Plasma levels of NE, EP, and DP in MP4-infected ICR mice without or with 6-OHDA treatment
Seven-day-old ICR mice were infected with LD50 of MP4 by injection i.p. Plasma levels of NE, EP, and DP were detected by ELISA. NE increased on day 2 and reached a peak on day 5 after infection (Fig. 1A). EP increased on day 2, but decreased slightly on day 4. EP reached the peak on day 5, similar to NE (Fig. 1B). DP increased on day 1 and reached a peak on day 3. The DP levels frequently changed between days 3 and 7. DP dropped on days 4 and 6, but elevated subsequently on days 5 and 7 (Fig. 1C). The NE and EP levels of the mock group had no significant peak-like virus group (Fig. 1A and 1B). The DP levels of the mock and virus groups had variable changes day by day. Thus, on day 4 after infection, MP4-infected mice were administered i.p. with 30 μg/g/100 μL 6-OHDA or an equivalent volume of the vehicle (0.9% saline, 0.02% ascorbate). After 1 h of 6-OHDA delivery on day 4, cardiac puncture blood collection was performed, and plasma was collected immediately. Plasma NE, EP, and DP levels were detected by ELISA and compared to their levels before the 6-OHDA injection. NE and DP decreased progressively after 6-OHDA treatment on day 4 (Fig. 2A). EP increased after 6-OHDA treatment from days 4 to 6, but decreased from days 6 to 8 (Fig. 2B). The NE, EP, and DA levels in MP4-infected and 6-OHDA-treated mice were lower than those in the MP4-infected and vehicle-treated mice (Fig. 2). Consequently, 6-OHDA reduced sympathetic overactivation on peripheral tissue and systemic compartments in MP4-infected mice.
6-OHDA treatment in the MP4-infected mouse model
Seven-day-old ICR mice were infected with LD50 of MP4 by injection i.p. On day 4 after infection, mock- and MP4-infected mice were administered 6-OHDA (20, 25, or 30 μg/g in 100 μL) or an equivalent volume of vehicle i.p. The survival rate of infected mice treated with 20 μg/g 6-OHDA (61.5%) or 25 μg/g 6-OHDA (66.7%) was higher than that of vehicle-treated mice (44.8%). No significant difference was found in 20 μg/g (P = 0.204) or 25 μg/g (P = 0.110) 6-OHDA-treated mice compared to vehicle-treated mice. The survival rate of infected mice treated with 30 μg/g 6-OHDA (76.9%) was elevated significantly (P = 0.009; Fig. 3). Mock- and MP4-infected mice were administered i.p. with 30 μg/g/100 μL of 6-OHDA or an equivalent volume of the vehicle on day 4 after infection. The clinical scores were recorded every day after infection. Mock-infected mice with or without 6-OHDA treatment were healthy and sacrificed until day 14. The clinical scores began to elevate on day 2 and reached the top on day 8. Most mice died from days 6 to 9, and the clinical scores were reduced gradually from days 8 to 14. The clinical scores of MP4-infected and 6-OHDA-treated mice (day 8: 3.02 ± 0.16, day 10: 2.91 ± 0.23, and day 12: 2.31 ± 0.20) were markedly lower than MP4-infected and vehicle-treated mice (day 8: 3.87 ± 0.16, day 10: 3.40 ± 0.20, and day 12: 3.17 ± 0.30) on days 8–12 (Fig. 4).
Cytokine expression in MP4-infected and 6-OHDA-treated mice
After 1 h of 6-OHDA delivery on day 4, cardiac puncture blood collection was performed, and plasma was separated immediately. Plasma was collected to measure IL-12p70, TNF, MCP-1, IL-6, IFN-γ, and IL-10 levels. After infection, the levels of IL-12p70 peaked on days 4 and 10. IL-12p70 diminished on day 8, whereas the clinical score reached the top (Fig. 5A). The levels of IL-12p70 had no significant change between 6-OHDA-treated mice (day 4: 14.97 ± 5.40, day 6: 7.36 ± 4.54, and day 8: 6.90 ± 3.80) and vehicle-treated mice (day 4: 20.60 ± 8.62, day 6: 10.59 ± 3.77, and day 8: 8.85 ± 4.04). The TNF and MCP-1 levels increased on day 2 and reached a peak on day 8. Then, TNF and MCP-1 were reduced gradually after days 10–14. The tendencies of TNF and MCP-1 were similar to the changes in clinical scores (Fig. 5B and 5C). On day 6, the TNF level was lower in 6-OHDA treatment (day 4: 78.26 ± 8.18 and day 6: 72.80 ± 11.05), but showed no statistical difference between vehicle treatment (day 4: 93.46 ± 10.52 and day 6: 108.53 ± 15.66; Fig. 5C). The IL-6 levels peaked on day 4, but dropped drastically on day 6 (Fig. 5D). IFN-γ peaked on day 4, but decreased progressively from days 6 to 14 (Fig. 5E). On days 4 and 8, IFN-γ expression was reduced significantly with 6-OHDA treatment (day 4: 27.89 ± 3.23, P = 0.016; day 6: 21.94 ± 0.73, P = 0.029; and day 8: 9.06 ± 1.45, P = 0.042) compared to the vehicle group (day 4: 58.50 ± 9.87, day 6: 32.39 ± 4.64, and day 8: 16.30 ± 3.35). The IFN-γ and IL-6 levels continued to decrease when the clinical scores were increased. The IL-10 level elevated from day 2 and was expressed constantly until day 14, but had no significant change between 6-OHDA-treated mice (day 4: 10.10 ± 6.41, day 6: 13.75 ± 6.45, and day 8: 6.53 ± 4.33) and vehicle-treated mice (day 4: 30.17 ± 9.41, day 6: 21.34 ± 9.17, and day 8: 17.79 ± 6.28; Fig. 5F).
Cell populations of PBMCs and splenocytes in MP4-infected and 6-OHDA-treated mice
Cell populations of PBMCs and splenocytes were collected and analyzed on day 6 after infection (Fig. 6). The absolute events of CD3+CD4+, CD3+CD8+, CD19+, CD3−NK1.1+, and CD3+NK1.1+ cells of PBMCs and splenocytes were lower after infection (Tables 1 and 2). The absolute events of CD3+CD4+, CD3+CD8+, and CD3+NK1.1+ cells of PBMCs were elevated significantly in MP4-infected and 6-OHDA-treated mice compared to MP4-infected and vehicle-treated mice (Table 1). The percentages of CD3+CD8+ cells of PBMCs were higher in MP4-infected and 6-OHDA-treated mice than in medium- and vehicle-treated mice (Table 1). In splenocytes, the absolute events of CD3−NK1.1+ and CD3+NK1.1+ cells of MP4-infected mice were increased significantly after 6-OHDA treatment (Table 2). Moreover, the percentages of CD3-NK1.1+ of splenocytes were higher in MP4-infected and 6-OHDA-treated mice than in medium- and vehicle-treated mice (Table 2). Therefore, cytotoxic T, NK, and NKT cells may contribute to infection after 6-OHDA treatment.