Experimental Study Of Substantia Nigra Hyperechogenicity Formation Mechanism In 6-OHDA-induced Rat Model Of Parkinson's Disease

Background and PurposeParkinson's disease(PD)is a severe neurodegenerative disease that mainly affects elderly individuals,and that is primarily characterized by progressive degeneration of the dopaminergic neurons in substantia nigra(SN).Around 90% of PD patients show substantia nigra hyperechogenicity(SNH)on transcranial sonography(TCS).This study aims to explain the mechanisms for the formation of sonographic features of PD using a 6-hydroxydopamine(6-OHDA)rat model of PD.TCS was performed to observe the SN echo signal of the brain.Immunofluorescence and specific iron staining were performed to observe the histological characteristics of the hyperechogenic area.The imaging findings were compared with the histopathological findings.Methods1.Rat models were created using stereotactic injections of 6-OHDA.TCS was performed to observe the SN echo signal of the brain,then immunohistochemical staining and Western-Blot experiments were performed to test whether PD model rats appeared PD characteristic pathological change,that is degeneration of the dopaminergic(DA)neurons in SN.2.Immunofluorescence and specific iron staining were performed to observe the histological characteristics of the hyperechogenic area in SN.The imaging findings were compared with the histopathological findings to clarify the SNH formation mechanism of PD model rats.Total iron content in SN of brain tissue was also measured to investigate the relationship between the SNH and iron.3.Iron chelator deferiprone(DFP)was used to intervene the PD model rats.DFP was administered intragastrically.TCS was performed to observe the effects of DFP on the SN echo signal of the brain.Then immunofluorescence and specific iron staining were performed to observe the effects of DFP on the histological characteristics of the PD model rats.4.Microglia proliferation inhibitor nitidine was used to intervene the PD model rats,nitidine was administered intraperitoneal injection.TCS was performed to observe the effects of nitidine on the SN echo signal of the brain.Then specific iron staining was performed to observe the effects of nitidine on the iron deposit in SN.Results1.Immunohistochemical staining showed that survived TH-labeled DA neurons in SN of the PD model rats were 24±4 percents,and there was significantly less than 97±2 percents in SN of the sham group rats.Western blot analysis also showed that a significant reduction the levels of TH in SN of the PD model rats.The above results indicated that the PD rat model built successful.TCS examination showed that a large area of hyperechogenicity was evident in the SN of the PD model rats,the area was 2.40±0.56 mm~2.Only the hyperechoic line of the needle pathway was found in some of the sham group rats.2.Specific iron staining revealed markedly increased ferric ion deposition in the SN area of the PD rat model,and numerous nuclei were evident where ferric ions aggregated.Ferric ion deposition was rare in the SN of the sham group rats,and the arrangement of the nuclei was no obvious difference between the SN and the surrounding area of SN.The total iron contents in SN of the PD model rats were 22.50±0.46 ng/mg,and that was notably higher than 14.20±1.14 ng/mg in the sham group rats.Immunofluorescence staining showed that marked Iba-1-labeled microglia and GFAP-labeled astrocyte activation and proliferation occurred.Western blot analysis also showed that the level of Iba-1 and GFAP was notably higher in the PD model rat than that of the sham group rats.3.The SNH area of the DFP interfere group rats was 1.92±0.38 mm~2,and it was significantly smaller than 2.92±0.92 mm~2 of the PD model rats.Compared with the PD model rats,ferric ion deposition was significantly less,total iron contents in SN were significantly less,also microglia and astrocyte proliferation was markedly inhibited in the DFP interfere group.Survived DA neurons were 59±19 percents in the SN of DFP interfere group,those were more than 21±5 percents in the SN of PD model rats.4.The SNH area of the nitidine interfere group rats was 1.73±0.20 mm~2,it was significantly smaller than 2.46±0.40 mm~2 of the PD model rats.Compared with the PD model rats,ferric ion deposition was significantly less,and the total iron contents in SN were significantly less in the SN of nitidine interfere group rats.ConclusionBoth iron aggregation and gliosis contribute to the formation of SNH in PD.DFP can reduce the hyperechogenicity area,thus inhibit ferric ion accumulation and the gliosis induced by 6-OHDA.This finding further demonstrated that ferric ions and gliosis was associated with hyperechogenicity in PD.DFP exhibited a neuroprotective effect by reducing the volume of ferric ions in the SN and inhibiting gliocyte proliferation.Evident SNH occured,obvious ferric ion deposited,and few DA neurons survived.The above results hinted that iron deposition and the SNH area were correlated with PD severity.We used microglia proliferation inhibitor nitidine to intervere the PD model rats,the result showed that nitidine significantly reduced the total iron content and attenuated iron deposition.Taken together,the above evidence suggested that activated microglia is part resource of iron following the 6-OHDA treatment of the PD model rats.