3.4 Differential responses of PrL and IL to microglial alterations induced by ICV-STZ.
Since neuroinflammation is regulated by glial cells in the central nervous system, we further observed the alteration of microglia in the PrL and IL to examine the potential target of 1-MT. Iba1 was considered as the microglia marker. As shown in Figure 5, the main effects of 1-MT treatment were observed in the Iba1-positive cell number (F(1,20)=5.118), levels of Iba1 protein (F(1,20)=11.20) and 3-HK (F(1,20)=11.97) from PrL. The post hoc test revealed that there was no significant difference in the fluorescent images of immunostaining for Iba1 (Figure 5A) and the count of microglia (Figure 5B) in the PrL of ICV-STZ rats administered saline or 1-MT. Similarly, the expression of Iba1 (Figure 5C) and microglial morphology in the PrL (Figure 5D-F) were not influenced by either ICV-STZ or 1-MT. Levels of 3-HK (Figure 5G) in the PrL was insignificant difference across the groups, although intra-PrL injection of 1-MT induced an increasing trend. In the IL, the main effects of ICV-STZ were observed in the Iba1-positive cell number (F(1,20)=8.092) and levels of Iba1 protein (F(1,20)=7.915); the main effects of 1-MT treatment were observed in the Iba1-positive cell branches (F(1,20)=8.960). Moreover, a significant interaction between ICV-STZ and 1-MT treatment was observed in the Iba1-positive cell number (F(1,20)=8.092), levels of Iba1 protein (F(1,20)=6.103), Iba1-positive cell branches (F(1,20)=37.13), Iba1-positive cell end-points (F(1,20)=7.035) and levels of 3-HK (F(1,20)=6.667). Fluorescent images of immunostaining for Iba1 (Figure 6A) and the count of Iba1-positive cells number (Figure 6B) presented that the number of microglia in the IL was significantly increased by ICV-STZ, while intra-IL injection of 1-MT recovered the density of Iba1-positive cells to normal. Western blot analysis also revealed that the expression of Iba1 in the IL was significantly increased by ICV-STZ, and reversed by intra-IL injection of 1-MT (Figure 6C). Subsequently, the analysis of morphological features in microglia (Figure 6D-F) indicated that ICV-STZ remarkably decreased the branch number (Figure 6E) and end-point number (Figure 6F) of Iba1-positive cells in the IL, and intra-IL injection of 1-MT prevented these decreases. On the other hand, the levels of 3-HK increased with microglia activation in the IL, and this increase was reversed by intra-IL injection of 1-MT (Figure 6G).