gondii is sustained through three biological stages of infection, namely tachyzoites, sporozoites, and bradyzoites. Tachyzoites are rapidly multiplying forms responsible for the acute phase of toxoplasmosis. After a few multiplication cycles, T. gondii tachyzoites convert into bradyzoites, which encyst in several organs, predominantly in the brain and muscle tissues [4]. Chronic toxoplasmosis remains subclinical in healthy individuals. However, tissue cyst reactivation can be clinically disastrous, causing Toxoplasma encephalitis (TE), a severe and potentially fatal condition in immunocompromised patients who are prone to lethal activation of dormant cysts [5][6].
Among the several effector immune mechanisms described against T. gondii infection, the role of nitric oxide (NO) has also been reported. Activation of the high-output NO pathway in macrophages is preferentially driven by a Type I immune response, characterized by substantial IFN-γ production [7][8].
Nitric oxide (NO) is a diatomic free radical whose duality in action enables both beneficial signalling and potential oxidative damage. This key effector molecule plays complex regulatory roles in physiology and pathophysiology [9], in addition to its anti-parasitic effects against both metazoa and protozoa [10]. Nitric oxide (NO) production is regulated by the family of nitric oxide synthases (NOS), which comprises three distinct isoforms: neuronal nNOS (NOS I), inducible iNOS (NOS II), and endothelial eNOS (NOS III). The NOS enzymes catalyze the oxidation of L-arginine to produce NO and L-citrulline [11].
Neuronal NOS and endothelial NOS, both calcium-dependent isoforms, are constitutively expressed at low levels under physiological conditions. In contrast, the calcium-independent iNOS is rapidly induced by Th1 cytokines, such as IFN-γ and TNF-α, which trigger macrophage activation and enhance NO production [8][11].
Neuronal NOS serves as the primary source of NO in both central and peripheral neurons. Within the central nervous system (CNS), NO produced by nNOS contributes to the regulation of blood pressure, in addition to its modulation of several physiological processes, including memory, learning, and neurogenesis [12]. Till now, the anti-parasitic functions of the neuronal enzyme (nNOS) have received less attention. Recently, nNOS proved protective during the enteral phase of experimental trichinosis. Intestinal expression of this isoenzyme enhanced the elimination of adult Trichinella spiralis (T. spiralis) worms [13]. Conversely, nNOS failed to clear Heterophyes heterophyes (H. heterophyes) worms from the intestinal sections of infected dogs [14].
In toxoplasmosis, nitric oxide plays a paradoxical role. Despite its reported protective anti-parasitic role during acute T. gondii infection, NO induces detrimental and potentially lethal impacts on host tissues, leading to necrosis and degenerative changes in the liver and bowel of infected mice [15]. In chronic toxoplasmosis, the generation of NO maintains control of the established infection. It acts as a critical modulator of long-term immunity against the parasite [16].
Of the three nitric oxide synthase isoforms, the inducible isotype (iNOS) has been extensively studied during both the acute and chronic phases of toxoplasmosis [15][16][17][18]. In contrast, knowledge is only beginning to emerge about the impacts of the neuronal enzyme (nNOS) on T. gondii infection. Building on this gap, this research pivoted toward understanding the less-explored activities of nNOS in chronic cerebral toxoplasmosis.

2. Materials and Methods

2.1. Toxoplasma gondii Strain

The Medical Parasitology Department, Faculty of Medicine, Alexandria University in Egypt, kindly provided the avirulent cystogenic (ME49) T. gondii strain. In order to establish chronic toxoplasmosis, the strain was consistently maintained by repeated inoculation of Swiss albino mice every 8 weeks with 0.1 mL of brain homogenate from previously infected mice containing approximately 100 tissue cysts/mL [19]. For experimental infection, mice were orally inoculated with 10 T. gondii cysts/0.1 mL/mouse [20].

2.2. Animals

Using the Epi-Info software programme version 7, the sample size was calculated to include 56 apparently healthy, laboratory-bred Swiss albino mice at a 95% confidence level and 80% power of test. The animals were 6 to 8 weeks old and weighed between 20 and 25 g each. Only male mice were selected to eliminate variables related to pregnancy and parturition. The mice were sourced from the animal facilities of Theodor Bilharz Research Institute (TBRI), Giza, Egypt. All breeding and experimental procedures were carried out at the Medical Parasitology Department, Faculty of Medicine, Zagazig University, in accordance with institutional and national rules for the care and use of laboratory animals. The mice were kept in individual, well-ventilated cages with the proper constant temperature (25 ± 2 °C), relative humidity (55–65%), and lighting (12 h light/12 h dark cycle). They were fed a commercial meal high in protein and fiber, with free access to water [19]. To exclude parasitic infections, faecal examination was performed [21].

