Ketogenic Metabolism Inhibits Histone Deacetylase Of Spinal Cord Tissues And Reduces Oxidative Stress After Acute Spinal Cord Injury

Ketogenic diet (KD) with high fat and low carbohydrate, or calorie restriction in the form of every-other-day fasting (EODF) resulted in ketone body metabolism and increase β-hydroxybutyrate (βOHB) level. Previous studies suggested that the ketone body metabolism is neuroprotective to many neurodegenerative diseases and acute neurotrauma models, and has been used in clinic as an effective therapy, particularly for some childhood epilepsies that are resistant to anticonvulsant medications. Previous studies discovered that KD or EODF improved outcomes after an incomplete cervical spinal cord contusion injury. Exciting new data show that βOHB is an endogenous histone deacetylase inhibitor, resulting in increase of gene expression of oxidative stress resistance in kidney. However, this effect of POHB in the spinal cord tissue has not been verified. This study is the first to compare the level of βOHB in serum with the level in CSF, and analysis that the effect of βOHB on inhibiting the histone deacetylation and oxidative stress resistance after spinal cord injury.Part I P hydroxybutyrate levels in serum and cerebrospinal fluid under ketone body metabolismsl.Backgrounds and ObjectivesBiochemical markers identified in the drained CSF are used to evaluate the treatments. β-OHB, the main component of ketone bodies, is carried by several monocarboxylic acid transporter (MCT) across the blood-brain barrier, and used as an energy source by the brain and spinal cord during ketone body metabolism. β-OHB level in the CSF may be affected by MCT upregulation or inhibitation. However, POHB levels in the cerebrospinal fluid (CSF) under ketogentic metabolism remain unkown. The objective of the present study was to measure POHB levels under standard diet, KD, EODF and EODKD, and compareβOHB levels among diets and between the serum and CSF2. MethodsTwenty-four male Sprague-Dawley rats were randomly divided into 4 groups fed with KD, EODF, EODKD and standard diet (SD) up to 2 weeks, respectively. A customized food with a ratio of 1:4 of carbohydrates to fats was used in the KD and EODKD groups. Samples of 200μl serum and 100μl CSF were collected in the end of the study, particularly on the fasting day for the EODF and EODKD groups. POHB levels were measured using ELISA kits. All groups had ad libitum supply of water. The control group had ad libitum standard food access while the KD group had ad libitum ketogenic food access. In the EODF group, there was no access to food (fasting) during the first 24 h, but access to food ad libitum on the alternating days (24-48 h,72-96 h,120-144 h). In the EODKD group, rats were fed with the same schedule as the EODF group, but the same food as the KD group.Food intake and body weight were daily monitored.3. ResultsThe serum βOHB levels in experimental groups were significantly higher than that in the SD group(62 μmol/L) (P<0.05).Particularly,The βOHB levels in the EODKD was the highest(161 μmol/L), and higher than the KD group (89 μmol/L) and EODF group (86 μmol/L) (P<0.05).. CSF POHB levels were averagely 69% of the serum βOHB levels There was a positive correlation between them (r=0.817, P=0.000). In addition, the diet intervention groups did not result in decrease of the body weight, but the standard diet led to increase of the body weight.4. ConclusionsThis study demonstrated KD, EODF and EODKD resulted in ketone body metabolism as POHB levels increased significantly compared to the standard diet. The CSF POHB level was lower than the serum βOHB level, which possibly due to the metabolism of central nervous system and blood-brain barrier. Our results suggested that the serum βOHB level was an indicator of the CSF βOHB level, paving a base for neuroprotection of ketone body metabolisms.Part II Effect of ketone body metabolism on histone acetylation and oxidative stress at rat in the spinal cord1. Backgrounds and ObjectivesPrevious studies suggested that the ketone body metabolism could ameliorate neurological disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, traumatic brain injury, sleep disorders, brain tumors, autism, multiple sclerosis, and has been successfully used for treatment of child-resistant epilepsy. In recent years, the Tetzlaff laboratory at University of British Columbia using two dietary interventions treat the acute contusive spinal cord injury in rats,and observed a significant neuroprotection in promoting the functional recovery of damaged limbs. Firstly, they used a way of CR-EODF to treat the incomplete cervical or thoracic spinal cord contusive injury, and observed a neuroprotection in motor function improvement. Because the CR-EODF clinic caused weight loess or the digestive disorders. they explored the ketogenic diet (KD, Ketogenic Diet), a similar physiological effects to the CR-EODF. KD was applied for the rat at 4 hours after the incomplete cervical spinal cord injury, and motor function recovery was improved signigicantly. Even though, many studies have shown CR and KD a clearly neuroprotection, the mechanism is still unknow. Exciting new data showed that the β-hydroxybutyrate was an endogenous histone deacetylase inhibitor (HDAC), resulting in an increase of widely histone acetylation and FOXO3A and MT2 gene expression of oxidative stress resistance in kidney. However, the effect of