Application of Fluoro-Jade C in acute and chronic neurodegeneration models: utilities and staining differences

Abstract

Recent neuropathological studies have shown that Fluoro-Jade C (FJC), an anionic fluorescent dye, is a good marker of degenerating neurons. However, those studies have mostly examined acute rather than chronic models of neurodegeneration. We therefore compared FJC staining using the intrastriatal 6-hydroxydopamine (6-OHDA)-injected rat as an acute model and the zitter rat as a chronic model, as both show dopaminergic (DA) neurodegeneration. In the 6-OHDA-injected rat, FJC-positive neurons were found in the substantia nigra pars compacta (SNc) before the loss of tyrosine hydroxylase (TH)-positive DA neurons. In the zitter rat, FJC-labeled fibers were first detected at 1 month old (1M) and were considerably increased in the striatum at 4M, whereas FJC-labeled cell bodies were found at 4M, but not at 1M in the SNc. Furthermore, FJC-labeled neurons of the zitter rat showed TH-immunoreactivity in fibers, but little in cell bodies, while those from the 6-OHDA-injected rat showed TH-immunoreactivity even in the cell bodies. These results demonstrate that FJC is a useful tool for detecting chronically degenerating neurons, and suggest that intracellular substances bound to FJC may accumulate in the cell bodies from fibers at a slower rate in the chronic model than in the acute model.

Keywords: 6-OHDA; Fluoro-Jade C; chronic neurodegenerative model; dopaminergic neurons; zitter rat.

Fig. 1

Comparison of FJC staining between KA-treated samples (A and C) and saline-treated control samples (B and D). FJC staining in the cerebral cortex, layer V (A and B) and hippocampus, CA3 (C and D). Bar=50 µm.

Fig. 2

Comparison of FJC staining (A and C) and TH immunostaining (B and D) between the contralateral side (A and B) and ipsilateral side (C and D) in the SN at 3 days after intrastriatal 6-OHDA injection. High magnification view of the highlighted box in C (inset). Cell bodies of the degenerating neurons induced by 6-OHDA were double labeled with IF using TH (E and H) and FJC (F and I) in the SNc and were round (E–G) or shrunken (H–J). G and J represent mergers of E and F, and H and I respectively. SN, substantia nigra; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticularis. Bars=100 µm (A–D) and 10 µm (inset in C and E–J).

Fig. 3

In the SD rat, no FJC-positive neurons are seen in the striatum (A) or SN (B) at 1M. Distribution of FJC-positive structures in the striatum (C, E, and G) and SN (D, F, and H) of the zi/zi rat at 1M (C and D), 4M (E and F), and 12M (G and H). Staining shows dot-shaped structures (white arrows), fiber-like structures (arrowheads) and cell body-like structures (yellow arrows). In the zi/zi rat, high magnification views show the distinct shaped fibers stained by FJC of the striatum at 1M (inset in C), 4M (inset in E), and 12M (inset in G) and those of the SN at 4M (left-side inset in F), and the cell bodies of the SN at 4M (right-side inset in F) and 12M (inset in H). SN, substantia nigra; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticularis. Bars=50 µm (A–H) and 10 µm (inset in C and E–H).

Fig. 4

Double labeling of FJC and IF using TH (A–H) or GFAP (I–P) in the striatum (A–D), SN (E–L), and hilus (M–P) of the zi/zi rat at 4M. Each high magnification view of the structure (arrow) in A, E, I and M is shown in B–D, F–H, J–L and N–P; double labeling of FJC (B, F, J and N) and IF using TH (C and G) or GFAP (K and O). D, H, L and P represent mergers of B and C, F and G, J and K, and N and O, respectively. Bars=50 µm (A, E, I and M) and 10 µm (B–D, F–H, J–L and N–P).