2.3. Drugs: Tested Compounds

The current research study was conducted to evaluate the potential impacts of the neuronal isoenzyme nNOS against T. gondii infection, using the following treatment lines; the nitric oxide donor, L-arginine that was applied to assess the main source of NO production, and the selective neuronal nitric oxide synthase NOS inhibitor 7-Nitroindazole (7-NI), which was chosen to investigate the potential protective/pathological impacts of nNOS isotypes during chronic toxoplasmosis.
The nitric oxide donor, L-arginine monohydrochloride (A5131, Sigma-Aldrich, St. Louis, MO, USA), was supplied as a powder and dissolved in normal saline before use. Mice received the drug at a dose corresponding to 2% of the total caloric value/animal, representing 10 mg of L-arginine/day, according to Viana et al. [22]. The drug was administered by oral gavage for seven consecutive days prior to infection [13][23].
The selective neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) (Cat. No. N7778) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Due to its poor water solubility, 7-NI was dissolved in dimethyl sulfoxide (DMSO) and administered intraperitoneally to allow efficient delivery to target sites [24]. The drug was given at a dose of 25 mg/kg [25], starting 42 days post-infection and continued for two weeks [18].

2.4. Experimental Design

The current experimental study involved 56 mice, which were equally divided into four groups (14 mice each), as follows: (G1) (normal control): non-infected, non-treated mice, each receiving 100 μL of phosphate-buffered saline (PBS) daily via gavage syringe. (G2) (infected control): The infected, untreated group comprised mice that were orally infected with 10 ME49 tissue cysts in 0.1 mL brain suspension per mouse, administered using gavage syringes fitted with blunt nozzles [20]. The third group (G3) included infected mice treated orally with L-arginine supplementation for seven days prior to infection, as described by Zheng et al. [23]. (G4) consisted of infected mice treated with 7-NI following the protocol implemented for the NOS inhibitor in chronic toxoplasmosis [18]. The drug was administered 7-NI for two weeks, beginning 42 days post-infection.
Nine weeks post-infection, the experiment was terminated as planned. Animals from different study groups were anaesthetized with isoflurane, followed by thoracotomy. Blood samples were collected from the chest cavity. For a subsequent biochemical test, serum was isolated and kept at −20 °C. After that, cervical dislocation was used to sacrifice every animal. Brain samples were swiftly extracted and cut longitudinally at the midline. For later histological and immunohistochemical examinations, half of each brain was preserved in 10% formalin. The second half was used for parasitological evaluation [26].

2.5. Assessment Measures

2.5.1. Parasitological Evaluation: Enumeration of Tissue Cysts

To prepare a brain suspension, the remaining unfixed brain half from each mouse was homogenized in 1.5 mL of saline (0.9% NaCl) using a mortar and pestle [18][20]. For cyst enumeration, 2 drops (20 μL, each) of brain homogenate were then dispensed onto glass slides, air-dried, and fixed with absolute methanol (Spectrum Chemical MFG. Corp., Compton, CA, USA). After fixation, the films were stained with Giemsa stain. Slides were examined microscopically under X40 objectives to determine cyst numbers. The count obtained was then multiplied by 25 to calculate the total number of tissue cysts per 1 mL (1000 μL) of brain homogenate [19]. Finally, the mean number of tissue cysts was determined for each study group.

2.5.2. Histopathological Assessment

For histology, brain samples from several research groups were fixed in 10% neutral buffered formalin, dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and embedded in paraffin (Embedding Station: Leica EG1160, Labexchange - Laboratory Equipment Exchange GmbH, Burladingen-Hausen, Germany) for further processing. Subsequently, a microtome was used to obtain serial brain sections of 5 μm thickness. The tissue microsections were then de-waxed, rehydrated, and stained with haematoxylin and eosin (H&E) [27]. Stained sections were examined under a light microscope (Olympus, Hamburg, Germany) to compare pathological changes between the tested groups. The inflammatory process was evaluated depending on the protocol implemented by Saad et al. [28]. The intensity of inflammation was scored in a semi-quantitative approach on a scale ranging from (+1 to +4), where +1 indicates mild changes, with less than one inflammatory cell focus per X100 field, +2 = moderate inflammatory changes, with 1–5 foci/X100 field, +3 = large inflammatory infiltrate, with >5 foci/100X field, and +4 corresponded to extensive inflammation, characterized by a widespread inflammatory reaction throughout the tissue. Five representative fields from each H&E section were examined at X100, X200 and X400 magnifications for histopathological scoring.

