*3*.*2*. *Ischemic Brain Injury and Neurological Deficits*

Figure 1 shows MCAO/Re-induced brain injury in the nondiabetic control, nondiabetic + AA, diabetic control, and diabetic + AA groups of rats. Examples of TTC staining in the coronal brain sections at 24 h after MCAO/Re are shown in Figure 1A. The infarct developed in the corpus striatum and cortex of the nondiabetic control rats. In the diabetic rats, the infarction zone was remarkably enlarged and extended to a large part of the left striatum and cortex. In contrast, the infarcts in the AA-supplemented groups were smaller than those in their respective controls. Quantitative assays revealed that the infarct volume and edema in the diabetic control group were significantly increased by approximately 2.5-fold and 2-fold, respectively, compared with those in the nondiabetic control group (Figure 1B,C). AA supplementation in the nondiabetic group significantly decreased infarction and edema. Furthermore, AA almost completely suppressed the exacerbation of brain damage by diabetes.

**Figure 1.** Effects of AA supplementation on infarction induced by MCAO/Re in the brain of nondiabetic and diabetic rats. (**A**) Representative photographs of staining of coronal brain sections from the rats of the nondiabetic + sham operation group and from distilled water-administered (Control) or AA (100 mg/kg)-supplemented nondiabetic or diabetic groups with MCAO with reperfusion (MCAO/Re). AA or water was orally administered once daily for 2 weeks; (**B**) Infarct volume in ischemic hemispheres of the diabetic and nondiabetic groups after MCAO/Re by TTC staining; (**C**) Edema volume in ischemic hemispheres of the diabetic and nondiabetic groups after MCAO/Re. Data are presented as means ± SD (*n* = 6–7). \*\* *p* < 0.01 compared with the nondiabetic control group. ## *p* < 0.01 compared with the diabetic control group. DM in the figure denotes diabetic, while non-DM denotes nondiabetic.

Consistent with the aforementioned results of the brain injury experiments, neurological deficits were exacerbated in the diabetic control group of rats (Figure 2). Compared with the diabetic control group, the diabetic + AA group showed significant alleviation of the neurological deficits.

**Figure 2.** Effects of AA supplementation on neurological deficits induced by MCAO/Re in nondiabetic and diabetic rats. Postischemic neurological deficits were evaluated on a 5-point scale at 24 h of reperfusion after 2 h of MCAO. Data are mean ± SD of 6–7 rats per group. \*\* *p* < 0.01 compared with the nondiabetic control group. ## *p* < 0.01 compared with the diabetic control group. DM in the figure denotes diabetic, while non-DM denotes nondiabetic.

*3*.*3*. *O2 – Production after Ischemia with Reperfusion* 

Figure 3 shows fluorescence intensity of DHE in the penumbral cortex, which depends on intracellular O2 – production. Representative histological images of DHE staining in the nondiabetic control, nondiabetic + AA, diabetic control, and diabetic + AA groups are shown in Figure 3A. DHE-positive cells with low fluorescence intensity were sparsely distributed in the cortex of sham-operated nondiabetic rats. In contrast, sham-operated diabetic rats had an increased number of DHE-positive cells with higher fluorescence intensity in the cortex, indicating basal augmentation of the generation of ROS in the brain of the diabetic rats. The intensity of DHE fluorescence was remarkably increased by MCAO/Re in the nondiabetic rats and was further augmented in the diabetic rats, suggesting that the exacerbated injury can be attributed to enhanced generation of ROS in diabetes. AA supplementation significantly reduced the fluorescence of DHE in the cortex of the nondiabetic and diabetic rats compared with that in their respective controls.

**Figure 3.** Effects of AA supplementation on production of O2 – after MCAO/Re in the brain of nondiabetic and diabetic rats. (**A**) Representative photographs of superoxide production detected by DHE staining in coronal sections of the cortex from the nondiabetic and diabetic rats; (**B**) Quantitative analysis of DHE fluorescence intensity in the cortex. The data are presented as mean ± SD (*n* = 3–4). \* *p* < 0.05, \*\* *p* < 0.01 compared with the nondiabetic control group. # *p* < 0.05, ## *p* < 0.01 compared with the diabetic control group. DM in the figure denotes diabetic, while non-DM denotes nondiabetic.

