Relationship between Neuronal Damage/Death and Astrogliosis in the Cerebral Motor Cortex of Gerbil Models of Mild and Severe Ischemia and Reperfusion Injury

Abstract

Neuronal loss (death) occurs selectively in vulnerable brain regions after ischemic insults. Astrogliosis is accompanied by neuronal death. It can change the molecular expression and morphology of astrocytes following ischemic insults. However, little is known about cerebral ischemia and reperfusion injury that can variously lead to damage of astrocytes according to the degree of ischemic injury, which is related to neuronal damage/death. Thus, the purpose of this study was to examine the relationship between damage to cortical neurons and astrocytes using gerbil models of mild and severe transient forebrain ischemia induced by blocking the blood supply to the forebrain for five or 15 min. Significant ischemia tFI-induced neuronal death occurred in the deep layers (layers V and VI) of the motor cortex: neuronal death occurred earlier and more severely in gerbils with severe ischemia than in gerbils with mild ischemia. Distinct astrogliosis was detected in layers V and VI. It gradually increased with time after both ischemiae. The astrogliosis was significantly higher in severe ischemia than in mild ischemia. The ischemia-induced increase of glial fibrillary acidic protein (GFAP; a maker of astrocyte) expression in severe ischemia was significantly higher than that in mild ischemia. However, GFAP-immunoreactive astrocytes were apparently damaged two days after both ischemiae. At five days after ischemiae, astrocyte endfeet around capillary endothelial cells were severely ruptured. They were more severely ruptured by severe ischemia than by mild ischemia. However, the number of astrocytes stained with S100 was significantly higher in severe ischemia than in mild ischemia. These results indicate that the degree of astrogliosis, including the disruption (loss) of astrocyte endfeet following ischemia and reperfusion in the forebrain, might depend on the severity of ischemia and that the degree of ischemia-induced neuronal damage may be associated with the degree of astrogliosis.

Keywords: S100; astrocyte endfeet; blood–brain barrier; cortical layer; glial fibrillary acidic protein; ischemia and reperfusion injury.

Figure 1

Change in spontaneous motor activity (SAM). SMA is evaluated in the traveled entire distance (meters) in 60 min at one day after tFI (n = 5 or 7, respectively; * p < 0.05, significantly different from the mild and severe sham group, and † p < 0.05, significantly different from the mild tFI group at the corresponding time). The bars indicate the means ± SEM.

Figure 2

CV staining in the motor cortex of mild sham (A,a1,a2), mild tFI (B–D,b1–d1,b2–d2), severe sham (E,e1,e2), and severe tFI (F–H,f1–h2,f2–h2) groups at one day (B,b1,b2,F,f1,f2), 2 (C,c1,c2, G,g1,g2) and five days (D,d1,d2,H,h1,h2) after tFI. At five days after tFI, CV-cells are apparently decreased and damaged in layers V and VI (d1,d2,h1,h2) in both of the tFI groups: more reduction and damage of CV-cells are shown in the severe tFI group (h1,h2) than the mild tFI group. Scale bars = 200 μm (A–H) and 50 μm (a1–h1,a2–h2).

Figure 3

(A,B) F-J B histofluorescence staining in layers V and VI of the motor cortex in the mild sham (Aa,Ae), mild tFI (Ab–Ad,Af–Ah), severe sham (Ba,Be), and severe tFI (Bb–Bd,Bf–Bh) groups at one day (Ab,Af,Bb,Bf), two days (Ac,Ag,Bc,Bg) and five days (Ad,Ah,Bd,Bh) after tFI. A few F-J B-cells are detected in the mild tFI group two and five days after tFI. In contrast, many F-J B-cells are found in the severe tFI group two and five days after tFI. Scale bars = 200 μm. (C,D) Numbers of F-J B-cells in layers V and VI, respectively (n = 7, respectively; * p < 0.05, significantly different from mild and severe sham group, # p < 0.05, significantly different from pre-time point group and † p < 0.05, significantly different from mild tFI group at corresponding time point). The bars indicate the means ± SEM.

