Fig. 2

Neuroinflammatory markers support the validity of the rotenone model. Increased tumor necrosis factor alpha (TNF-alpha, green in A and B) specific signal density (SSD) in glial fibrillary acidic protein (GFAP; red in A and B)-expressing astrocytes was detected in the substantia nigra pars compacta (SNpc) as shown in graph C also. No TNF-alpha immunoreactivity was observed in ionized calcium binding adaptor molecule 1 (IBA1)-containing microglia (white in A and B). Inducible nitric oxide synthase (iNOS, red in D and E) immunoreactivity was detected only in very few IBA1 (white in A and B)-containing SNpc microglia in control rats (boxed area and red arrowhead in D). Rotenone treatment increased the number of iNOS immunoreactive (-ir) IBA-immunopositive microglia (inset in E) that occasionally formed active cell clusters (yellow arrowheads in E). Some faintly iNOS-ir nerve cell bodies (blue arrowheads in E and graph G) were also found and occasionally, TH-ir neurons also appeared to show weak iNOS positivity (see inset in E). IBA1 (green in H and I) and cluster of differentiation 68 (CD68, red in H and I) double-labeling revealed that upon rotenone treatment (I), the microglial cells in the centrally-projecting Edinger–Westphal nucleus (EWcp) co-express CD68 (J) suggesting their reactivity. Black bars: vehicle (oil) injected rats, red columns: rotenone-treated group. *p < 0.05, **p < 0.01, ***p < 0.001 in Student’s t test, n = 6. Bars: 50 µm. Scatter plot K demonstrates the negative correlation between the TH immunoreactivity in the SNpc and EWcp microglial activity scores. Scatter plot L illustrates the inverse relationship between SNpc/TH immunoreactivity and astroglial activity scores