Studying The Effects Of Exogenous β-hydroxybutyrate On Anti-epilepsy

Epilepsy is a group of neurological disorders characterized by recurrent epileptic seizures,seriously affecting the quality of human life.In China,the morbidity of epilepsy is about 3.6%-4.4%.Especially,the children and adolescents under the age of 16 are easily affected.Although a variety of anti-epileptogenic drugs have been used to treat the epilepsy,-30%epilepsy is still difficult to be controlled with drugs.Therefore,it is essential for exploring other effective treatments.Currently,the ketogenic diet(KD)has been widely applied to treat epilepsy.However,the mechanisms of KD on epilepsy remain unclear.Most studies suggested that beta-hydroxybutyrate(BHB)was an ideal indicator for monitoring the anti-epileptogenic effect of KD,and may be the main substance of the anti-epileptogenic effects of KD.BHB is the most important component of the ketone bodies,and its neuroprotective and anti-epileptogenic effects have been focused by researchers.Previous studies demonstrated that the application of exogenous BHB had neuroprotective effects on some epileptic models and other neurological diseases.The mechanisms of BHB on neuroprotective and anti-epileptogenic effects mainly inculded:1)the regulation of neurotransmitter concention in central nervous system;2)the fluence on energy metabolism and neuronal membrane potential;3)the inhibition of apoptosis and oxidative damage;4)the reduction of calcium ions flow.Taking into account the limitation of the KD application on clinical treatment,exogenous BHB may replace KD as a novel treatment for refractory epilepsy and other neurological diseases.In the present study,we aimed to explore the anti-epileptogenic effects of exogenous BHB from the following three parts.Part 1 The effects of exogenous β-hydroxybutyrate on the contents ofβ-hydroxybutyrate and blood glucose in vivoObjective:The aim of this study was to explore the effects of exogenousβ-hydroxybutyrate(BHB)on the contents of BHB and blood glucose in vivo.Methods:1.A total of 32 male Wistar rats were randomly divided into four groups:A-C,which rats were treated with 2,4 and 8 mmol/kg BHB(n = 9 in each group);D,which rats were treated with 4 ml/kg normal saline(NS)(n = 5).2.At 0,15,45,75 and 90 min after BHB administration,～1 ml blood was collected from angular veins of the rats under ether anesthesia.3.The concentration of BHB in the serum was detected using a BHB assay kit according to the manufacturer’s protocol.4.A total of 20 rats were used to detect the glucose concentration,treated with either 4 mmol/kg of BHB or 4 ml/kg of NS(n = 10 in each group).5.At 0,15,45,75 and 90 min after BHB administration,peripheral blood was collected from rat tail,then glucose concentration was tested using a glucose meter.6.Statistical analyses were performed utilizing SPSS software 20.0.Results:1.Exogenous BHB administration significantly increases the concentration of BHB in the blood.Prior to BHB administration,the concentration of BHB in the blood was-0.57±0.01 mmol/l(there were no differences among each group).The concentration of BHB in the blood increased to 1.35-2.37 mol/l after BHB administration for 15 min,and this level was maintained for the next 75 min.Compared with the control group,BHB concentrations were significantly higher in the BHB groups(p<0.05)at all time points.There was no significant difference in the blood BHB concentration between the rats administrated with 2,4,or 8 mmol/kg BHB at 15 min;however,at 45,75,and 90 min,there were significant differences in the blood concentration between the rats administrated with 2,4,or 8 mmol/kg BHB.In addition,it was observed that the BHB concentration in the blood was relatively stable at 1-2 mmol/1 after the rats were administrated 4 mmol/kg BHB,which was most similar to the BHB level after rats had been on KD.There were significant differences among different times in 4 mmol/kg BHB group compared with the control group(p<0.05).2.Exogenous BHB administration has no significant effect on blood glucose.The concentration of glucose in the blood initially increased after BHB administration for 15 min and then decreased in the subsequent next 75 min.However,there were no significant differences for glucose concentrations in the BHB groups compared with the control group(p>0.05).Additionally,there were also no significant differences for the glucose concentration among different times in each group(p>0.05).Conclusions:1.Exogenous BHB administration significantly increases the concentration of BHB in the blood.2.The BHB concentration in the blood increased to the level which was most similar to the BHB levels after rats had been on KD or starved when the rats were administrated 4 mmol/kg BHB.3.Intraperitoneal injection of BHB has no significant effect on the blood glucose concentration of rats.Part 2 The protective effect of exogenous P-hydroxybutyrate on kainic acid-induced epilepsyObjective:The aim of this study was to investigate the protective effect of exogenous BHB on kainic acid(KA)-induced epilepsy.Methods:1.The establishment of KA-induced seizure model:A total of 50 rats received intraperitoneal injection of KA.The seizure behavior of animals was then analyzed after KA injection for 2 h according to the scale provided by Racine:Stage I,facial clonus;Stage Ⅱ,head nodding and wet dog shaking;Stage Ⅲ,forelimb clonus;Stage IV,forelimb with rearing;Stage V,rearing,jumping and falling.Then 10%chloral hydrate(400 