|Year : 2014 | Volume
| Issue : 1 | Page : 103-114
Parasitological, histopathological, and immunohistochemical assessment of nitric oxide synthase inhibitor: aminoguanidine versus albendazole in the treatment of experimental murine toxocariasis
Nashaat A. Nassef1, Wafaa M. El-Kersh2, Nadia S. El-Nahas2, Salwa A. Shams El-Din1, Salwa F. Oshiba1, Mona M. Nosseir2
1 Department of Parasitology, Faculty of Medicine, Menoufiya University, Menufia, Egypt
2 Department of Pathology, Theodor Bilharz Research Institute, Giza, Egypt
|Date of Submission||07-Sep-2013|
|Date of Acceptance||30-Oct-2011|
|Date of Web Publication||20-May-2014|
Salwa A. Shams El-Din
Department of Parasitology, Faculty of Medicine, Shibin El-Kom-YassinAbd El-Ghaffarstreet 32511
Source of Support: None, Conflict of Interest: None
The study was intended to evaluate the effect of aminoguanidine compared with that of albendazole on mice infected with eggs of Toxocara canis by parasitological, histopathological, and immunohistochemical studies.
T. canis is a widely distributed parasite. The nematode can cause toxocariasis in man. The infection takes place after swallowing fully embryonated eggs with larvae inside.
Materials and methods
The study was conducted on 117 albino mice classified into four groups: GI (the control group with subgroups Ia, Ib, and Ic); GII (infected with T. canis eggs); GIII (infected with T. canis eggs and treated with albendazole); and GIV (infected with T. canis eggs and treated with aminoguanidine). Sera from different groups of mice were collected for nitrite assay. The lung and brain tissues were taken for larval recovery and histopathological and immunohistochemical [inducible nitric oxide synthase (iNOS)] studies.
The mean larval count decreased significantly from 7th to 45th day post infection (d.p.i), with albendazole and aminoguanidine, which was more effective in decreasing larval count. Aminoguanidine treatment caused early improvement in histopathological lesions initiating from the second d.p.i.; however, albendazole improvement was observed on the seventh d.p.i. The grade of iNOs expression in GII and GIII was high as compared with that in GIV. There was significantly positive correlation between serum nitrite and the grade of iNOS expression, especially on early d.p.i only in groups II and III.
Both albendazole and aminoguanidine treatment for T. canis-infected mice caused decrease in larval count and improvement in histopathological lesions. Aminoguanidine was more effective with early response and decreased tissue damage.
Keywords: Albendazole, aminoguanidine, immunohistochemistry, nitric oxide synthase, Toxocara spp
|How to cite this article:|
Nassef NA, El-Kersh WM, El-Nahas NS, Shams El-Din SA, Oshiba SF, Nosseir MM. Parasitological, histopathological, and immunohistochemical assessment of nitric oxide synthase inhibitor: aminoguanidine versus albendazole in the treatment of experimental murine toxocariasis. Menoufia Med J 2014;27:103-14
|How to cite this URL:|
Nassef NA, El-Kersh WM, El-Nahas NS, Shams El-Din SA, Oshiba SF, Nosseir MM. Parasitological, histopathological, and immunohistochemical assessment of nitric oxide synthase inhibitor: aminoguanidine versus albendazole in the treatment of experimental murine toxocariasis. Menoufia Med J [serial online] 2014 [cited 2020 Feb 26];27:103-14. Available from: http://www.mmj.eg.net/text.asp?2014/27/1/103/132778
| Introduction|| |
Toxocara canis is a widely distributed parasite that can cause toxocariasis/visceral larva migrans (VLM) in man and many other hosts ,. VLM can be acquired from dog (T. canis) or cat (Toxocara cati) .
The seroprevalence of human VLM was found to be 14% in the USA , 31.6% in Argentina , and 51.6% in Brazil . However, it was about 11.1% in children in Brazil  and 35.6% in Peruvian children . The infection rates were 7.6, 25, and 44.6% among children in Swaziland, Turkey, and Iran, respectively ,,.
VLM seropositivity was recorded to be 24.2% in different rural areas in Egypt . Recently, T. canis eggs were identified in the hair samples of 26.6% stray dogs and 10.7% domestic dogs in Kafr el-Sheikh .
