Dehydroascorbic acid, a blood-brain barrier transportable form of vitamin C, mediates potent cerebroprotection in experimental stroke

Abstract

Neuronal injury in ischemic stroke is partly mediated by cytotoxic reactive oxygen species. Although the antioxidant ascorbic acid (AA) or vitamin C does not penetrate the blood-brain barrier (BBB), its oxidized form, dehydroascorbic acid (DHA), enters the brain by means of facilitative transport. We hypothesized that i.v. DHA would improve outcome after stroke because of its ability to cross the BBB and augment brain antioxidant levels. Reversible or permanent focal cerebral ischemia was created by intraluminal middle cerebral artery occlusion in mice treated with vehicle, AA, or DHA (40, 250, or 500 mg/kg), either before or after ischemia. Given before ischemia, DHA caused dose-dependent increases in postreperfusion cerebral blood flow, with reductions in neurological deficit and mortality. In reperfused cerebral ischemia, mean infarct volume was reduced from 53% and 59% in vehicle- and AA-treated animals, respectively, to 15% in 250 mg/kg DHA-treated animals (P < 0.05). Similar significant reductions occurred in nonreperfused cerebral ischemia. Delayed postischemic DHA administration after 15 min or 3 h also mediated improved outcomes. DHA (250 mg/kg or 500 mg/kg) administered at 3 h postischemia reduced infarct volume by 6- to 9-fold, to only 5% with the highest DHA dose (P < 0.05). In contrast, AA had no effect on infarct volumes, mortality, or neurological deficits. No differences in the incidence of intracerebral hemorrhage occurred. Unlike exogenous AA, DHA confers in vivo, dose-dependent neuroprotection in reperfused and nonreperfused cerebral ischemia at clinically relevant times. As a naturally occurring interconvertible form of AA with BBB permeability, DHA represents a promising pharmacological therapy for stroke based on its effects in this model of cerebral ischemia.

Figure 1

DHA accumulation. Effects of cerebral ischemia on the accumulation (at 120 min) of DHA, AA, and sucrose in the brain (n = 3/cohort). The data are expressed as percentage of injected dose (ID)/g of brain tissue. DHA, but not AA or sucrose, accumulated similarly in cerebral tissue of animals that underwent a sham procedure and in animals that were not subjected to ischemia (*, P < 0.05; **, P < 0.01).

Figure 2

Effects of low- and intermediate-dose DHA administration before ischemia. Reperfused cerebral ischemia cohort (low dose: n = 21; intermediate dose: n = 16; AA: n = 8; vehicle: n = 17). Nonreperfused cerebral ischemia cohort (intermediate dose: n = 21; AA: n = 14; vehicle: n = 14). (A) Dose-dependent improvements were demonstrated in cerebral blood flow at the time of death (24 h) after reperfused cerebral ischemia (*, P < 0.05 for either DHA doses vs. AA or vehicle). There was a significant increase in cerebral blood flow at death in nonreperfused animals treated with intermediate-dose DHA (*, P < 0.05 vs. both AA and vehicle). (B) Dose-dependent improvements were demonstrated in infarct volume after reperfused cerebral ischemia (Left, **, P < 0.01 for both DHA doses vs. AA and vehicle). Improvement was demonstrated in infarct volume after nonreperfused cerebral ischemia (Right, *, P < 0.05 for intermediate DHA dose vs. vehicle). (C) There was a slight decrease in neurological deficit scores after reperfused cerebral ischemia (Left) in the intermediate-dose DHA-treated animals compared with AA- and vehicle-treated animals (P = NS). There was a significant decrease in neurological deficit scores after nonreperfused cerebral ischemia (Right) in the intermediate-dose DHA-treated animals compared with AA-treated animals (P < 0.05). (D) There was a significant decrease in mortality after reperfused cerebral ischemia (Left) in the low- and intermediate-dose DHA-treated animals compared with vehicle-treated animals (P < 0.05). There was a 50% decrease in mortality after nonreperfused cerebral ischemia (Right) in the intermediate-dose DHA-treated animals compared with vehicle-treated animals (P = NS).

Figure 3

Delayed intermediate- and high-dose DHA administration. Intermediate-dose DHA (n = 9) and high-dose DHA (n = 8) were compared with vehicle (n = 11) and high-dose AA (n = 10) given 3 h after MCAO in nonreperfused cerebral ischemia. (A) There was a significant reduction in infarct volume in high-dose DHA animals (*, P < 0.05) compared with both AA and vehicle animals. (B) There was a significant reduction in neurological deficit scores in intermediate-dose DHA animals (*, P < 0.05) and high-dose DHA animals (*, P < 0.005) compared with both AA and vehicle animals. (C) There was a dose-dependent decrease in mortality in DHA-treated animals compared with AA- and vehicle-treated animals (P = NS).