Experimental Study Of Ketogenic Diet In Reducing Diffuse Axonal Injury

Diffuse axonal injury(DAI),a kind of traumatic brain injury(TBI),is characterized by demyelination and delayed axonal rupture following mechanical force.With regard to the mechanism of delayed axonal disconnection in DAI,it is generally believed that it is the result of complex pathophysiological changes secondary to initial shear force.Studies have shown that demyelination deprive the trophic and metabolic support of the axons,and the glucose hypometabolism following TBI will further aggravate the energy defect of axons.Based on these,we hypothesize that energy defect may be an important factor contributed to secondary axonal injury.Ketogenic diet(KD)can provide monocarboxylic acid,which can be used by axons through monocarboxylic acid transporter 2(MCT2).Thus,the present study intends to establish an animal model of DAI to explore the protective effect of ketogenic diet on DAI from the level of myelin-axon functional unit.In our experiment,176 adult male SD rats were randomly divided into three groups:control group(control rats with standard diet,n=16),DAI+SD group(DAI rats with standard diet,n=80)and DAI+KD group(DAI rats with ketogenic diet,n=80).The DAI model was established according to the Marmarou method,and the rats were fed with ketogenic diet(KD)or standard diet(SD)following DAI.The upper part of the brainstem was selected as the observation site.The enzyme-linked immunosorbent assay(ELISA)was used to detect fat acid metabolism of heart blood and cerebral levels of energy metabolites.The myelin injury was evaluated by Luxol Fast Blue(LFB)staining and myelin basic protein(MBP)expression.The axonal injury was evaluated by examine of the expression ofβ-amyloid precursor protein(APP)and phosphorylated high molecular weight neurofilament subunit(p-NF-H)by Western blotting.The ultrastructural changes of axon and myelin,and the morphological changes of mitochondria were further observed by transmission electron microscope.In final,the expression changes of protein of dynamin-related protein 1(DRP1),mitofusin2(Mfn2),nuclear respiratory factor 1(NRF1)and mitochondrial transcription factor A(mt TFA)were detected to monitor mitochondrial mass.Our results showed that:(1)Within 3 days of dietary intervention with KD,blood levels of total cholesterol(TC),triglyceride(TG)and free fatty acid(FFA)were significantly elevated compared to DAI+SD group.The plasmaβHB was consistently increased in the KD-fed rats,which peaked in the 2nd week.Compared with DAI+SD group,blood glucose levels and body weight decreased significantly in DAI+KD group at 3 days,7 days and 14 days after injury.(2)The contents of energy metabolites ATP and ADP levels were significantly decreased from day1 post-injury,and further decreased with the continuous extension of injury time.NAD~+levels were significantly reduced at days 3,7 and 14 post-DAI versus the control rat.Compared with the DAI+SD group,KD-fed rats showed a robust increase in ATP,ADP and NAD~+levels from day 3 following DAI,while AMP,NADP~+and NADPH showed on statistically significant.(3)The results of LFB staining and MBP immunohistochemical staining showed that the average optical density of myelin in brainstem significantly reduced on day3 and day7 following DAI,which suggested myelin loss.MBP immunoblotting analysis further confirmed that the levels of MBP were deceased from day2 to week2compared to control group.By contrast,DAI rats fed with KD displayed significantly higher levels of LFB intensity and MBP expression compared with DAI+SD group.(4)APP was significantly up-regulated within 14 days after DAI,and reached the peak on the 3rd day.P-NF-H increased significantly at day 3,7 and 14 following DAI.By contrast,administration of KD resulted in a significant decrease in levels of APP and p-NF-H compared to with DAI+SD group.(5)The results of ultrastructural electron microscopy showed that the brainstem in control rats exhibited compact myelin sheath with regularly organized cytoskeleton included microtubules and filaments in the axon.Following DAI,rats fed with SD showed focal disorganization of the myelin sheath appeared on day 1 post-DAI.Myelin lamellae became delaminated,extended either outside or inside,and separated from the axolemma while the axonal cytoskeleton was generally organized by day 3 post-DAI.Myelin separated diffusely and disconnected locally and axon degenerated at day 7 post-DAI.At day 14 after injury,most myelin-axon unite collapsed or even dissolved and disappeared,and the remaining myelin sheaths were thinner.KD treatment ameliorated the myelin and axon degeneration,as evidenced by confined ultrastructural axonal damage and an overall increased mean g-ration revealed thicker myelin.In addition,the morphology of mitochondria was observed.In DAI+SD group,the number of mitochondria in axons significantly increased,with higher proportion of small and round mitochondria,while the rats in DAI+KD group showed a higher proportion of long tubular mitochondria.(6)Changes of mitochondrial mass:a significant increase of mitochondrial fission DRP1 expression was found at day 3 and 7 after DAI,but it was reversed by KD treatment.KD elevated the levels of mitochondrial fusion Mfn2 throughout the first 2 weeks after injury in the brain stem.The marker of mitochondrial biogenesis NRF1 expression was significantly increased throughout the first 2 weeks in DAI rats fed with SD,and increased mt TFA expression was only observed at day 3 after injury.Interestingly,KD in the DAI animals did not alter the protein expression of NRF1 or mt TFA.The above data suggested that ketogenic diet induces high ketogenic metabolism in DAI rats and significantly increases brain energy availability.Moreover,KD inhibits DAI-induced excessive mitochondrial fission and promote mitochondrial fusion,and protects the integrity of myelin-axon units.This study indicates a role of energy metabolism in the pathogenesis of DAI and provide insights into novel therapeutic strategies.