Ketogenic Diet Enhances Neuroplasticity After Stroke By Modifying Intestinal Flora

Objective: At present,there are few treatment methods for stroke,and the most effective method is acute thrombolytic therapy and intravascular thrombectomy.However,due to the limitation of time window,many patients will leave neurological defects,which will greatly reduce the quality of life of patients and bring great harm to the family and even the society.Therefore,it is necessary to actively seek a treatment method beyond the time window.Stroke not only causes direct damage to the brain,but also affects various organs including gastrointestinal tract.There are many kinds of microorganisms living in the gastrointestinal tract,which play an important role in maintaining the intestinal environment stability and immune regulation.Currently,more and more studies have shown that gut microbes can change after stroke,and may even make the intestinal epithelial barrier integrity was broken,the intestinal immune cells into the peripheral blood,and through the damage of blood brain barrier into the central nervous system,increase intracranial inflammation after stroke and neurological deterioration,and gut microbes change also will increase the risk of stroke events.Therefore,gut microbes could be a potential target for stroke treatment.Ketogenic diet is a diet of low carbohydrate,high fat and appropriate proportion of protein,which is widely used in neurological diseases,most commonly refractory epilepsy,and has also been studied in neurodegenerative diseases.Compared with glucose for energy,ketone bodies require less oxygen to produce the same energy,and neurons,glial cells and other cells can fully use ketone bodies,which is now widely used in neurological diseases.On the one hand,stroke is due to atherosclerosis caused by vascular stenosis,resulting in ischemia and hypoxia of brain cells.In this environment,glucose will undergo anaerobic glycolysis to produce a large amount of lactic acid,further aggravating the damage,and the ketone body only needs a small amount of oxygen to produce a large amount of energy.On the other hand,the ketogenic diet can affect the intestinal immunity by changing the intestinal flora,and ultimately has an impact on intracranial inflammation.In this study,we conducted ketogenic diet intervention in rats for 8 weeks to detect its effects on intestinal microflora composition and metabolites,and to observe its effects on neuroplasticity(neurogenesis and axonal regeneration)and neurological function recovery after stroke,so as to explore the possible molecular mechanism.The purpose of this study was to explore the internal relationship and related mechanism of ketogenic diet by altering intestinal flora,intestinal immunity and neurological recovery,and to reveal that intestinal flora can be a potential target for stroke treatment.In conclusion,our data point to a novel and highly complex phenomenon of interaction between the brain and gut microbiota,in which the microbiota is an intermediate link that influences stroke outcomes by modulating immunity.Methods:1.During the experiment,rats were divided into the following four intervention groups: Normal Diet + Sham group(ND+SHAM group),Normal Diet + Ischemia group(ND+ISC group),Ketogenic Diet +SHAM group(KD+SHAM group)and ketogenic Diet + Ischemia group(KD+ISC group).2.Firstly,16 s RNA sequencing was used to identify the microflora of intestinal contents of rats in each intervention group.3.The rats in each intervention group were tested for intestinal microbe untargeted metabolism to determine the most significant differential metabolites and metabolic pathways of ketogenic diet.4.Neurological function recovery of rats were evaluated using common behavioral tests,namely the trapezoidal balance beam test and the cylinder test.5.TTC staining was used to observe the infarct volume of cerebral infarction model rats.6.Survival analysis was used to evaluate the survival rate of ketogenic diet and normal diet after MCAO operation.7.The effect of ketogenic diet on weight loss was evaluated by measuring the weight of rats in each intervention group every 3 days.8.The corticospinal tract was sequentially labeled by injection of Biotinylated Dextran Amine(BDA)into the cerebral motor cortex,and 5 Ethynyl 2 ’deoxy uridine,The regeneration of axons in cervical enlargement plane and neurogenesis of lateral ventricle in rats after ischemia were observed by immunofluorescence staining with EDU labeled new proliferating cells.9.As for the molecular mechanism