The Experimental Study Of PD-like Pathology And Abberant Intestinal Microbiota In The ?-syn^+/-?A53T? Transgenic Mice Combined With Rotenone Treatment

Objective:Parkinson's disease is characterized by the massive loss of dopaminergic?DA?neurons in the substantia nigra?SN?of the midbrain and the aggregation of lewy body?LB?with synuclein as the main component.The patients of PD often show typical motor symptoms such as resting tremor,gait instability and muscle rigidity.Before the onset of motor symptoms,they are often accompanied by constipation,nausea,hyposmia,cognitive impairment and other non-motor symptoms.The etiology of PD have not been conclusive yet,and it may involve multiple factors such as gene,environment,and age.In this study,the?-syn^+/-?A53T?transgenic mice of different ages were intragastrically administrated with rotenone for two months to establish the different ages-h?-syn^+/-?A53T?transgenic-rotenone PD mouse model to study the combined effects of heredity,aging and environment on behavior,PD-like pathology in different brain areas and intestinal microbiota in mice.Methods1.Rotenone treatment.When the wild type mice and?-syn^+/-?A53T?transgenic mice were 3 months old,9 months old and 15 months old,they were intragastrically given rotenone for two months,5 days a week and 2 days at intervals.After rotenone treatment,the mice were fed for another month,then the behavioral test was performed and mice were sacrificed,small intestinal and brain tissues were collected.2.Behavior tests.The spontaneous activity of the mice was evaluated by open field test.The movement coordination and exercise ability were tested by pole descent.The fatigue wheel test was used to assess the exercise endurance.The olfactory test was used to evaluate the olfactory function.The three-box experiment was used to evaluate the social cognitive function.3.PD-like pathology in related brain areas.The numbers of DA neurons in the SNc,as well as the activation of microglia in SN,striatum and olfactory bulb?OB?were investigated by immunohistochemical staining.The expression of h?-syn expression in SN,striatum,and OB,the level of fibrotic protein and?-H₂AX positive particles in SN and OB were detected by immunofluorescence.The?-galactosidase staining was used to evaluate the aging of SN nerve cells and OB mitral cells.The protein expression of P16 and?-H₂AX were observed by Western blot.4.Changes in intestinal microbiota and function.The fecal total DNA was extracted and the number of beneficial bacteria and harmful bacteria were detected by real time RT-PCR.Water content of fecal pellets was detected by measuring the dry and wet weight,and the level of FITC-dextran4 in serum was detected to evaluate the change in intestinal mucosal permeability.5.Changes in Peyer patch's lymphocyte subtypes.Lymphocyte were extracted from Peyer patch,and lymphocyte subsets were detected by flow cytometry.Results1.Assessment of PD related motor behaviors in?-syn^+/-?A53T?transgenic mice with retenone treatment of different ages.Compared with wild type?WT?group,wild type treated with rotenone?WT+R?group and transgenic?h?-syn?group,transgenic combined with rotenone?h?-syn+R?group mice exhibited a distinct decline in motor function.18-month-old h?-syn+R group mice displayed more significant decrease in spontaneous activity,motor coordination,and exercise endurance,as well as olfactory dysfunction and recognition dysfunction.2.Rotenone induced PD-like pathology in related brain regions of?-syn^+/-?A53T?transgenic mice of different ages.Compared with WT group,WT+R group and h?-syn group,h?-syn+R group mice related brain regions appear PD-like pathology.18-month-old h?-syn+R mice showed more significant PD-like pathology,and the number of DA neurons in SN was significantly reduced;the expression of h?-syn in the SN,striatum,and OB and the level of fibrotic proteins in the SN and OB in18-month-old h?-syn+R mice were increased.3.Rotenone induced the senescence of nerve cells in related brain regions of?-syn^+/-?A53T?transgenic mice of different ages.Compared with WT group,WT+R group and h?-syn group,the aging of nerve cells in SN and OB of h?-syn+R mice increased.18-month-old h?-syn+R mice showed the number of aging SN nerve cells significantly increased,and?-H₂AX positive particles in SN increased.The aging mitral cells significantly increased,the protein expression of P16 and?-H₂AX increased in OB.4.Rotenone induced inflammation in related brain regions of?-syn^+/-?A53T?transgenic mice of different ages.Compared with WT group,WT+R group and h?-syn group,the activation of microglia in SN,striatum and OB increased.18-month-old h?-syn+R mice showed more significant activation of microglia.5.Rotenone induced abberant intestinal microbiota changes in?-syn^+/-?A53T?transgenic mice of different ages.Compared with WT group,WT+R group and h?-syn group,h?-syn+R group showed the number of beneficial bacteria decreased,while the harmful bacteria increased.18-month-old h?-syn+R mice showed lower numbers of beneficial bacteria and higher numbers of harmful bacteria.6.Rotenone-induced the changes in Peyer patch's lymphocyte subtypes of?-syn^+/-?A53T?transgenic mice of different ages.6-month-old h?-syn+R group showed a decrease in the number of CD4⁺T lymphocytes,CD8⁺T lymphocytes,CD19⁺B lymphocytes and CD4⁺CD25⁺Treg cells,while 12-month-old h?-syn+R group showed different degrees of increase.7.Rotenone-induced changes in intestinal function of?-syn^+/-?A53T?transgenic mice of different ages.Compared with WT group,WT+R group and h?-syn group,the h?-syn+R mice showed water content of fecal pellets decreased,intestinal mucosa permeability increased and intestinal dysfunction.18-month-old h?-syn+R mice showed more prominent intestinal dysfunction.Conclusions1.Rotenone induces PD-like motor and non-motor behavior changes in?-syn^+/-?A53T?transgenic mice of different ages.The PD-like motor and nonmotor behavior changes were more significant with the increase of age.2.Rotenone induces decreased the DA neurons in SN,high expression of h?-syn in brain,aging of nerve cells and increased microglia activation in transgenic mice of different ages,with typical age dependence.3.Rotenone induces the dysregulation of intestinal microbiota,the changes of small intestinal lymphocytes subtypes,and small intestinal dysfunction in?-syn^+/-?A53T?transgenic mice of different ages,with obvious age dependence.