A Guide to the Generation of a 6-Hydroxydopamine Mouse Model of Parkinson's Disease for the Study of Non-Motor Symptoms

Abstract

In Parkinson's disease (PD), a large number of symptoms affecting the peripheral and central nervous system precede, develop in parallel to, the cardinal motor symptoms of the disease. The study of these conditions, which are often refractory to and may even be exacerbated by standard dopamine replacement therapies, relies on the availability of appropriate animal models. Previous work in rodents showed that injection of the neurotoxin 6-hydroxydopamine (6-OHDA) in discrete brain regions reproduces several non-motor comorbidities commonly associated with PD, including cognitive deficits, depression, anxiety, as well as disruption of olfactory discrimination and circadian rhythm. However, the use of 6-OHDA is frequently associated with significant post-surgical mortality. Here, we describe the generation of a mouse model of PD based on bilateral injection of 6-OHDA in the dorsal striatum. We show that the survival rates of males and females subjected to this lesion differ significantly, with a much higher mortality among males, and provide a protocol of enhanced pre- and post-operative care, which nearly eliminates animal loss. We also briefly discuss the utility of this model for the study of non-motor comorbidities of PD.

Keywords: 6-hydroxydopamine; Parkinson’s disease; basal ganglia; behavior; bilateral; mouse; non-motor symptoms; prodromal; rodent surgery; striatum.

Figure 1

Timeline showing perioperative steps implemented during standard and enhanced care protocols. EC (lower panel) contains additional steps that increase animal welfare (see text for further information). W, week.

Figure 2

Bilateral partial striatal lesion with 6-OHDA. (a) Schematic illustration of the stereotaxic apparatus employed to inject 6-OHDA into the dorso-lateral part of the striatum (STR, blue). The coronal section on the right shows the targeted positions (scale 1 mm). (b) Representative coronal sections showing immunolabelling of dopaminergic fibers by TH-antibody in sham and 6-OHDA-lesion mice. Raw images of slices surrounding the injection site are presented with inverted color (scale: 1 mm, position from bregma: 1.42, 0.60 and 0.26 mm, in rostro-caudal order). (c) Percentage of TH-loss in 6-OHDA lesion mice compared with sham-lesion mice as quantified by Western blot. Values were normalized to sham group. Left, data obtained from bilateral free-hand dissection of whole striatum (full STR), showing a 56.4 ± 4.73% reduction in TH (n = 8 sham and 14 lesion mice; unpaired t-test, one-tailed, p < 0.0001, t = 11.92, df = 20). Middle, TH levels in whole left and right striata from 6-OHDA lesion mice, in comparison to sham striata (n = 8/12/12 per group, in order as shown in the graph; one-way ANOVA, Dunnett’s vs. sham, p < 0.0001). Note the similar decrease in left and right striata (left vs. right, ratio paired t-test, two tailed, p = 0.5267, t = 0.6538, df = 11). Right, comparison between the level of TH immunoreactivity in punches from dorsal and ventral striatum of sham and 6-OHDA lesion mice (n = 20/24/24 per group, in order as shown in the graph; one-way ANOVA, Sidak’s sham vs. lesion^dorsal p < 0.0001; sham vs. lesion^ventral, p = 0.0091; lesion dorsal vs. ventral, p < 0.001). Lower panels show representative Western blots of TH immunoreactivity. (d) Coronal view of lesion site, showing percentage of TH-fiber loss in STR, as quantified via microscopy and section mapping. Total depletion is shown in yellow (n = 5 mice/group). (e) Representative images of TH immunolabelling of cells within SNc (orange shade) and VTA (blue shade) of sham and lesion mice (scale 500 µm, −3.08 mm from bregma). Division of the two areas was based on the axon bundle (medial lemniscus) separating SNc and VTA. (f) Bar graphs show cell count quantifications as % of sham with 59.5 ± 6.8% reduction in TH-positive cells in SNc (n = 4 mice per group/8 sections each, unpaired one-tailed t-test, p < 0.0001, t = 8.757, df = 6) and 10.8 ± 5.3% reduction in TH-positive cells in VTA (n = 4 mice per group/8 sections each, unpaired one-tailed t-test, p = 0.0434, t = 2.041, df = 6). All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. 6-OHDA: 6-hydroxydopamine, TH: tyrosine hydroxylase, SNc: substantia nigra compacta, VTA: ventral tegmental area.

Figure 3

Enhanced perioperative care improves survival following bilateral injection of 6-OHDA. (a) Pie charts showing the post-surgical outcomes of SC and EC—the percentage of animals that survived and were then used for experimentation (light grey) or were lost during post-surgical recovery (dark grey/black). (b) Left, SC survival curves (%; Kaplan–Meier method lines and asymmetrical SEM, shadow zones) of sham (grey, n = 176), female^lesion (red, n = 77) and male^lesion (blue, n = 409) mice. Mantel-Cox test (p < 0.0001) followed by log-rank analyses of pairs showed higher mortality risk for male^lesion group (sham vs. male^lesion, p < 0.0001, sham vs. female^lesion, p = 0.2871). Right, EC survival curves of sham (grey, n =155), lesion female (red, n = 181) and male^lesion (blue, n = 256) mice. Dashed line represents male^lesion from SC protocol (SC-male^lesion). Mantel-Cox test (p = 0.0749) followed by log-rank analyses of pairs showed reduction in mortality risk for the EC-male^lesion group (EC-sham vs. male^lesion, p = 0.0243, EC-male^lesion vs. SC-male^lesion, p < 0.0001, sham vs. female^lesion, p = 0.1487). * p < 0.05, *** p < 0.001.

Figure 4

6-OHDA lesion-induced weight variability affects survival. (a) Bar graphs showing the number of animals lost in the weeks (W) following striatal lesion in SC and EC groups. SC loss 91/662 and EC loss 30/592. (b) Survival probability for female^lesion (red, n = 122 and 286) and male^lesion (blue, n = 448 and 310) mice receiving SC and EC, calculated by Logistic Regression Logit, showing difference in predictor factor ‘age’ before and after the implementation of EC protocol. (c) On the week prior to surgery, EC protocol promotes weight gain in males (blue, n = 91) but not females (red, n = 51) as compared to control (normal chow; CTRL, n = 12). Data are presented as mean ± SEM (Kruskal–Wallis test, p < 0.0001, statistic = 32.27. Dunn’s rank difference, CTRL vs. EC-female p > 0.9999, CTRL vs. EC-male p = 0.0066). (d) Line graphs (mean ± SD) showing weight (g) on surgery day, W1 and W4 post-surgery, in mice treated with EC which survived (female^sham n = 73 ± 6; female^lesion n = 120 ± 24; male^sham n = 25 ± 4; male^lesion n = 139 ± 20). Females (left) and males (right). Females show comparable weight throughout the whole period (two-way ANOVA, interaction F_{(2, 576)} = 3.175, p = 0.0425, Holm–Sidak’s surgery day p = 0.001, W1 p = 8148, W4 p = 8148), whereas male^lesion mice lose weight during the post-surgery recovery period (two-way ANOVA, interaction F_{(2, 486)} = 8.884, p = 0.0002, Holm–Sidak’s surgery day p = 0.7425, W1 p = 0.0015, W4 p < 0.0001). ** p < 0.01, *** p < 0.001.