There were statistically significant differences between group mean scores as determined by one-way ANOVA (F (3,210) = 10.08, p = .001). The results showed that, disease score was significantly lower in P and T1 groups than the control group (p = 0.01). Mice in P and T1 had trivial sign of paralysis which developed slowly, however, the scores over than 3 were not observed. No significant difference was found in clinical scores between T2 and control groups (p > 0.05) (Fig. 1). Peaks of EAE were shorter and fewer in P and T1 groups than the control group. Otherwise, EAE developed faster and reached to score 5 in T2 group, and we missed one mouse same as the control group. After injection of eggs on day 18 in T2 group, remission was observed compared to the control group, but signs and symptoms were presented very quickly after 1 week.
In the control group, EAE was not seen in only one mouse. In T1 and P groups, just 2 mice from 8 mice in each group afflicted to the EAE. 50% decrease of cumulative incidence in the two mentioned groups was attributed to protective effect of Dicrocoelium eggs immunization and treatment. Dicrocoelium eggs immunization and treatment significantly reduced cumulative incidence rate in prophylaxis and treatment 1 groups (p = 0.0), and caused a delay of about 5 days in the onset of EAE in the mentioned groups. (Control n = 4), (Prophylaxis n = 8), (Treatment 1 n = 8), and (Treatment 2 n = 8). EAE developed in most mice in two groups (the control and treatment 2 groups) on day 10 [mean day of onset] but in prophylaxis and treatment1 groups, EAE developed just in 25% of mice on day 15 after induction (Fig. 2).
There were no statistically significant differences between group mean weight changes at baseline as determined by one-way ANOVA (F (3,20) = 1.995, p = 0.147). Mauchly’s test indicated the violation in the assumption of sphericity, χ 2(35) = 93.578 p < .001, therefore degrees of freedom were corrected using the Greenhouse-Geisser estimates of sphericity (ε = 0.429). The result showed that, there was no significant difference in weight changes between the groups in four times of measurement (weight * groups), F (10.28, 65.1), p = .0.115. But, helminthic therapy with Dicrocoelium eggs could inhibit severe weight loss in prophylaxis and treatment1 groups. In the control group, after induction of EAE, mean weight of mice reduced dramatically from 19 gr on day 10 to 17.5 gr on day 20. During 10 days, the mean weight loss was about 1.5 gr. In treatment group 2, mean weight loss from 20.5 gr on day 5 reached to 17.8 gr on day 15. Approximately, a 2.8 -gr weight loss was observed in this group. However, the maximum weight loss was estimated lower than 0.5 gr after induction of EAE in other two groups (Prophylaxis, treatment1groups) (Fig. 3).
Evaluation of Neuroglia Cell Number and Density in Corpus Callosum
To evaluate the effect of Dicrocoelium egg administration on the number of neuroglia cells in corpus callosum, H&E and TB staining were investigated by light microscope. The neuroglia cells were counted in a Scale bar of 200 µm.
The results demonstrated that, the quantity of neuroglia cells in corpus callosum is similar in 4 groups (P > 0.05; Fig. 4). The LFB stained sections showed more density in treatment 1 group compared to the control group (P = 0.03). No significant difference was observed between prophylaxis, treatment 2, and control groups (P > 0.05, Fig. 5).
Brains from each mouse (collected on day 40 post-immunization) were fixed, and were embedded in paraffin, sections (5-µm) were prepared, and then the tissues were stained with H&E (Fig. 4A) and TLB (Fig. 4B) to count the number of neuroglia cells (as presented in the left side of Fig. 4A and 4B). The quantification of neuroglia cells is shown in the right side of Fig A and B. (Control n = 4), (Prophylaxis n = 6), (Treatment 1 n = 6), and (Treatment 2 n = 4). No significant difference was found between 4groups (P > 0.05).
Brains from each mouse tissues were stained with LFB to assess the extent of density of brain tissue. Histologic features were scored semi quantitatively as presented in the right side of Fig. 5 (control, n = 4), (Prophylaxis, n = 6), (Treatment 1, n = 6), and (Treatment 2, n = 8). The quantification of brain tissue density is shown in the left side of Fig. 5. No significant difference was found between prophylaxis, treatment 2, and control groups (P > 0.05). There was a significant difference in EAE incidence between treatment1 and control groups (P = 0.03, *p < 0.05).
Evaluation of IFN- γ and IL-4 m RNA Expression
There were no statistically significant differences between group means of IFN- γ and IL-4 as determined by one-way ANOVA (F (3, 19) =.90, p = .459) (F (3,19) =1.210, p = .333). In prophylaxis and treatment 1 groups, the treatment induced a great up-regulation of IL-4, compared to the control group. The highest level of IL-4 was observed in the treatment 1 group (RQ=2.65). The IL-4 mRNA gene expression level was lowest in the treatment 2group (RQ= 0.92), also, different levels of mRNA gene expression were observed in the IFN- γ. The mRNA gene expression level of IFN- γ was at highest copies in the control (RQ=8.09) and T2 (RQ= 5.91) groups, whereas the mRNA gene expression of IFN- γ was lower in the prophylaxis (RQ=4.03) and treatment 1 groups (RQ=0.37) than the control group (RQ=8.09) (Figure 6).
Relative mRNA gene expression of genes was determined compared to the housekeeping gene, β2 microglobulin.