**1. Introduction**

Diabetes mellitus is a metabolic disorder associated with chronic hyperglycemia, which is known to enhance systemic oxidative stress, predisposing to diabetic complications. Diabetes is a major risk factor for atherosclerotic diseases such as acute brain ischemia [1,2]. Moreover, it increases the risks of morbidity and mortality after stroke [3,4]. Oxidative stress plays an essential role in the pathogenesis of transient cerebral ischemic injury [3–5]. In particular, reperfusion after a long period of vessel occlusion triggers the explosive generation of reactive oxygen species (ROS), such as superoxide radical (O2 – ), hydroxyl radical, hydrogen peroxide, *etc*., which causes apoptosis and delayed death of cells through oxidative damage to lipids, proteins, and DNA in the ischemic penumbral region [6–9]. In addition to apoptotic cell death, inflammatory neurodegeneration is another crucial process contributing to cerebral damage after ischemia and reperfusion [10]. ROS have been shown to activate nuclear factor-κB, which enhances the transcription of the genes encoding proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), leading to inflammatory responses [11]. Myeloperoxidase (MPO) expressed by microglia, a histopathological marker of inflammation, generates cytotoxic ROS and leads to further inflammatory damage in the ischemic tissue [12]. Accumulating evidence indicates that hyperglycemia in diabetes is associated with a decrease in the antioxidant potential and an increase in ROS generation [13–15]. In addition, diabetes has been shown to be a proinflammatory state that increases the risk of vascular complications [16,17]. Thus, the enhanced oxidative stress and inflammatory responses in the diabetic state may substantially contribute to the aggravation of cerebral injury caused by transient ischemia and subsequent reperfusion.

L-ascorbic acid (AA) is an essential antioxidant for scavenging free radicals in the brain. AA participates not only in sustaining the normal function of the central nervous system (CNS) but in ameliorating the damage induced by pathological conditions that increase the generation of ROS [18]. The CNS maintains relatively high concentrations of AA, indicating a neuroprotective role for AA [19]. The transport of AA from the plasma to the CNS is mainly mediated by

sodium-dependent vitamin C transporter 2 (SVCT2). In addition, glucose transporter 1 (GLUT1) is located in the endothelial cells of the blood–brain barrier (BBB) and transports oxidized AA ([dehydroascorbic acid (DHA)) as another source of AA to the brain. Upregulation of SVCT2 [20] and GLUT1 [21] expression has been demonstrated in rats with cerebral ischemic injury, which suggests that AA is necessary for protection against oxidative neuronal injury. The concentration of AA is considered to reflect oxidative stress in animal tissues sensitively [22]. Patients with ischemic stroke or diabetes have lower concentrations of AA in the plasma, suggesting that a systemic decrease in AA is a consequence of enhanced consumption of AA by elevated oxidative stress [23,24]. In a previous study, we demonstrated that chronic supplementation with AA attenuates oxidative stress in both the plasma and the brain and alleviates cerebral injury induced by middle cerebral artery occlusion and reperfusion (MCAO/Re) in rats with streptozotocin (STZ)-induced diabetes [25]. We showed that the activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) is decreased, and lipid peroxidation is accelerated in the brain of diabetic rats; those detrimental oxidative processes are inhibited by AA supplementation. These data suggest that the enhanced oxidative stress in the diabetic state causes the functional impairment of antioxidant enzymes, and the resulting diminution in antioxidative defense can cause further enhancement of the generation of ROS and subsequent neuronal apoptosis and inflammatory neurodegeneration in the ischemic brain. Nonetheless, there is a shortage of studies so far on the antiapoptotic and anti-inflammatory effects of AA in cerebral ischemia with diabetes.

In the present study, we evaluated the effects of chronic oral pretreatment with AA on the production of O2 – and on apoptosis in the brain after MCAO/Re in rats with STZ-induced diabetes. To test whether AA suppresses inflammatory responses induced by MCAO/Re, we also examined the effects of AA on the expression of TNF-α, IL-1β, and MPO in the brain. Although the transport of AA to the CNS is an important factor for its neuroprotection, changes in expression of SVCT2 and GLUT1 proteins in response to AA supplementation and/or cerebral ischemia in diabetic state have not been reported. Therefore, we investigated the effects of AA supplementation on the expression of SVCT2 and GLUT1 after MCAO/Re in the brain of diabetic rats.
