3.5 Differential responses of PrL and IL to astrocyte alterations induced by ICV-STZ.
Then, we evaluated the expression and function of astrocytes in the PrL and IL separately. GFAP was considered as the astrocyte marker. In the PrL, the main effects of ICV-STZ were observed in the intersection number (12 μm: F(1,20)=5.075; 15 μm: F(1,20)=7.447), levels of GLAST (F(1,20)=8.530), GFAP (F(1,20)=8.883) and KA (F(1,20)=20.43); the main effects of 1-MT treatment were observed in the intersection number (3 μm: F(1,20)=5.057; 9 μm: F(1,20)=4.648; 12 μm: F(1,20)=7.488; 15 μm: F(1,20)=18.27; 18 μm: F(1,20)=24.75; 21 μm: F(1,20)=17.39; 24 μm: F(1,20)=7.569), levels of GLT-1 (F(1,20)=9.437), GLAST (F(1,20)=5.884), GFAP (F(1,20)=10.77) and KA (F(1,20)=38.04). A significant interaction between ICV-STZ and 1-MT treatment was observed in the GFAP-positive cell number (F(1,20)=21.19), intersection number (12 μm: F(1,20)=5.959; 15 μm: F(1,20)=8.523; 18 μm: F(1,20)=5.719; 21 μm: F(1,20)=7.213), levels of GLAST (F(1,20)=14.21) and KA (F(1,20)=17.85). ICV-STZ significantly reduced the number of GFAP-positive cells in the PrL, which was reversed by intra-PrL injection of 1-MT (Figure 7A, B). Sholl analysis showed an attenuated number of intersections, suggesting the atrophy of astrocytes. Specifically, the ICV-STZ rats significantly decreased at steps 9 and 12 compared with the vehicle rats, and a significant increase in the number of intersections was found in the intra-PrL injection of 1-MT rats at steps 9 to 24 compared with ICV-STZ rats (Figure 7C, D). Consistent with above results, western blot analysis (Figure 7E) also revealed that ICV-STZ markedly decreased the expression of GFAP (Figure 7H) in the PrL, accompanied by dysfunctional changes including the reduction of GLT-1 (Figure 7F) and GLAST (Figure 7G). All these defects were restored by intra-PrL injection of 1-MT (Figure 7E-H). Levels of KA decreased with astrocyte deficits in the PrL, and this decrease was reversed by intra-PrL injection of 1-MT (Figure 7I). In the IL, the main effects of ICV-STZ were observed in the intersection number (12 μm: F(1,20)=5.809; 21 μm: F(1,20)=4.424) and levels of KA (F(1,20)=10.61); the main effects of 1-MT treatment were observed in the levels of GFAP (F(1,20)=17.08). ICV-STZ and intra-IL injection of 1-MT had no effect on the number of astrocytes per unit area (Figure 8A, B). In addition, there was no significant distinction in the number of intersections for all groups (Figure 8C, D). Western blot analysis (Figure 8E-H) did not reveal a significant difference in the expression of GLT-1 (Figure 8F), GLAST (Figure 8G) and GFAP (Figure 8H) in the IL across the groups. Intriguingly, the levels of KA also decreased in the IL, and did not reverse by intra-IL injection of 1-MT (Figure 8I).