Stereotactic infusion of rotenone into the SN induced a late-stage model of Parkinson's disease

The establishment of an in vivo model for Parkinson's disease (PD) that accurately mimics late-stage clinical features is crucial for elucidating disease pathogenesis and developing novel therapeutic strategies. To establish this model, rotenone (ROT) was stereotactically infused into the substantia nigra (SN). Motor behavior in PD mice was assessed using the pole test and rotarod test. Apomorphine (APO)-induced rotation and dystonia were also evaluated. Alterations in anxiety, depression, and cognitive function were examined using the open field test, sucrose preference test, Y-maze test, and Morris water maze test, respectively. Gastrointestinal (GI) motility was determined by examining the timing of pink stool passage. Changes in gut microbiota composition were analyzed via 16S rRNA sequencing. The expression and localization of nucleotide-binding oligomerization domain-leucine-rich repeat-pyrin domain-containing 3 (NLRP3), caspase-1, and α-synuclein (α-syn) within astrocytes were determined using immunofluorescence. Peripheral organ toxicity induced by ROT was investigated using hematoxylin and eosin (HE) staining. Experimental results demonstrated that the number of APO-induced rotations in the ROT group exceeded 210 rpm at 4 weeks and decreased significantly by 36 weeks, reflecting progressive disease characteristics. Specifically, the incidence of APO-induced dystonia was significantly higher in the ROT group at 36 weeks compared to 4 weeks (P < 0.05). Compared to the vehicle group, ROT-treated mice exhibited prolonged pole test descent time and reduced rotarod latency. The open field test revealed decreased center duration and crossings. Morris water maze showed fewer platform crossings and reduced target quadrant time. However, no statistically significant differences were observed between groups in the sucrose preference test at either 4 or 36 weeks. By 36 weeks, ROT-treated mice demonstrated a significantly lower spontaneous alternation rate compared to vehicle controls. ROT infusion also reduced overall gut microbiota abundance and altered microbial composition. Specifically, the ROT group showed decreased abundance of Allobaculum, Atopobium, Sneathia, Rikenella, Aestuariispira, Bacillus, Barnesiella, Escherichia/Shigella, Klebsiella, Eisenbergiella, and Parasutterella. Immunostaining analysis revealed that compared with the vehicle group, NLRP3, cleaved caspase-1, and α-syn expression was increased in astrocytes in the ROT group. The ROT-based model reproduced the clinical features of the early and late stages of PD, particularly dystonia and inflammasome activation, thus providing a reliable framework for studying late-stage PD.