*3*.*4*. *Apoptosis Induced by Ischemia with Reperfusion* 

Activation of caspase-3, a key mediator of the execution phase of apoptosis, was determined by immunostaining for cleaved caspase-3, which is an activated form of this enzyme, in the ischemic penumbral cortex of the four experimental groups after MCAO/Re (Figure 4). Compared with the nondiabetic control group, the number of cleaved caspase-3 positive cells was remarkably increased by MCAO/Re in the diabetic control group. AA significantly attenuated the MCAO/Re-induced activation of caspase-3 in the nondiabetic and diabetic groups.

**Figure 4.** Effects of AA supplementation on cleaved caspase-3 after MCAO/Re in the brain of nondiabetic and diabetic rats. (**A**) Representative photographs of cleaved caspase-3 immunostaining in the cortex coronal sections of nondiabetic and diabetic rats; (**B**) Quantitative analysis of cleaved caspase-3 positive cells (fluorescence intensity in the cortex). The data are presented as mean ± SD (*n* = 3–4). \* *p* < 0.05, \*\* *p* < 0.01 compared with the nondiabetic control group. ## *p* < 0.01 compared with the diabetic control group. DM in the figure denotes diabetic, while non-DM denotes nondiabetic.

*3*.*5*. *Expression of IL-1β*, *TNF-α*, *and MPO in the Cortex*

To assess the effects of AA supplementation on the expression of proinflammatory cytokines, we performed immunohistochemical staining for IL-1β and TNF-α. This experiment confirmed upregulation of the protein level of these cytokines as a result of MCAO/Re and diabetes (Figures 5 and 6). Quantification of the immunostaining data showed that the sham-operated diabetic control group had a significant increase in IL-1β and TNF-α expression compared with the sham-operated nondiabetic control group, suggesting basal augmentation of the inflammatory response in the

diabetic brain. MCAO/Re significantly increased the expression levels of those proinflammatory cytokines in the nondiabetic cortex. The ischemia-induced upregulation of those cytokines was markedly accelerated by diabetes: the diabetic control group showed a 15.9- and 21.0-fold increase in IL-1β and TNF-α expression, respectively, compared with the nondiabetic control group. AA supplementation significantly suppressed the basal and ischemia-enhanced expression of these cytokines in diabetic rats.

**Figure 5.** Effects of AA supplementation on expression of IL-1β in the penumbral cortex after MCAO/Re in the brain of nondiabetic and diabetic rats. (**A**) Representative photographs of IL-1β immunostaining (red fluorescence) and staining of nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of nondiabetic and diabetic rats; (**B**) Quantitative analysis of IL-1β fluorescence intensity in the cortex. The data are presented as mean ± SD (*n* = 3–4). \*\* *p* < 0.01 compared with the nondiabetic control group. # *p* < 0.05 compared with the diabetic control group. DM in the figure denotes diabetic, while non-DM denotes nondiabetic.

**Figure 6.** Effects of AA supplementation on expression of TNF-α in the penumbral cortex after MCAO/Re in the brain of nondiabetic and diabetic rats. (**A**) Representative photographs of TNF-α immunostaining (red fluorescence) and staining of nuclei by TOPRO-3 (blue fluorescence) in the cortex coronal sections of nondiabetic and diabetic rats; (**B**) Quantitative analysis of TNF-α fluorescence intensity in the cortex. The data are presented as mean ± SD (*n* = 3–4). \*\* *p* < 0.01 compared with the nondiabetic control group. # *p* < 0.05 compared with the diabetic control group. DM in the figure denotes diabetic, while non-DM denotes nondiabetic.

In the nondiabetic rat cortex, the protein expression of MPO was markedly upregulated after MCAO/Re (Figure 7). AA supplementation abrogated the ischemia-induced increase in MPO expression. Compared with the nondiabetic rats, the basal level of MPO expression increased only slightly in the diabetic rats. MCAO/Re upregulated the expression of MPO to the level similar to that in the nondiabetic group. AA supplementation had no effect on the ischemia-induced increase in the MPO level in the diabetic rats.

**Figure 7.** Effects of AA supplementation on MPO expression after MCAO/Re in the brain of nondiabetic and diabetic rats. (**A**) Representative photographs of MPO immunostaining in the cortex coronal sections of nondiabetic and diabetic rats; (**B**) Quantitative analysis of MPO fluorescence intensity in the cortex. The data are presented as mean ± SD (*n* = 3–4). \* *p* < 0.05 compared with the nondiabetic control group. DM in the figure denotes diabetic, while non-DM denotes nondiabetic.