The disease showed hypergammaglobulinemia, anemia, leukocytosis, and marked eosinophilia, together with the positive result of serological tests . The symptoms were found to decrease by antihelminthic drugs .
Albendazole was able to decrease the larval count of Toxocara spp. in the liver and lung and to prevent migration of larvae to the mice brain . It acts by binding to the colchicine-sensitive site of tubulin-inducing degenerative alterations in the tegument and intestinal cells of the worm. Degeneration of the endoplasmic reticulum and mitochondria of the germinal layer and the subsequent release of lysosomes result in decreased production of adenosine triphosphate, which is the energy required for the survival of the helminthes. Because of diminished energy production, the parasite is immobilized and eventually dies ,.
It is known that microorganisms increase inducible nitric oxide synthase (iNOs) in humans and animals. Nitric oxide (NO), a free radical important in inflammation and immunity , is produced by macrophages, endothelial cells, and neurons. Studies suggested that NO participates in antiparasite defense, for example trypanosomiasis and malaria [19-21], but it is responsible for some pathological disorders . NO can affect the living cells through oxidation of iron-containing proteins such as ribonucleotide reductase and aconitase .
Nitric oxide synthases are a family of eukaryotic enzymes (NOs) that act by conversion of O 2 and L-arginine to NO and L-citrulline in the presence of NADPH and dioxygen (O 2 ) . iNOS limits parasite development in the vectors . Phagocytes are stimulated by interferon-gamma or by tumor necrosis factor to form iNOS . In contrast, transforming growth factor-β inhibits iNOs, whereas interleukin-4 (IL-4) and IL-10 provide weak inhibitory signals with resultant decrease in tissue injury .
The control of NO synthesis by affecting a number of important biological processes has been implicated in the treatment of a variety of diseases . Expression of iNOS, which synthesizes nitric oxide, leads to elimination of parasites . iNOS can be affected by aminoguanidine, a selective iNOs inhibitor . Increased expression of transforming growth factor β-1 by aminoguanidine improves wound healing and preserves the ultrastructure of collagen . Aminoguanidine can also inhibit osteoarthritis by suppressing the production of iNOs, and it decreases the apoptosis of some cells .
The aim of the present study was to evaluate the effect of aminoguanidine as compared with that of albendazole on mice infected with T. canis eggs by parasitological, histopathological, and immunohistochemical studies and to correlate these effects with serum nitric oxide level in different groups on different days.
| Materials and methods|| |
In all, 117 albino mice were used and classified into four experimental groups. Group 1 (GI) included 27 mice that were divided into three subgroups: GIa contained noninfected nontreated mice (negative control), GIb contained noninfected mice receiving albendazole (as a drug control subgroup), and GIc contained noninfected mice receiving aminoguanidine (as a drug control subgroup). GII included 30 mice infected with T. canis eggs. GIII included 30 mice infected with T. canis eggs then treated with albendazole. GIV included 30 mice infected with T. canis eggs then treated with aminoguanidine. On 2, 7, and 45 days post infection (d.p.i.), three animals from the control subgroups and 10 animals from each group (GII, GIII, and GIV) were killed . Tissue and serum samples were collected for detection of serum nitrite and parasitological, histopathological, and immunohistochemical studies.
Male Balb/c albino mice with an age of 3 months and weight of 25-30 g were used in the current study. The mice were kept in the animal house in Faculty of Medicine, Menoufiya University, exposed to 12 h light/12 h dark, and fed ad libitum on standard diet and tap water.
T. canis eggs were obtained from the intestines of adult female stray dogs from Cairo and Giza Provinces. The eggs were purified by straining through a sieve with 0.5 mm pores, washed with saline several times, and then kept in 0.5% formol saline solution (99.5 ml physiological saline and 0.5 ml formaldehyde 40%) for 4-8 weeks in petridishes at 28-30°C to induce embryonation. Maturation of the eggs was checked every day after aeration and shaking of eggs to enhance maturation and to prevent sticking. Then, mature embryonated eggs were kept at 4°C until used. Each mouse was inoculated with about 1000 viable T. canis eggs counted by hemocytometer through the oral route .