involved in ketogenic diet promoting axonal regeneration and neurogenesis after ischemia in rats,we used GEO database data to conduct bioinformation analysis,and then obtained metabolic pathways with significant differences by KEGG pathway enrichment analysis.10.The inflammatory indexes of IL-17 and TGF-β in peripheral blood were detected by ELISA.11.Finally,RT-q PCR was used to detect the peripheral tissues of cerebral infarction in rats,and the detection indexes included IL-17α,IFN-γ,IL-10,IL-2,IL-33,TGF-β,IL-4,IL-6,CCL1,CCL3,CCL4,CCL5,CCL6,CCL9,TNF-α,etc.to indirectly reflect the ratio of Th17 to Treg cells during ischemia.Results: Intestinal contents were sequenced using 16 s RNA technology.The results showed that the total OTU of rat microorganisms in KD+ISC group was significantly lower than that in ND+SHAM group(P<0.001),and also lower than that in ND+ISC group(P<0.01).The abundance and α diversity of bacteria in KD+ISC group were decreased.In terms of microbial composition,the relative abundance of Firmicutes in ND+ISC group was higher than that in the other two groups,but there was no statistical difference.The relative abundance of Proteobacteria in KD+ISC group was significantly higher than that in the other two groups.The relative abundance of Bacteroides decreased compared with the other two groups.After ketogenic diet treatment,many different species were found at both genus level and species level.The analysis of different species found that the upregulation of Lactobacillus in Firmicutes was significant in ND+ISC group compared with ND+SHAM group.KD+ISC group compared with ND+ISC group,there were significant differences in four gate levels.Significantly upregulated Odoribacter,Adlercreutzia,Prevotella,Parabacteroides,Bacteroides,Alistipes(another clade),Oscillospira(Fibrillation)of Firmicutes,and Sutterella(Proteobacteria)of Proteobacteria,which have been found to be closely associated with reducing intestinal inflammation.The results of intestinal flora metabolism showed that the inosine content of microbial differential metabolites increased significantly after ketogenic diet,and the biosynthesis and purine metabolism of linoleic acid,pantothenic acid and Co A were significantly affected.During the MCAO model preparation of each intervention group,it was found that the survival rate of rats in the ketogenic diet group was significantly increased,and their body weight was significantly decreased compared with that in the normal diet group.It was found that the rats in the KD+ISC group had a good performance in neurological function recovery,and the balance beam footstep error rate was smaller,the use frequency of both upper limbs was more frequent,and the voluntary movement was increased.Immunofluorescence staining showed that BDA-labeled axon regeneration and EDU/DCX labeled neurogenesis in KD+ISC group were significantly higher than those in ND+ISC group.Bioassay of peripheral blood GEO database suggested that the changes in peripheral blood after stroke were related to IL-17 signaling pathway.Subsequent Elisa and RT-QPCR results showed that the IL-17 content in peripheral blood of ND+ISC group was increased and TGF-βcontent was decreased,while the KD+ISC group was on the contrary.In ND+ISC group,IL-17 α secreted by Th17 was increased,IFN-γ secreted by Th1 was increased,IL-10,IL-33 and TGF-β secreted by Treg were decreased,while the results obtained in KD+ISC group were opposite,suggesting that T cells were involved in the process of ketogenic diet promoting the recovery of neurological function.Conclusion:1.Ketogenic diet can modify intestinal microflora,resulting in a decrease in the abundance and diversity of intestinal microflora,and the composition of species was significantly different from that of the control group.The analysis of different species found that the content of many bacteria that inhibit intestinal inflammation was significantly increased.2.After ketogenic diet,inosine content in microbial metabolites increased significantly,and linoleic acid metabolism and Co A biosynthesis pathway were significantly affected.3.Ketogenic diet promoted axonal regeneration of corticospinal tract and neurogenesis of lateral subventricular region after cerebral ischemia in rats.4.Ketogenic diet can promote the recovery of neurological function after cerebral ischemia in rats.5.Ketogenic diet promoted the recovery of nerve function after cerebral ischemia by enhancing the expression of anti-inflammatory cytokines and inhibiting the expression of pro-inflammatory cytokines.