Gut Microbiota From Essential Tremor Patients Aggravate Tremor In Mice

Background and Objective:Essential tremor(ET)pertains to the concept of Tremor in Traditional Chinese Medicine and it is one of the most common movement disorders in adults.The disease is still lack of effective treatment since the mechanism is unclear.Gut microbiota is believed to play an important part in human physiology and pathology,and affect the occurrence and development of several neurological diseases,which include Parkinson’s Disease(PD)and Autism spectrum disorders(ASD).Our previous study found an ET patient who received Washed Microbiota Transplantation was unexpectedly relieved in her ET symptoms,which suggested a possible undiscovered link between gut microbiota and essential tremor.Thus,our research aim at investigating the effects and mechanism of the gut microbiota on the occurrence of essential tremor.Methods:Feces were collected from patients who meet the diagnostic criteria of essential tremor and Tremor in TCM,and also from their sex-matched and age-matched healthy controls.SPF(male C57BL/6J)mice were divided into 2 groups,both were gavaged three times a week for 3 weeks,one with mixed fecal suspension from 6 patients with ET,the other with mixed fecal suspension from 6 healthy controls.After gavage,feces from both groups of mice were collected for 16 S ribosomal DNA gene sequencing analysis.Fecal pellets count in 15 minutes of each mouse was also recorded.Initial behavioral tests,which include open-field test,pole test,grip strength test and rotarod test,were performed on all mice before modeling.After initial behavioral tests,each mouse was injected with 10mg/kg of Harmaline Hydrochloride Dihydrate to create models of essential tremor.The duration of tremor was recorded,and the scores of tremor was rated every 30 minutes.Behavioral tests were carried out in specific point in time.The extent of Qi,blood,Yin and Yang in each mouse were calculated by using the methods and formulas of mouse TCM syndrome science,then the syndrome differentiation of each mouse was obtained according to the syndrome differentiation standard,and finally the final syndrome differentiation result of each mouse was obtained comprehensively.At the same time,the extent of Qi,Blood,Yin and Yang in the two groups were also analyzed.After scoring and all behavioral tests,mice were all harvested.The length of large intestine was all measured,and intestinal tissues from small intestines and colons were collected for histology.The extent of inflammation,epithelial damage,edema,goblet cell loss,and mucosal hyperplasia was rated for colon tissues.Villus height,crypt depth and the villus height to crypt depth ratio was measured for small intestine tissues.The feces samples of mice were analyzed using 16 Sr DNA sequencing.ASV analysis was performed to calculate Alpha diversity and Beta diversity,significance test of community structure difference between groups and species analysis of significant difference between groups were performed,correlation analysis was performed between microbial community data and environmental factor data,and flora function prediction was performed using PICRUSRt2.Results:(1)Mice receiving fecal suspension from ET patients had worse performance in initial behavior tests,compared to mice receiving microbiota from healthy controls.(2)After Harmaline injection,an acute tremor was generated in all mice.We observed a significantly higher level of tremor(P <.05)at the 150 min timepoint and a significantly longer duration(P <0.01)in mice fed with fecal suspension from ET patients,as well as significantly worse motor performance(P < 0.05).(3)TCM syndrome evaluation showed that the degree of Qi and Yin in ET group mice was significantly lower than that in HC group mice(P<0.05).The degree of Blood and Yang shows a downward trend compared to that of HC group mice(P>0.05).(4)Mice fed with fecal suspension from ET patients also had significantly lower body weight and lower fecal pellets count in 15 minutes(P <.05).(5)Colons from mice fed with fecal suspension from ET patients showed higher level of inflammation(P<0.01),goblet cell loss(P<0.01),and mucosal hyperplasia(P<0.001)than mice fed with fecal suspension from controls.Small intestines from mice fed with fecal suspension from ET patients showed lower higher crypt depth(P<0.001)and lower villus height to crypt depth ratio(P<0.05).(6)16S ribosomal DNA gene sequencing analysis of feces from mice showed 2 groups of mice had different microbiota composition,which proved the effectiveness of bacterial flora transplantation.There was a significant correlation between Alpha diversity and phylum and genus level species and environmental factors,suggesting that the flora might be related to tremor score,duration and motor performance of mice.The results of functional prediction showed that in ET group the abundance of enzyme which synthesizes γ-aminobutyric acid was decreased,while the abundance of enzyme which metabolize γ-aminobutyric acid was increased.Conclusions:(1)We found feeding fecal suspension from ET patients alone can induce worse performance in behavioral tests,compared with feeding fecal suspension from healthy controls.(2)In Harmaline model of tremor,mice fed with fecal suspension from ET patients presented a longer tremor duration and higher tremor scoring as well as worse motor performance.(3)TCM syndrome evaluation showed that mice from the ET group have an overall higher degree of weakness compared with HC group.(4)Nutritional status and GI function of mice receiving fecal suspension from ET patients also altered.(5)Histology showed higher level abnormality in colons and decreased digestion and absorption capacity in small intestine from mice fed with fecal suspension from ET patients.(6)The composition and richness of intestinal flora were different between mice receiving ET flora and mice receiving healthy control flora.The microflora may be associated with the tremor score,duration and exercise schedule in mice.Changes in the abundance of some key enzymes involved in gamma-aminobutyric acid synthesis and metabolism may cause changes in gamma-aminobutyric acid and glutamic acid concentrations,which may account for the tremor and behavioral differences.This study is the first study to confirm the important role of gut microbiota in the occurrence and development of essential tremor through animal experiments.It provides new evidence for the connection between gut microbiota and central nervous system,and may hint a new treatment for ET patients.