Albendazole in the form of Alzental (Epico Pharm Co., 10 th Ramadan city, Egypt) as a white suspension of 100 mg/5 ml was given orally at a dose of 100 mg/kg once daily for 5 consecutive days, diluted in 0.1 ml distilled water on the first day of infection .
Aminoguanidine in the form of aminoguanidine bicarbonate as a white powder (Sigma Chemicals, Cairo, Egypt) dissolved in distilled water was given intraperitoneally at a dose of 50 mg/kg on 1, 3, 5, 8, 10, 12, 14, 16, 18, 22, 27, 32, 37, 40, and 44 d.p.i. .
Parasitological study for T. canis larval recovery
Samples from the lung and brain tissues (0.5 g each) were sliced and digested in 50 ml pepsin-HCl solution (2.5 g pepsin, 3.5 ml HCl, and 500 ml water) and incubated at 37°C for 24 h to examine the larval recovery . The digest in petridish and larvae were counted at × 40 magnifications under an inverted microscope (Olympus, Tokyo, Japan) . Larvae in the brain were counted directly, after squashing small amounts of fresh unstained brain tissue between two slides to calculate parasite load in 1 g .
Serum nitrite determination 
Serum samples (50 μl) collected from each mouse in different groups on the tested days were incubated in Griess reagent (1% sulphanilamide, 0·1% naphthylethylenediamide dihydrochloride in distilled water, and 2·5% orthophosphoric acid) for 10 min. The absorbance was determined for individual sample with an automated spectrophotometer at 540nm wave length. The nitrite concentration was calculated with reference to a sodium nitrite linear standard curve .
The lung and brain tissues of each mouse were fixed in 10% formalin, embedded in paraffin, and sectioned and stained with hematoxylin and eosin stain  [Figure 1],[Figure 2],[Figure 3],[Figure 4] and [Figure 5].
Detection of inducible nitric oxide synthase by immunohistochemical stain (iNOS enzyme in the tissue)
Briefly, paraffin-embedded tissues were deparaffinized and blocked with 3% H 2 O 2 (Merck, Taufkirchen, Germany). Then, they were washed and incubated in serum-blocking reagent G (mouse CTS002-kits) [Figure 6],[Figure 7],[Figure 8],[Figure 9] and [Figure 10]. Thereafter, the specimens were incubated with avidin-blocking reagent (SP2001; Vector, Burlingame, California, USA), washed, and then incubated with biotin-blocking reagent (SP2001; Vector). The specimens were incubated with primary antibody (X0907; Dako, Carpinteria, California, USA) and washed, and again incubated with biotinylated secondary antibody [NOS2 (N-20) sc-651, H2306, 200 μg/ml rabbit polyclonal IgG (Santa Cruz Biotechnology Inc., Dallas, Texas, USA) and washed. Then, the specimens were incubated with horseradish peroxidase and washed. The specimens were incubated with DAB chromogen solution, washed, and counterstained with Mayer's hematoxylin . The grading of iNOS expression in tissues was carried out on the basis of the number of cells showing immunopositivity according to Othman et al.  [Figure 11],[Figure 12],[Figure 13],[Figure 14] and [Figure 15].
Animals were housed and scarified according to the international guides to use of laboratory animals.
Results were collected, tabulated, and statistically analyzed by IBM personal computer and statistical package for the Social Science program for Windows (version 13; SPSS Inc., Chicago, Illinois, USA). Two types of statistics performed are as follows.
It included percentage (%), mean, and SD.
The Student t-test, the paired t-test, the F-test (analysis of variance), the c2 -test, the Z-test (z), and Pearson's correlation test were performed. The data were considered statistically significant when P-value was less than 0.05*  [Figure 16],[Figure 17],[Figure 18],[Figure 19] and [Figure 20].
| Results|| |
Data in [Table 1] show that T. canis larvae were detected in the lung tissue of GII, GIII, and GIV on 2 d.p.i. and their number was significantly decreased up to 45 d.p.i. T. canis larvae were not detected in the brain on 2 d.p.i.; however, it increased significantly on the following days in GII. There was a statistically significant difference in the number of T. canis larvae in the brain and lung tissues in GII, GIII, and GIV on 7 d.p.i. The number of T. canis larvae in the lung and brain tissues decreased significantly when compared on 7 and 45 d.p.i. in all groups (data not shown) [Figure 21],[Figure 22] and [Figure 23].