β-hydroxybutyrate to inhibit the histone deacetylase in the spinal cord tissue has not been investigated. The objective of the present study was to demonstrate the effect of βOHB on promoting the histone acetylation and improving the capability of ant-oxidative stress in spinal cord tissues under KD, EODF and EODKD, in order to study their mechanisms of neuroprotection in spinal cord.2. MethodsTwenty-four male Sprague-Dawley rats were randomly divided into 4 groups (n=6) fed with KD, EODF, EODKD and standard diet (SD) up to 2 weeks. with a ratio of carbohydrates to fats of the KD cookie used in the KD and EODKD groups is 1:4. The C5 spinal cord samples were harvested on the fasting day, tissue homogenates for HDACs activity assay, western bolt to detect the expression of the Histone acetylation protein (AcH3K9,ACH3K14) and the anti-oxidative stress protein (MN-SOD,FOXO3A,Catalase). Levels of anti-oxidative stress genes Foxo3a and MT2 was detected by QPCR.3. ResultsCompared to dietary intervention groups or standard diet (SD), the dietary intervention animals displayed significantly increased POHB levels in serum and CSF. HDACs activity from the experimental groups was significantly inhibited, and was decreased 31%-43%.the expression of AcH3K9, AcH3K14, markers of histone acetylation levels, the dietary intervention groups were significantly increasing. Moreover, the anti-oxidative stress-related proteins including Mn-SOD, FOXO3A, Catalase, have the similar trend. Levels of anti-oxidative stress genes Foxo3a and MT2 was also increasing by responsed to βOHB.4. ConclusionsWe demonstrated for the first time that the βOHB as a product of the metabolism of ketone bodies,which could inhibition of histone deacetylase (HDAC) activity, increasing histone acetylation in spinal cord and increasing anti-oxidative stress genes expression,playing a role of anti-oxidative stress,and pave a foundation for neuroprotective mechanisms of ketone body metabolism.Part Ⅲ Neuroprotective effects of Pre-conditions ketobodies metabolism in rats after acute spinal cord injury1. Backgrounds and ObjectivesSpinal cord injury (SCI) is a serious health problem whit high care cost on families and society. However, in currenty, there is no universally accepted and effective method for treating SCI in clinic. In the acute phase, besides maintaining the vital signs of patient, and orthopedic surgical decompression and stabilization of the spine, the more common way is to give methylprednisolone (MP) shocking treatment within 8 hours after injury, by reducing inflammation in order to achieve neuroprotection.But It’s very difficult to applicate, because many patients often out of the MP treatment time window. Moreover, MP effection is not certain and have a huge side effects. So, more and more researchers to explore a new way to treatment the spinal cord injury. ketogenic diet (KD) defined as a high fat and low carbohydrate diet resulted in ketone body metabolism and increase β-hydroxybutyrate (βOHB) level in blood. KD have been long recognized to ameliorate neurological disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, traumatic brain injury, sleep disorders, brain tumors, autism, multiple sclerosis, and has been successfully used for treatment of Child-resistant epilepsy. Previously study have shown that EODF or KD improves neurological recovery in rats following cervical and thoracic SCI. But even given KD immediately after spinal cord injury, the body’s metabolism of glucose converted into ketone bodies at least 24h. Therefore, if the ketone body metabolism body can inhibit oxidative stress and protect the spinal cord nerve cells is still a lack of research, is still needed to understand the neuroprotective effect of per-condition ketone body metabolism after spinal cord injury. The present study was aimed to investigate effects of prophylactic ketobodies metabolism on anti-oxidant and anti-apoptotic after acute spinal cord in rats.2. MethodsSevern-two male Sprague-Dawley rats were randomly assigned into 4 groups fed with KD,EODF,EODKD and standard diet (SD)(n=18) for up to 2 weeks. Levels of blood ketones was measured for each rat at 2 weeks. All rats were subjected to a 1.5 mm contusion injury at 500 mm/s on the C5 spinal cord using an electromagnetic-servo material testing machine. Samples of spinal cord tissues were harvested at 3h and 24h after injury. Protein expression of Bax, caspase-3, Bcl-2 and CD68, indicating apoptosis and inflammation, were tested using double immunofluorescence. Contents of superoxide dismutase (SOD) and malondialdehyde (MDA), reflecting oxidative stress, were measured using ELISA.3. ResultsExpression of Bax, Bcl-2,caspase-3 and CD68 were positive after injury. A significant difference in these expression between groups was seen at 3h and 24h after injury. The content of SOD in the KD group was higher than that in the SD group at 3h after injury, and there was a significant difference between groups at 24h (281 pg/mL) (P<0.05). Change of levels of MDA was opposite to the change of SOD. The level of MDA in the Dietary intervention groups was significantly lower than the level in the SD group (855 pg/mL) (P<0.05).4. ConclusionsThe present results indicated that a pre-exist high level of blood ketones reduce oxidative stress and apoptosis in the spinal cord after acute contusion injury, suggesting neuroprotective for acute spinal cord injury under ketobodies metabolism.