mg/kg)was administered intraperitoneally to stop seizure behavior if the status epilepticus continued over 90 min.2.A total of 50 Wistar rats were randomly selected and divided into BHB+KA and NS+KA groups(n = 25 each group).Rats in the BHB+KA group were administered with 4 mmol/kg BHB intraperitoneally 30 min prior to KA injection.Rats in the NS+KA group were administered with 4ml/kg NS intraperitoneally prior to KA injection,and this group was used as a control.3.The onset time of stage IV or V of seizure behaviors were recorded and calculated.4.The stage IV or V degree of seizure behaviors were recorded for 2 h following KA administration.5.Statistical analyses were performed using SPSS software 20.0.Results:1.Exogenous BHB administration significantly affected the onset time of seizure.There were no significant differences for the weight of rats between the NS+KA(54.61±0.78 g)and BHB+KA(54.80±2.12 g)groups.The onset time of seizure in the BHB+KA group was 63.31±4.050 min,which was significantly longer than that of the NS+KA group(37.08±1.958 min)(p<0.05).2.Degree of seizure behavior did not differ significantly after rats treated with exogenous BHB.There was no abnormal seizure behavior for the rats treated with the BHB or normal saline solely,while seizure behaviors occurred in the rats treated with KA.The average degree of seizure behavior in the BHB+KA group was 4.54±0.100,which was slightly lower than that of the NS+KA group(4.671±0.098).However,this difference was not significant(p>0.05).Conclusion:Exogenous BHB administration prolonged the onset time of seizure induced by KA,but could not reduce the degree of seizure behavior.Part 3 The antiepileptic effects of exogenous β-hydroxybutyrate on kainic acid-induced epilepsyObjective:The aim of the current study was to explore the antiepileptic effects of exogenous BHB on KA-induced epilepsy.Methods:1.Wistar rats were randomly divided into BHB+KA(n = 25),BHB(n = 20),NS+KA(n = 25),and NS(n = 20)groups.Rats in the BHB and NS groups were administered with 4 mmol/kg BHB and 4 ml/kg NS,respectively.Rats in the BHB+KA group were administered with 4 mmol/kg BHB and 10 mg/kg KA.Rats in the NS+KA group were administered with 4 ml/kg NS and 10 mg/kg KA.2.Rats were sacrificed and their skulls were then immediately cut open at 1,3,7,and 14 days following KA administration.Then the left hemisphere was used for preparing the paraffin sections,while the right hemisphere was used for obtaining the hippocampus tissues.The hippocampus tissues were immediately stored at-80℃.3.The Nissl staining was performed to observe neuronal loss and damage in the hippocampus tissues using the Nissl staining kit.4.The Timm staining was performed to observe mossy fiber sprouting(MFS)in the hippocampus tissues.5.The glutathione(GSH)content in the hippocampus tissues was detected using the GSH ELISA assay kit.6.The γ-aminobutyric acid(GABA)content in the hippocampus tissues was detected using the GABA ELISA assay kit.7.The adenosine triphosphate(ATP)content in the hippocampus tissues was detected using the ATP ELISA assay kit.8.The expression of neuro-specific enolase(NSE)and glial fibrillary acidic protein(GFAP)were detected by double immunofluorescence labeling method.9.Statistical analyses were performed using SPSS software 20.0.Results:1.Neuronal damage in the hippocampus was alleviated in a BHB-pretreated KA-induced seizure model.At the 3rd day after the KA injection in rats,rhe hippocampus tissues were stained using Timm staining method.The results The results of Nissl staining demonstrated that there was no neuronal loss in the hippocampus in NS-and BHB-treated rats.However,typical neuronal loss and damage in the CA1 and CA3 region were found in the KA-induced seizure model that did not receive BHB pretreatment.Moreover,neuronal loss was attenuated in the BHB+KA group compared with the NS+KA group.Furthermore,the content of GSH and GABA in the BHB+KA group was significantly increased compared with the NS+KA group.These results indicated that the BHB application alleviated the neuronal damage of rats.2.MFS was alleviated in a BHB-pretreated KA-induced seizure model.At the 14th day after the KA injection in rats,the hippocampus tissues were stained using TTimm staining method.The results demonstrated that the average Timm score in NS+KA group was significantly higher than that in the NS,BHB,and BHB+KA groups(p<0.05)Furthermore,there was no significant difference in the average Timm score between the NS(0.13±0.06)and BHB groups(0.23±0.06,p>0.05).Compared with the NS+KA group(3.67±0.15),the average Timm score of the BHB+KA group(1.5±0.50)was significantly decreased(p<0.05),indicating that MFS was alleviated in the KA-induced seizure model group receiving BHB pretreatment.3.BHB may reduce the damage of neurons caused by KA and promote the compensatory hyperplasia of astrocytes.At the 3rd and 7th days after the KA injection in rats,the neuron amounts were reduced in the NS+KA group compared with the NS group,while the astrocytes amounts were significantly increased.Besides,the results showed that the neuron amounts were increased in the BHB+KA group compared with the NS+KA group,while the change of astrocytes amounts were decreased.These results indicated that BHB showed a protective role for the KA-induced rats through alleviating the neuron damage and the compensatory hyperplasia of astrocytes.Conclusions:Neuronal damage in the hippocampus tissues were alleviated after the application of BHB.