Minocycline Alleviates Cluster Formation of Activated Microglia and Age-dependent Dopaminergic Cell Death in the Substantia Nigra of Zitter Mutant Rat

Abstract

Microglial activation is a component of neurodegenerative pathology. Here, we examine whether activated microglia participate in age-related dopaminergic (DA) cell death in the substantia nigra pars compacta (SNc) of the zitter (zi/zi) rat, a mutant characterized by deletion of the attractin gene. Confocal microscopy with double-immunohistochemical staining revealed activated microglia-formed cell-clusters surrounding DA neurons in the SNc from 2 weeks after birth. An immunoelectron microscopic study showed that the cytoplasm of activated microglia usually contains phagosome-like vacuoles and lamellar inclusions. Expression levels of the pro-inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were increased in the midbrain of 2-month-old zi/zi rats. Chronic treatment with the anti-inflammatory agent minocycline altered the morphology of the microglia, reduced cluster formation by the microglia, and attenuated DA cell death in the SNc, and reduced the expression of IL-1β in the midbrain. These results indicate that activated microglia, at least in part and especially at the initial phase, contribute to DA cell death in the SNc of the zi/zi rat.

Keywords: activated microglia; dopamine neuron; minocycline; pro-inflammatory cytokines; substantia nigra.

Fig. 1.

Immunohistochemical staining for Iba1 in the SNc at the age of 2M. (A, B) Distribution of Iba1 immunoreactive microglia in zi/+ (A) and zi/zi (B) rats in low magnification photomicrographs. (C–E) Double immunohistochemical staining for Iba1 (green) and TH (red) in low magnification photomicrographs of the SNc in zi/zi rats. (C) Immunostaining for Iba1: the area of SNc is delineated by a white line. Clusters of Iba1-positive microglia evident in the SNc of the zi/zi rat are indicated with arrows. (D) Immunostaining for TH. (E) Merged image. (F, G) Three-dimensional confocal photomicrographs of the SNc of zi/zi rats. Clusters of Iba1-positive microglia surround the TH neurons in the SNc of zi/zi (F) but few are observed in zi/+ (G) rats. The nuclei were stained using DAPI (blue). Bars = 200 μm (A–E) and 10 μm (F, G). (H) The number of Iba1-positive cell clusters in the SNc of SD (black bars), zi/+ (checkerboard bars) and zi/zi (white bars) rats at different ages (n = 4 each group). * P < 0.05, ** P < 0.001.

Fig. 2.

Ultrastructural characterization of Iba1-immunoreactive microglia (m) in the SNc of 2M zi/zi rats. (A) Microglia with thick enlarged processes in the neuropil of the SNc. The broadened cytoplasm of these cells contained several phagocytosis products (asterisks). (B) Activated microglia showing abnormal lamellar structures (indicated by an arrow). (C) Three microglia (m) with phagosome-like vacuoles of various sizes were observed adjacent to the neuron (n). (D) Iba1-immunoreactive microglia (m) in the SNc of zi/zi rats following minocycline administration for 2 months. Bars = 1 μm (A, B), 5 μm (C), and 2 μm (D).

Fig. 3.

Effect of minocycline on the microglia in the SNc of zi/zi rats. (A–D) Iba1-immunoreactive microglia in vehicle- (A, C) and minocycline-treated (B, D) zi/zi rats for 2 months (A, B) and 6 months (C, D). Bar = 500 μm. (E) The number of Iba1-immunoreactive cell clusters in this area after different minocycline administration periods. The white bars show vehicle-treated zi/zi rats and the grid bars show minocycline-treated zi/zi rats (n = 3 each group). * P < 0.05, ** P < 0.01.

Fig. 4.

The effects of minocycline treatment on TH-immunoreactive neurons in the SN of zi/zi rats. (A–D) A comparison of TH-immunoreactive neurons in the SN of vehicle- (A, C) and minocycline-treated (B, D) zi/zi rats at 2M (A, B) and 6M (C, D). The right corners in panels A–D are high magnification images of the dendrites of TH-immunoreactive neurons in the SNr. Bars = 500 μm and 50 μm (corners). (E) The number of TH-immunoreactive neurons in the SNc from different age groups. The white bars show vehicle-treated zi/zi rats and the hatched bars show minocycline-treated zi/zi rats (n = 3 each group). * P < 0.005.

Fig. 5.

Expression of IL-1β, TNF-α, and iNOS mRNA in the midbrain of 2M SD (black bars), and zi/zi rats treated with vehicle (white bars) or minocycline (hatched bars) (n = 4 each group). The mRNA levels are presented as a fold change (mean ± SEM). * P < 0.01 vs. SD, # P < 0.01 vs. vehicle-treated zi/zi rats.