Introduction
Ketones, R-beta-hydroxybutyrate (BHB) and acetoacetate (AcAc) are known to be essential alternate energy substrates to glucose for most tissues, especially brain, under conditions of starvation, early development, fasting and heavy exercise, and a high fat diet. Clinicians and investigators have been interested in their use as alternate energy substrates to glucose, such as therapeutic agents for the treatment of hypoglycemia, seizure disorders, Alzheimer's and Parkinson's disease and as alternates to high lipid parenteral and enteral feedings 1 . Treatment with ketone body precursors, such as 1,3-butanediol, has shown promising evidence towards ameliorating brain damage due to ischemia and/or reperfusion injury2, 3. The proposed biochemical mechanism is thought to be through ketosis. Therefore, we studied the effects of diet-induced ketosis on reperfusion injury in ketotic rat brain following reversible middle cerebral artery occlusion (MCAO).
Methods
Male Wistar rats (n=19), 28 days old (100 g), were fasted 24 h to deplete glycogen stores and initiate a state of ketosis. Rats were then divided into 3 groups fed either: standard lab-chow (control; n=5, STD), ketogenic (high fat, no carbohydrate; n=7, KG), or carbohydrate (high carbohydrate, low fat; n=7, CHO), or diet for three weeks. Rats were anesthetized with 2% halothane/O2/N2O and MCAO was performed using a monofilament model 5 , occluded for 2 h; rats were sacrificed at 24 h of reperfusion and the infarct volumes determined by TTC staining. In another set of experiments, brain ketone uptake and regional blood flow were measured using a dual label unidirectional tracer method, which allows for quantitation of substrate (beta-hydroxybutyrate) at the endothelial boundary of the blood-brain barrier, as well as a simultaneous determination of blood flow 5 .
Results
The infarct volumes at 24 h of reflow after 2 h occlusions for each of the diet groups were 45.9 ± 21.5, 30.2 ± 5.1 and 14.4 ± 9.6% (mean ± SD) of the ipsilateral hemisphere for STD, CHO, and KG, respectively. The magnitude of the decrease in KG diet was significantly different (p<0.05) from those of STD (68.7 ± 22.1%) and CHO (52.4 ± 11.0%). There was no difference in infarct volumes of the CHO relative to the STD diet group. Regional ketone uptake was significantly elevated (40 fold) in brain of the KG rats compared to STD and CHO; whereas, there were no differences in blood flow among each of the diet groups.
Conclusion
Ketosis induced by KG diet reduced the infarct volume by 70% compared to STD diet and CHO groups. It appears that in brain, ketones may be neuroprotective against reperfusion injury that is not related to changes in blood flow. The precise mechanism remains to be determined, but may be hypothesized through restoration of energy status via regulation of glycolytic intermediates and/or mitochondrial function.