|Table 1: Comparison between mean number of Toxocara canis larvae in the lung and brain tissues on different days in different mice groups|
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[Table 2] shows that serum NO decreased significantly from 2 to 45 d.p.i. in GII, GIII, and GIV. NO level was also significantly lower in GIV than in GII and GIII on all tested days.
|Table 2: Comparison of serum nitric oxide level between different groups on different days|
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Graph 1[Additional file 1] shows that, in the lung tissues, no significant difference was recorded in the grades of iNOS expression between the studied groups on the second d.p.i. However, on the seventh d.p.i., the percentage of grade II iNOS expression was significantly higher among GIV (P < 0.001***), whereas the percentage of grade III iNOS expression was significantly higher among GIII than among other groups (P < 0.001***). On the 45 th d.p.i., the percentage of grade I iNOS expression was significantly higher among GIV (P < 0.001***), whereas the percentage of grade III iNOS expression was significantly higher among GII and GIII than among GIV (P < 0.001***). In Graph 2 [Additional file 2] concerning the brain tissues, the percentage of grade I iNOS expression was significantly higher among GIV than among other groups, whereas the percentage of grade II iNOS expression was significantly higher among GII than among other groups on the seventh d.p.i. (P < 0.001***). On the 45th d.p.i., the percentage of grade 0 iNOS expression was significantly higher among GIV and the percentage of grade I iNOS expression was significantly higher among GIII, whereas the percentage of grade III iNOS expression was significantly higher among GII than among other groups (P < 0.001***).
In [Table 3] and Graph 3 [Additional file 3], there was significant positive correlation between serum NO level in μmol/l and grade of iNOs expression in the lung on 2 d.p.i. (P < 0.05*).
|Table 3: Correlation between serum NO level and grade of iNOs expression on different days post infection for group II|
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[Table 4] and Graph 4 [Additional file 4] show significant positive correlation between serum NO level in μmol/l and grade of iNOs expression in the lung on 2 d.p.i. (P < 0.001***).
|Table 4: Correlation between serum NO level and grade of iNOS expression on different days for group III|
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[Table 5] shows no significant positive correlation between serum NO level in μmol/l and grade of iNOS expression in the lung and brain in GIV.
The lung tissues were moderately inflamed on the second d.p.i., but severe inflammation was recorded in the lung tissues on the seventh and 45th d.p.i. The brain tissue was normal on the second and seventh d.p.i., but on the 45th d.p.i. some of the brain tissues were mildly inflamed.
|Table 5: Correlation between serum NO level in m mol/l and grade of iNOS expression on different days for group IV|
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After albendazole treatment, mild improvement in histopathological lesions initiated on the seventh d.p.i., whereas evident improvement occurred on the 45th d.p.i. After aminoguanidine treatment, improvement in histopathological lesions was observed from the second d.p.i. and increased on the seventh d.p.i., and the maximum improvement was evident on the 45th d.p.i. compared with the infected nontreated group.
Immunohistochemical results of this study indicated that the grade of iNOS expression in different tissues among GII (infected nontreated mice) was high. Albendazole treatment for T. canis-infected mice caused increased expression of iNOS in different tissues especially lung, whereas aminoguanidine treatment caused diminution of iNOS enzyme in all tissues. There was significant positive correlation between serum nitric oxide level and the percentages of grade of iNOS expression in some tissues of different groups, especially on early d.p.i.
| Discussion|| |
This study was conducted on 117 albino mice that were classified into four experimental groups: the control group GI [control negative, (a); drug control for albendazole, (b); and drug control for aminoguanidine, (c)], GII that was infected with embryonated T. canis eggs, GIII that was infected with embryonated T. canis eggs and treated with albendazole, and GIV that was infected with embryonated T. canis eggs and treated with aminoguanidine.
In the current study, the recovery of T. canis larvae initiated in the lung tissue on the second d.p.i. and decreased significantly on the following days in GIII and GIV until disappeared. In contrast, T. canis larvae were not detected on the second d.p.i. in the brain tissues but appeared on the seventh and decreased on the 45th d.p.i. Similar results were reported by Othman et al. , Taira et al. , and Caldera et al.  who found that larval recovery showed progressive increase over the course of infection, with special predilection for the central nervous system.
