Absence of Delayed Neuronal Death in ATP-Injected Brain: Possible Roles of Astrogliosis

Abstract

Although secondary delayed neuronal death has been considered as a therapeutic target to minimize brain damage induced by several injuries, delayed neuronal death does not occur always. In this study, we investigated possible mechanisms that prevent delayed neuronal death in the ATP-injected substantia nigra (SN) and cortex, where delayed neuronal death does not occur. In both the SN and cortex, ATP rapidly induced death of the neurons and astrocytes in the injection core area within 3 h, and the astrocytes in the penumbra region became hypertropic and rapidly surrounded the damaged areas. It was observed that the neurons survived for up to 1-3 months in the area where the astrocytes became hypertropic. The damaged areas of astrocytes gradually reduced at 3 days, 7 days, and 1-3 months. Astrocyte proliferation was detectable at 3-7 days, and vimentin was expressed in astrocytes that surrounded and/or protruded into the damaged sites. The NeuN-positive cells also reappeared in the injury sites where astrocytes reappeared. Taken together, these results suggest that astroycte survival and/or gliosis in the injured brain may be critical for neuronal survival and may prevent delayed neuronal death in the injured brain.

Keywords: astrogliosis; brain injury; delayed neuronal death.

Fig. 1

Time-dependent behavior of astrocyte in the ATP-injected SN. ATP (100 nmol in 2 µl PBS) was unilaterally injected into SNpc (^*, injection sites), and brains were obtained at the indicated times after the injection. Brain sections (30 µm thickness) of the midbrain including the entire SN were prepared, and every sixth serial section was selected. (A) Sections were stained with GFAP antibodies, and visualized with biotin-conjugated secondary antibodies. Photographs of the most damaged sections were taken. The contralateral side (contra) and PBS-injected rat brain sections were used as control. (B) Adjacent sections were stained with GFAP and vimentin antibodies and visualized as for (A). The lower panels show the higher magnification of the boxed areas (a~c) in the upper panels. (C) Sections were double-labeled with GFAP and vimentin antibodies, and visualized using Alexa Fluor555- and Alexa Fluor488-conjugated secondary antibodies, respectively. Nuclei were labeled with DAPI. Area 'a' is adjacent to the damage core, and area 'b' is in the undamaged area. Scale bars, 500 µm (A, B upper panel); 100 µm (B lower panel); 10 µm (C). All data are representative of at least three independent experiments.

Fig. 2

Astrocyte proliferation in ATP-injected SNpc. (A) Serial sections were obtained at the indicated times after ATP injection into the SN (^*, injection sites) and processed for Ki67 immunostaining, as described for Fig. 1. Photographs of the most damaged sections were obtained. The lower panel represents higher magnification of the indicated area in the upper panel. Arrows indicate Ki67+ cells. (B) Sections obtained at 2 d after injection were double-labeled with GFAP/Ki67 or vimentin/Ki67. GFAP and vimentin were visualized with biotin-conjugated secondary antibodies and purple-color reaction, and Ki67 were visualized with biotin-conjugated secondary antibodies and brown-color reaction. Inset: higher magnification of the boxed area. Scale bars, 500 µm (A, upper panel); 50 µm (A, lower panel); 50 µm (B); 20 µm (B, inset). All data are representative of at least three independent experiments.

Fig. 3

Time-dependent behavior of astrocyte and neurite in ATP-injected cortex. ATP (1,000 nmol in 2 µl PBS) was unilaterally injected into the cortex, and brains were obtained at the indicated times. Brain sections (30 µm thickness) were prepared, and every sixth serial section was selected. (A) Adjacent sections were stained with GFAP and MAP-2 antibodies, and visualized with biotin-conjugated secondary antibodies. Photographs of the most damaged sections were taken. The contralateral side (contra) was used as the control. (B) Sections were double-labeled with GFAP/NeuN. GFAP was visualized with a purple-color reaction (white arrows), and NeuN with a brown-color reaction (thin black arrows). The black arrow heads and thick black arrows in 'h' indicate unhealthy neurons and astrocytes, respectively. The lower panels show the higher magnification of the boxed areas in the upper panels. All data are representative of at least three independent experiments.

Fig. 4

Increase in vimentin expression and Ki67+ cells in ATP-injected cortex. Sections obtained at the indicated times after ATP injections were processed for vimentin (A) and Ki67 (B) immunostaining. Photographs of the most damaged sections were obtained. The lower panel s in (B) represents higher magnification of the indicated area in the upper panel. Arrows in (B) indicate Ki67+ cells. All data are representative of at least three independent experiments.