Figure 4

(A–H) GFAP immunohistochemistry in layers V and VI of the motor cortex in the mild sham (A,a1,a2), mild tFI (B–D,b1–d1,b2–d2), severe sham (E,e1,e2), and severe tFI (F–H,f1–h2,f2–h2) groups on day 1 (B,b1,b2,F,f1,f2), day 2 (C,c1,c2,G,g1,g2) and day 5 (D,d1,d2,H,h1,h2) after tFI. In the mild tFI group, GFAP immunoreactivity is increased from 2 days after tFI. However, GFAP immunoreactivity in the severe group is significantly enhanced from one day after tFI. On day 5 after tFI, the cell bodies and processes of GFAP-astrocytes are larger and thicker than those of the sham groups, showing that GFAP immunoreactivity in the severe tFI group is higher than that in the mild tFI group. Scale bars = 200 μm (A–H) and 50 μm (a1–h2). (I,J): ROD as % of GFAP-immunoreactive structures in layers V and VI (n = 5 or 7, respectively; * p < 0.05, significantly different from mild or severe sham group, # p < 0.05, significantly different from corresponding former time group, and † p < 0.05, significantly different from mild tFI group at the corresponding time). The bars indicate the means ± SEM.

Figure 5

(A) Representative blot image and quantitative analysis of GFAP protein level in the motor cortex of the mild sham, mild tFI, severe sham and severe tFI groups on days 1, 2 and 5 after tFI. (B) Densitometric analysis of GFAP protein level by normalization to the level of β-actin. The bars indicate the means ± SEM (n = 5, respectively; * p < 0.05, significantly different from mild or severe sham group, # p < 0.05, significantly different from corresponding former time group, and † p < 0.05, significantly different from mild tFI group at the corresponding time). The bars indicate the means ± SEM.

Figure 6

(A,B) S100 immunohistochemistry in layers V and VI of the motor cortex in the mild sham (Aa,Ae), mild tFI (Ab–Ad,Af–Ah), severe sham (Ba,Be), and severe tFI (Bb–Bd,Bf–Bh) groups at one day (Ab,Af,Bb,Bf), two days (Ac,Ag,Bc,Bg) and five days (Ad,Ah,Bd,Bh) after tFI. S100-astrocytes are significantly increased in numbers from two days after tFI. Scale bar = 200 μm. (C,D) Mean numbers of S100-astrocytes in layers V and VI (n = 5 or 7, respectively; * p < 0.05, significantly different from mild or severe sham group, # p < 0.05, and significantly different from corresponding former time group). The bars indicate the means ± SEM.

Figure 7

Double immunohistofluorescence for AEf around blood vessels in layers V and VI using anti-GFAP (green) and GLUT-1 (red) in the mild (A) sham (Aa,Ae), mild tFI (Ab–Ad,Af–Ah), severe (B) sham (Ba,Be), and severe tFI (Bb–Bd,Bf–Bh) groups at one day (Ab,Af,Bb,Bf), two days (Ac,Ag,Bc,Bg) and five days (Ad,Ah,Bd,Bh) after tFI. In all sham groups, GFAP-AEf (arrows) contact GLUT-1-endothelial cells. On day 5 after tFI, GFAP-AEf (arrowheads) around GLUT-1-endothelial cells apparently disappear in both groups. Scale bars = 50 μm.

Figure 8

Electron micrographs of AEf around a blood vessel using TEM in the mild sham (A), mild tFI (B–D), severe sham (B), and severe tFI (F–H) groups at one day (B,F), two days (C,G) and five days (D,H) after tFI. In both sham groups, AEf has many mitochondria and surrounds the basal membrane of endothelial cells (EC). AEf becomes damaged with time after tFI: in the severe tFI group, the damage is more significant. On day 5 after tFI, AEf looks like a vacuole and no mitochondria were in both groups: in the severe group, EC and pericyte (P) are severely damaged. Scale bar = 2.5 μm.

Figure 9

Experimental procedure. Gerbils are given five- and 15-min tFI, respectively, for mild and severe ischemia and reperfusion injury. Open field test (for change of motor activity) was performed on day 1 after tFI, and the gerbils were sacrificed on days 1, 2 and 5 after tFI for the examination of histopathological and biochemical changes.