*3*.*6*. *Expression of SVCT2 and GLUT1 in the Cortex*

The expression and transport activity of SVCT2 as a specific transporter of AA in the CNS have been reported to be enhanced after MCAO/Re in mice [20]. In addition, DHA is known to be transported across the BBB via GLUT1 [19]; thus, we investigated effects of AA supplementation on changes in the expression of SVCT2 and GLUT1 mRNA in diabetic animals with or without MCAO/Re (Figure 8). There was no significant difference in the basal expression levels of SVCT2 mRNA between the nondiabetic and diabetic groups or between each of them and their AA-supplemented groups. In the nondiabetic and nondiabetic + AA groups, the expression level of SVCT2 mRNA was equally upregulated at 24 h after MCAO/Re. In contrast, the upregulation of ischemia-induced SVCT2 mRNA expression was not observed in the diabetic control group, whereas it was observed in the diabetic + AA group. Compared with the nondiabetic control group, the diabetic state significantly downregulated the expression of GLUT1 mRNA in the cortex. AA supplementation increased the expression of GLUT1 in the nondiabetic and diabetic rats. In the nondiabetic and diabetic control groups, MCAO/Re had a stimulating effect on the expression of GLUT1 mRNA that was similar to the effect of AA supplementation. Further enhancement by ischemia was not detected in the AA-supplemented groups.

**Figure 8.** Effects of AA supplementation on SVCT2 and GLUT1 mRNA expression in the ischemic penumbral cortex of nondiabetic and diabetic rats. Expression levels of SVCT2 mRNA (*Slc23a2*) (**A**) and GLUT1 mRNA (*Slc2a1*) (**B**) were assessed using real-time PCR analysis of the penumbral cortex of nondiabetic and diabetic rats after MCAO/Re. The data are presented as mean ± SD (*n =* 3–6). **†** *p* < 0.05, **††** *p* < 0.01 compared with the respective sham-operated controls. \* *p* < 0.05, \*\* *p* < 0.01.

To assess the effects of AA supplementation on the expression of SVCT2 protein after MCAO/Re in detail, the localization and the expression levels of SVCT2 in the penumbral cortex were examined by immunohistochemical staining (Figure 9A). Double immunofluorescence staining with antibodies of anti-SVCT2 and anti-NeuN, a biomarker for neurons, revealed that majority of the cells exhibiting SVCT2 expressed NeuN in the sham-operated nondiabetic and diabetic rat cortex (Figure 9A). In addition, the cells exhibiting the endothelial cell maker RECA1 showed a low level of SVCT2-immunoreactivity (Figure 9B). These expression levels in the neurons and capillary endothelial cells of SVCT2 were upregulated by MCAO/Re in the nondiabetic rats, but not in the diabetic rats. In agreement with the results from the mRNA measurement, quantification of the immunofluorescence revealed that the expression levels of SVCT2 immunoreactivity were significantly increased in response to MCAO/Re both in the nondiabetic control and AA-supplemented nondiabetic groups (Figure 9C). This reaction against MCAO/Re was abrogated in the diabetic control group, whereas the AA-supplemented diabetic rats showed an increased expression of SVCT2 in the cortex after MCAO/Re.

**Figure 9.** Effects of AA supplementation on SVCT2 and GLUT1 expression in the ischemic penumbral cortex of nondiabetic and diabetic rats. Expression levels of SVCT2 (**A**–**C**) and GLUT1 (**D**,**E**) were assessed using immunohistostaining of the penumbral cortex of nondiabetic and diabetic rats after MCAO/Re. Immunohistochemical expression of SVCT2 (red fluorescence) merged with NeuN (**A**) or with RECA1 (**B**) (green fluorescence); (**C**) Quantitative analysis of SVCT2 fluorescence intensity in the cortex. # *p* < 0.05, ## *p* < 0.01. \* *p* < 0.05, \*\* *p* < 0.01 compared with the respective sham-operated nondiabetic controls; (**D**) Immunohistochemical expression of GLUT1 (red fluorescence) merged with RECA1 (green fluorescence); (**E**) Quantitative analysis of SVCT2 fluorescence intensity in the cortex. \* *p* < 0.05, \*\* *p* < 0.01 compared with the respective sham-operated nondiabetic controls. **†** *p* < 0.05, **††** *p* < 0.01 compared with the respective sham-operated diabetic controls.

**(A) SVCT2**

**50 m**

**(D) GLUT1**

On the other hand, GLUT1 immunoreactivity was predominantly colocalized with RECA1 (Figure 9D). Some of the cells exhibiting NeuN showed a weak GLUT1-immunoreactivity (data not shown). In the sham-operated nondiabetic and diabetic groups with AA supplementation, the expression levels of GLUT1 in the capillary endothelial cells were remarkably upregulated. Additionally, MCAO/Re showed a stimulating effect on the GLUT1 expression both in the nondiabetic and diabetic cortex. Quantitative analyses of immunofluorescence in the sections revealed that the sham-operated diabetic control rat cortex had a lower expression level of GLUT1 compared with the nondiabetic rat cortex, and AA supplementation or MCAO/Re caused a significant increase in the expression of GLUT1 (Figure 9E).
