Study On Ultrasound Imaging Features And Mechanisms Of Parkinson’s Disease Rat Model

Parkinson’s disease(PD)is an age-related neurodegenerative disease with complex,multifactorial etiologies.Its pathological characters can be summarized as the progressive loss of dopaminergic neurons in the midbrain and the denervation of the nigrostriatal pathway.As 1.7 million people in China suffer from PD,prevalence of PD in China is similar to the one in developed countries.And there are difficulties in early diagnosis of PD because the symptoms may not be typical in the early stage.Since Becker et al.discovered the relationship between substantia nigra hyperechoic(SNH)PD,the application of transcranial sonography(TCS)has been gradually accepted.SNH is characteristic in 70% to 98% of PD patients and is independent of the severity of the motor symptoms or the stage of the disease.Studies have also reported that PD patients may exhibit large SNH areas at early stage and do not change during disease progression.SNH can be seen on the brain stem planes in the axial scanning of TCS,and the shape of the hyperechogenicity is usually patchy or banded.Grading of SNH is a semi-quantitative method based on comparison with surrounding tissue on midbrain planes: SNH is divided into isoechogenic,mildly echogenic or hyperechogenic contrast to brainstem.Not only that,the rating is also accurate and reliable in combination with the measurement results of the SNH area,and there is almost no difference between the measurers.Previous research results proposed an optimal cutoff value of 0.24cm~2,using the Siemens Antares system(Siemens,Germany).Berg,D,etc.used Sonoline Elegra(Siemens,Germany)to rate the size of the echogenic area.The size of SNH area between0.20 and 0.25 cm~2 was divided into moderate increase,and the size above 0.25 cm~2 was divided into significant increase.SNH related researched were limited due to the difficulty of obtaining living specimens.This study turned to laboratory animal disease models,and aimed to establish a 6-OHDA-induced unilateral PD model which is widely used in PD research to observe if the typical SNH emerged under the TCS.Through this research,we evaluated the application value of the model in TCS related research,explored the relationship between SNH and PD,provided a new medium for SNH related research.Due to the mechanism of SNH formation is not clear,we aimed to provide a theoretical basis for the value of TCS in diagnosis of PD by exploring SNH formation mechanism.The main research methods and results are as follows:1.Under the TCS,the 6-OHDA-constructed rat unlateral PD model showed characteristic SNH in substantia nigra.We constructed a rat unlateral PD model by stereotactic injection.Rats were divided into model group(6-OHDA injection)and vehicle group(vehicle injection).TCS detection was performed 15 days after the operation.Vevo2100 was used for the examination.The TCS images were compared with the rat brain atlas,and the striatum plane,thalamus plane,midbrain plane,and fourth ventricle plane were located to find the midbrain substantia nigra(SN).The results showed that the echogenicity of the ipsilateral SN of the model group rats was significantly enlarged,which was similar to the SNH observed in PD patients.This result provides evidence to the application of 6-OHDA stereotactic injection PD model in SNH-related studies.2.The survival of dopamine(DA)neurons in SN is correlated to the SNH.In order to explore the relationship between SNH and dopaminergic neuron survival,we divided the rats into three groups.We applied neuroprotective drug Nitidine to restrain neurodegeneration in 6-OHDA-induced PD model rats which named Nitidine group despite the model group and the vehicle group.TCS examination was performed after15-days’ treatment.Immunofluorescence staining was performed to observe the survival of dopaminergic neurons in the substantia nigra,and the relationship between the dopaminergic neurons and the sizes of SNH area and gray values was analyzed statistically.The results showed that the SNH area was negatively correlated with the number of surviving dopaminergic neurons and the OD value of TH-ir dopaminergic neurons,and the gray value of the SN region was negatively correlated with the OD value(no significant correlation with the number of surviving neurons).These result suggest the value of TCS in assessing the survival of PD dopaminergic neurons,even disease progression.3.Microglial activation might contribute to SNH formation.Since many researches have reported the importance glial activation in neurodegenerative progression,in this study we explored the role of glial activation in SNH formation.Immunofluorescence staining and Western blot were used to observe the activation of microglia in the c substantia nigra of the rats with SNH.And after nitidine treatment,both microglia activation and the size of SNH were limited.Result also showed that the average branch length decreases compared with vehicle group,but the number of branches showed no significant changes.Combining with previous researches which reported that SNH formation was related to iron deposition in the SN and that microglia obtained higher iron content,our result indicate that microglial activation might contributed to SNH formation.