Challenges and Opportunities in Exploring Non-Motor Symptoms in 6-Hydroxydopamine Models of Parkinson's Disease: A Systematic Review

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of midbrain dopaminergic neurons, leading to motor symptoms such as tremors, rigidity, and bradykinesia. Non-motor symptoms, including depression, hyposmia, and sleep disturbances, often emerge in the early stages of PD, but their mechanisms remain poorly understood. The 6-hydroxydopamine (6-OHDA) rodent model is a well-established tool for preclinical research, replicating key motor and non-motor symptoms of PD. In this review, we systematically analyzed 135 studies that used 6-OHDA rodent models of PD to investigate non-motor symptoms. The review process adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Our analysis highlights the growing use of 6-OHDA PD models for experimental research of non-motor symptoms. It also reveals significant variability in methodologies, including choices of brain target, toxin dosage, lesion verification strategies, and behavioral assessment reporting. Factors that hinder reproducibility and comparability of findings across studies. We highlight the need for standardization in 6-OHDA-based models with particular emphasis on consistent evaluation of lesion extent and reporting of the co-occurrence of non-motor symptoms. By fostering methodological coherence, this framework aims to enhance the reproducibility, reliability, and translational value of 6-OHDA models in PD non-motor symptom research.

Keywords: 6‐OHDA; Parkinson's disease; anxiety; depression; preclinical; rodent; sleep; social interaction; striatum; substantia nigra.

FIGURE 1

Systematic flowchart and publication year of articles included in this meta‐analysis review. (a) PRISMA flow diagram illustrates the process of article selection, detailing the steps from identification and screening to inclusion in the review. (b) Histogram distribution of included articles by publication year, highlighting trends in research focus over time.

FIGURE 2

An overview of 6‐OHDA‐based methodologies for modeling Parkinson's disease: Considerations for subject selection and neurotoxin infusion parameters. (a) Total number of articles reviewed (purple), number of 6‐OHDA approaches identified (gray), distribution of rodent choice (blue) and the breakdown by sex (last bar). (b) Proportion of studies using rat (r) and mouse (m) as well as preferred strain. (c) Distribution of selected 6‐OHDA injection sites. (d) Number of 6‐OHDA injections per hemisphere in unilateral and bilateral approaches. (e) Schematic representation of 6‐OHDA injection sites based on reported stereotaxic coordinates. Scale bar, distance between bregma and lambda reference points, as defined by Franklin and Paxinos (2019) and Paxinos and Watson (2007). (f) Profile of injection site variability for rats. Heatmap depicts 6‐OHDA concentration (μg/μL), and injection site stereotaxic position (top panel; mediolateral, ML. bottom panel; dorsoventral, DV) by anteroposterior (AP) axis. (g) Average amount of 6‐OHDA mass and volume infused per brain region. Data are presented as mean ± SEM.

FIGURE 3

Most common motor tests used in 6‐OHDA lesion articles. (a) Tests used for motor phenotype analysis. (b) Self‐report of motor phenotype as a confounder to non‐motor symptoms evaluation.

FIGURE 4

Diversity in 6‐OHDA animal models of Parkinson's Disease: Limited replication and prevalence of cognitive and affective symptom evaluation. (a) Grouping of experimental approaches based on parameters used to generate the lesion. Each model is characterized by specific choices of species/strain, 6‐OHDA injection coordinates, number of injections, and 6‐OHDA concentration. A total of 125 unique models were identified, with only 12 having been replicated. (b) Distribution of model usage frequency across studies. Each circle represents a unique model, with the color coding indicating the number of replications. (c) Number of models tested (count, n) for each class of non‐motor symptom, including both positive and negative findings. (d) Venn diagram depicting the overlap between the two most commonly evaluated classes of non‐motor symptoms. Models presenting cognitive deficits (red circles), affective deficits (green), or both (brown) are shown inside the diagram. Models in which these deficits were absent or not assessed are displayed outside the diagram (gray). (e) Venn diagram showing the subcategories of cognitive and affective symptoms, with areas proportional to the number of models presenting each phenotype. (f) Bar graphs displaying the distribution of cognitive and affective symptoms evaluated per infused nigrostriatal region (MFB, SN, STR) and lesion laterality (bilateral vs. unilateral). (g) Heatmap illustrating the conditional probabilities for all pairs of behavioral features reported across models.

FIGURE 5

Low similarity of references and lack of cross‐citation in studies using 6‐OHDA models of Parkinson's Disease. (a) Shared references between article pairs. The index (x‐axis) measures how similar two articles are based on the references they share relative to the total set of references they cite. The similarity score of each article pair is a value between 0 and 1, where 0 indicates no similarity (no shared references) and 1 indicates complete similarity (all references are shared). The y‐axis represents the percentage of article pairs with shared bibliographies. (b) Number of cross‐citations between reviewed articles (at least one cross‐citation required). Node color indicates if an article cites (dark blue) or is cited (light blue). Only three articles cite and are cited by other articles in the review list. Right panel, citation node graph. Arrows help depict citation direction, and article ID was used to identify each node. Article ID and respective DOI‐reference can be found in Table S1.