Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor

Abstract

Nigrostriatal dopaminergic neurons undergo sprouting around the margins of a striatal wound. The mechanism of this periwound sprouting has been unclear. In this study, we have examined the role played by the macrophage and microglial response that follows striatal injury. Macrophages and activated microglia quickly accumulate after injury and reach their greatest numbers in the first week. Subsequently, the number of both cell types declines rapidly in the first month and thereafter more slowly. Macrophage numbers eventually cease to decline, and a sizable group of these cells remains at the wound site and forms a long-term, highly activated resident population. This population of macrophages expresses increasing amounts of glial cell line-derived neurotrophic factor mRNA with time. Brain-derived neurotrophic factor mRNA is also expressed in and around the wound site. Production of this factor is by both activated microglia and, to a lesser extent, macrophages. The production of these potent dopaminergic neurotrophic factors occurs in a similar spatial distribution to sprouting dopaminergic fibers. Moreover, dopamine transporter-positive dopaminergic neurites can be seen growing toward and embracing hemosiderin-filled wound macrophages. The dopaminergic sprouting that accompanies striatal injury thus appears to result from neurotrophic factor secretion by activated macrophages and microglia at the wound site.

Fig. 1.

Top. Photomicrographs of periwound reactive glia. A, MAC-1 immunostained resting microglial cell. Note the small cell body, long processes, and relatively faint immunoreactivity. B, MAC-1 immunostained activated microglial cell. Note the relatively large cell body, short, retracted processes, and intense immunoreactivity. C, MAC-1 immunostained wound macrophage at day 1. D, MAC-1 immunostained wound macrophage at 1 month. Note, the diameter has significantly increased, and the cell is filled with hemosiderin.E, NSE-positive macrophage (30 d). F, GFAP-positive periwound reactive astrocyte. Scale bar, 10 μm.

Fig. 2.

Middle. Line graphs showing the changing number of MAC-1-positive macrophages, NSE-positive macrophages, and MAC-1-positive microglia in groups of four mice at 0, 1, 3, 7, 14, 30, and 120 d after striatal injury. All values are the mean number of cells in five representative sections through the wound (1 section through the middle of the wound and 2 sections either side). Error bars indicate SEM.

Fig. 4.

Colocalization of neurotrophins to activated microglia and macrophages. A, MAC-1 immunostained section 7 d after injury. DAB is unintensified, and activated microglia and macrophages are stained yellow-brown.B, MAC-1 immunostained section 7 d after injury in which BDNF in situ hybridization has also been performed. Note the intense silver grain deposits over MAC-1 immunostained cells. C, High-power photomicrograph showing the colocalization of the BDNF in situ signal (black silver grain deposits) with MAC-1 immunolabeled activated microglia. D, Section stained for NSE 30 d after injury. Macrophages appear as large red cells within the wound area. E, Section stained for NSE 30 d after injury in which GDNF in situhybridization has also been performed. The silver grains of thein situ signal are colocalized with the NSE-positive macrophages. F, High-power photomicrograph ofE. Scale bar: A, B,D, E, 100 μm; C,F, 25 μm.

Fig. 9.

Photomicrographs showing DAT-positive sprouting dopaminergic fibers growing toward and around hemosiderin-filled wound macrophages. A, Low-magnification image showing bundles of dark DAT-positive fibers sprouting in the periwound margin. B, High-magnification image showing sprouting fibers approaching and growing around hemosiderin-containing macrophages. C–E, Higher magnification images showing the intimate association of sprouting dopaminergic fibers to a number of individual hemosiderin-filled (arrow) wound macrophages. F, Photomicrograph demonstrating that hemosiderin (arrow) is located within macrophage cytoplasm (arrowhead) (NSE stain). G, NSE-positive macrophage containing prussian blue deposits of iron-containing hemosiderin. Scale bar:A, 375 μm; B, 25 μm;C–G, 10 μm.