Characterization of graded 6-Hydroxydopamine unilateral lesion in medial forebrain bundle of mice

Abstract

Parkinson's disease (PD) is the second most common age-related neurodegenerative disease, with a progressive loss of dopaminergic cells and fibers. The purpose of this study was to use different doses of 6-hydroxydopamine (6-OHDA) injection into the medial forebrain bundle (MFB) of mice to mimic the different stages of the disease and to characterize in detail their motor and non-motor behavior, as well as neuropathological features in the nigrostriatal pathway. MFB were injected with 0.5 μg, 1 μg, 2 μg of 6-OHDA using a brain stereotaxic technique. 6-OHDA induced mitochondrial damage dose-dependently, as well as substantia nigra pars compacta (SNpc) tyrosine hydroxylase-positive (TH⁺) cell loss and striatal TH fiber loss. Activation of astrocytes and microglia in the SNpc and striatum were consistently observed at 7 weeks, suggesting a long-term glial response in the nigrostriatal system. Even with a partial or complete denervation of the nigrostriatal pathway, 6-OHDA did not cause anxiety, although depression-like behavior appeared. Certain gait disturbances were observed in 0.5 μg 6-OHDA lesioned mice, and more extensive in 1 μg group. Despite the loss of more neurons from 2 μg 6-OHDA, there was no further impairment in behaviors compared to 1 μg 6-OHDA. Our data have implications that 1 μg 6-OHDA was necessary and sufficient to induce motor and non-motor symptoms in mice, thus a valuable mouse tool to explore disease progression and new treatment in PD.

Keywords: 6-Hydroxydopamine; Gait analysis; Glial cell activation; Medial forebrain bundle; Mitochondrial dysfunction; Motor behavior; Non-motor behavior; Parkinson’s disease.

Figure 1

Experimental design, graphical abstract, body weight changes and apomorphine-induced rotation. Time sequence of mice receiving different behavioral tests and other tasks (A). Body weight for the 14 consecutive days post lesion plotted per group (C). Apomorphine induced the turning behavior of animals with different doses of 6-OHDA (D). ***P < 0.001 compared with control, ^###P < 0.001 compared with 6-OHDA-0.5, ^{^^^}P < 0.001 compared with 6-OHDA-1.

Figure 2

Dose-dependent damage of 6-OHDA in the nigrostriatal system of mice. Representative photomicrographs of brain slices stained for TH⁺ (A). Quantification of the striatum/NAc average optical density (% control) and SNpc/VTA TH⁺ cells (% control) were shown (B–E). ^***P < 0.001 compared with control, ^#P < 0.05 compared with 6-OHDA-0.5, ^##P < 0.01 compared with 6-OHDA-0.5, n = 4, upper panel scale bars = 500 μm, larger version scale bars = 200 μm.

Figure 3

Motor behavioural assessment of 6-OHDA degree of lesion. Different doses of 6-OHDA reduced the total distance traveled by mice in the open field test (A) Different doses of 6-OHDA caused significant impairment of motor coordination in the rotarod test in mice (B). Different doses of 6-OHDA reduced the score of mice in the balance beam test (C). Different doses of 6-OHDA increased the time of mice in pole test (D).^***P < 0.001 compared with control, ^###P < 0.001 compared with 6-OHDA-0.5, ^{^}P < 0.05, ^{^^}P < 0.01 compared with 6-OHDA-1, n = 8.

Figure 4

Anxiety- and depressive-like behavior assessment of 6-OHDA degree of lesion. 6-OHDA did not change the time and number of mice entering the open arm in the elevated plus maze test (A, B). 6-OHDA did not change the time of open-field test in the central area of mice (C). 6-OHDA reduced the number and time of mice exploring new objects in novelty exploration test (D, E). 6-OHDA increased the immobility time of mice in tail suspension test and forced swimming test (F, G). 6-OHDA reduced the preference of mice to sucrose in the sucrose preference test (H). ^**P < 0.01, ^***P < 0.001 compared with control, ^#P < 0.05, ^##P < 0.01 compared with 6-OHDA-0.5, n = 8.

Figure 5

Gait parameters of 6-OHDA mice. Graphical representation of gait parameters. The upper panel (green prints in blue background) shows the digitized prints, while the lower panel (with colorful phase lags) shows the stance phase duration of each individual paw in a single step cycle (A). Blue, RF; red, RH; yellow, LF; green, LH. Four walking patterns including control (B), 6-OHDA-0.5 (C), 6-OHDA-1 (D), or 6-OHDA-2 (E) group. 6-OHDA increased the run duration and number of steps in the gait analysis of mice (F, I). 6-OHDA reduced the run average speed and cadence in gait analysis of mice (G, H). 6-OHDA reduced the stride length of limbs in gait analysis of mice (J). 6-OHDA reduced the print area and max contact area of the right limb in the gait analysis of mice (K, L). 6-OHDA increased the base of support of hind limbs in gait analysis of mice (M). 6-OHDA increased the stand, swing duration and step cycle of limbs in gait analysis of mice (N, O, Q). 6-OHDA reduced the swing speed, stand index and body speed of the limbs in the gait analysis of mice (P, R, S).^**P < 0.01 compared with control, ^***P < 0.001 compared with control, ^#P < 0.05 compared with 6-OHDA-0.5, ^##P < 0.01 compared with 6-OHDA-0.5, ^###P < 0.001 compared with 6-OHDA-0.5, n = 8.

Figure 6

TEM analysis of mitochondria in the striatum and SNpc regions of from 6-OHDA or control. The representative images are shown and red arrows indicate nucleus, yellow arrows indicate mitochondria (A). Quantification of the striatum and SNpc disrupted mitochondria (%) were shown (B, C). ^**P < 0.01 compared with control,^***P < 0.001 compared with control, n = 4.

Figure 7

The activation of astrocytes in striatum or SNpc after MFB injection 6-OHDA. Immunofluorescence representative micrographs showed labelling of TH⁺ (green) and of GFAP⁺ from the striatum and SNpc of 6-OHDA-lesioned mice (A, B). 6-OHDA increased GFAP⁺ (red) and decreased TH⁺ (green) in the striatum and SNpc of mice. Quantification of the striatum or SNpc coverage by GFAP⁺ or TH⁺ staining were shown (C–F). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with control, ^#P < 0.05 compared with 6-OHDA-0.5, ^###P < 0.001 compared with 6-OHDA-0.5, n = 4, upper panel scale bars = 200 μm, larger version scale bars = 50 μm.

Figure 8

The activation of microglial in striatum or SNpc after MFB injection 6-OHDA. Immunofluorescence representative micrographs showed labelling of TH⁺ (green) and of Iba-1⁺ from the striatum and SNpc of 6-OHDA-lesioned mice (A, B). 6-OHDA increase the mean area of Iba-1⁺ and Iba-1⁺ cells as well as decrease Iba-1⁺ total branch length in the striatum and SNpc of mice. Quantification of the striatum or SNpc mean area of Iba-1⁺, Iba-1⁺ cells and Iba-1⁺ total branch length were shown (C–H). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with control, ^#P < 0.05 compared with 6-OHDA-0.5, ^##P < 0.01 compared with 6-OHDA-0.5, ^###P < 0.001 compared with 6-OHDA-0.5, n = 4, upper panel scale bars = 200 μm, larger version scale bars = 50 μm.