T. canis larvae detected in the lung and brain tissues were significantly lower in the aminoguanidine-treated group (GIV) than in the albendazole-treated group (GIII) and in the infected group (GII) on days 7 and 45 d.p.i., respectively [Table 2]. Albendazole exhibited antihelminthic effect on the brain (0 larva/mouse) and lung . The study by Fernando et al.  coincided with the present study and showed that albendazole treatment in patients with toxocariasis caused clinical improvement.
Serum NO was significantly reduced with the chronicity of infection in GII, GIII, and GIV. NO level was significantly reduced also in GIV than in GII and GIII on all tested days. Lin et al.  reported decreased NO level on 28 d.p.i. with T. canis and continued decreasing on the following days. Also, NO level was drastically reduced in thegroup treated with iNOs inhibitor: L-NIL then infected with T. canis.
The lung tissues showed infiltration with inflammatory cells and focal peribronchial and intrabronchial inflammation. The blood vessels showed dilatation, congestion, and interstitial hemorrhage. These manifestations progressed from moderate to severe in GII. Oryan et al.  showed similar pathological findings in infected lung tissues.
Production of iNOs during T. canis infection causes direct host damages and is strongly related to the oxidative stress. We propose that larval NO can also be effective in larval migration . These pathological manifestations progressed from moderate to mild in GIII after albendazole treatment. In GIV treated with aminoguanidine, the lung pathology ranged from mild to moderate. L-NIL treatment resulted in large, irregularly shaped granulomas with suppressed collagen contents at 4 weeks post infection (w.p.i). but not at 8 w.p.i. The suppressed collagen contents might have been related to decreased serum NO and Th2-type cytokine of IL-4 but not to Th1-type cytokine of interferon-gamma expression . Enhanced iNOs expression seemed to play a certain role in pathological damage. A potential therapeutic strategy for treatment of nematodes is through manipulation of iNOs expression .
The brain tissue of GII contained few scattered larvae and showed congestion and inflammation that progressed from normal to mild. In GIII also, the pathological manifestation progressed from normal to mild after albendazole treatment. The brain tissue was nearly normal in GIV in which mice were treated with aminoguanidine. Inhibition of iNOs has protective effect on the brains of infected mice, and T. canis infection could be related to oxidative stress; hence, NO production and iNOs inhibition can protect the tissue from damage in this infection .
Expression of iNOs reported in the lung tissues in the current study changed from grade I to grade III in GII and GIII. In the aminoguanidine-treated GIV, iNOs expression ranged from grade I to II. This is in agreement with the study by Othman et al. , who reported increased expression of iNOs in T. canis-infected tissues. These changes were maximal in the chronic stage of infection or were increased over time.
Expression of iNOs reported in the brain tissues of GII was found to be increased from grade 0 to grade III then decreased again to grade 0. Othman et al.  observed that iNOS was expressed by the glial cells of Toxocara spp.-infected mice at 2 w.p.i but not in the age-matched control group of uninfected mice. Moreover, there was a significant increase in its expression with increasing duration of infection in GIII. Aminoguanidine treatment decreased iNOs expression from grade III to 0 in GIV. Hamilton et al.  showed increased expression of iNOs in murine toxocariasis. These results have implications for the role of iNOs in the cerebral establishment of T. canis and in the cerebral pathology reported during infection. Inducible NOs expression was enhanced in infected and L-NIL-treated mice at 4 w.p.i. but declined at 8 w.p.i. as shown by immunohistochemical study .
There was a significant correlation between serum NO level and grade of iNOS expression in the lung of GII and GIII on 2 d.p.i. only.
| Conclusion|| |
Serum NO level can be a marker for suspected cases of visceral larvae migrans, mainly in early -d.p.i. iNOs inhibition with aminoguanidine resulted in less damaged tissues and diminished the deleterious effects of the parasite. Aminoguanidine can be used alone for the treatment of VLM, as it was effective in decreasing larval count especially in the brain tissue and it improved histopathological changes in the tissues. Finally, combinations of albendazole and aminoguanidine for treatment of VLM should be further studied as a more effective therapy for VLM.
| Acknowledgements|| |
Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]