2.5.3. Immunohistochemical Assessment

In the current study settings, the avidin–biotin–peroxidase complex (ABC) method was applied to examine the distribution of the neuronal marker (nNOS) in the brain sections of infected mice, as described by Czarnewski et al. [29]. Briefly, 4- to 5-μm paraffin sections were prepared on poly-L-lysine-coated slides, deparaffinized for 5 min in each of three xylene series, and rehydrated through descending grades of ethyl alcohol and distilled water. Sections were deposited in citrate buffer (pH 6.0; Thermo Fisher Scientific Inc., San Diego, CA 92121, USA, Cat. No. 005000) in a microwave oven for 20 min after being treated with tris-buffered saline (TBS) for five minutes in order to retrieve antigens. Sections were treated with 3% hydrogen peroxide in absolute methanol for seven minutes at room temperature in order to inhibit endogenous peroxidase activity. Tissue sections were incubated with the primary antibody, mouse monoclonal anti-nNOS (1:200 dilutions; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. No. sc-5302) according to the manufacturer’s recommendations. Sections were incubated with horseradish peroxidase (HRP)-conjugated mouse anti-rabbit secondary antibody (San-ta Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat. No. sc-2357) following washing with phosphate-buffered saline (PBS). Diaminobenzidine (DAB) substrate (DAKO Corp., Fremont, CA, USA) was applied for five minutes in order to visualize the reaction. For two minutes, Mayer’s hematoxylin was applied as a counterstain. The same method was used to process negative controls, except that the primary antibody step was omitted [30].
Immunostaining of the nNOS isoenzyme was evaluated semi-quantitatively following the grading scheme of Hao et al. [31], which considers both the intensity and extent of staining. Positive cells were identified by the presence of brownish cytoplasmic colouration. A scale of 0 to 3 was used to measure the strength of the staining; 0 denoted no staining, 1 mild staining, 2 moderate staining, and 3 noticeable intensities. The extent of staining was graded as 0 for less than 5%, 1 for 5–25%, 2 for 25–50%, 3 for 50–75%, and 4 for more than 75% of cells. The intensity and extent values were multiplied to determine the final score, which ranged from 0 to 12. Scores were categorized as high (9–12), moderate (5–8), and negative or low (0–4). The immunoreactivity for nNOS was assessed microscopically in five representative fields for each tissue section using the Leica Qwin 500 C Image Analyzer system (Leica Imaging Systems Ltd., Cambridge, UK).
To minimize observer bias, all histopathological and immunohistochemical tissue sections were randomized, coded, and examined blindly.

2.5.4. Biochemical Assessment: Serum Nitric Oxide (NO) Levels

Concentrations of nitrites (NO2−), indicative of NO production, were measured in the serum samples of the tested mice using the colourimetric nitric oxide assay kit (Abcam Inc., Waltham, MA, USA, Cat. No. ab65328) following the directions of the manufacturer. A microtiter plate was filled with 85 μL of each serum sample, a set of prepared standards, and a sample blank. There were two steps in the test methodology. In order to convert nitrate to nitrite, 5 μL of each nitrate reductase mixture and enzyme cofactor were added to the sample and standard wells. This was followed by an incubation period of 60 min. The plate was then incubated for ten minutes after 5 μL of the enhancer was applied. To create a deep purple azo molecule from nitrite, 50 μL of each Griess reagent (R1 and R2) was added in the second stage. The amount of the azochromophore reflects the concentrations of NO in the samples. Absorbance was measured at 450 nm. All measurements for the standards, controls, and samples were performed in triplicate and reported in micromolar (μM) units.

2.6. Statistical Analysis

IBM SPSS software version 25 (IBM, Armonk, NY, USA) was used to conduct statistical analyses. Standard deviation (SD), mean, and range were used to report numerical data. The analysis of variance (ANOVA) F-test, followed by Tukey’s post hoc analysis, was applied to compare variables across the study groups. The non-parametric Chi-square (χ2) test was employed to compare categorical variables [32]. Treatment efficacy was determined using the following formula: Efficacy (%) = 100 × (mean cyst number in controls minus mean cyst number in treated mice) divided by mean cyst number in controls [33]. Statistical significance was defined as a p-value less than 0.05 in all analyses [34].

2.7. Ethical Statement

The Scientific Research Ethical Committee of the Faculty of Medicine, Zagazig University (Egypt) (ZU–IACUC) approved the study under ethical code (ZU–IACUC/3/F/43/2024). All animal experiments adhered to the research protocols and complied with the recommendations and guidelines of the Declaration of Helsinki. Animals had unrestricted access to food and water. Upon completion of the studies, euthanasia was performed using isoflurane, and all possible measures were taken to minimize their suffering.

This entry is adapted from: https://doi.org/10.3390/diseases14070240

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