Quantitative evaluation of motor function before and after engraftment of dopaminergic neurons in a rat model of Parkinson's disease

Abstract

Although gait change is considered a useful indicator of severity in animal models of Parkinson's disease, systematic and extensive gait analysis in animal models of neurological deficits is not well established. The CatWalk-assisted automated gait analysis system provides a comprehensive way to assess a number of dynamic and static gait parameters simultaneously. In this study, we used the Catwalk system to investigate changes in gait parameters in adult rats with unilateral 6-OHDA-induced lesions and the rescue effect of dopaminergic neuron transplantation on gait function. Four weeks after 6-OHDA injection, the intensity and maximal area of contact were significantly decreased in the affected paws and the swing speed significantly decreased in all four paws. The relative distance between the hind paws also increased, suggesting that animals with unilateral 6-OHDA-induced lesions required all four paws to compensate for loss of balance function. At 8 weeks post-transplantation, engrafted dopaminergic neurons expressed tyrosine hydroxylase. In addition, the intensity, contact area, and swing speed of the four limbs increased and the distance between the hind paws decreased. Partial recovery of methamphetamine-induced rotational response was also noted.

Figure 1

Effect of dopaminergic depletion and embryo stem cell derived dopaminergic neuron implantation. Gait parameters were assessed at 4 weeks after injection of 6-OHDA (6-OHDA) or saline (SHAM) at 8 weeks after transplantation of ES-derived dopaminergic neurons (6-OHDA-SC-8w) or injection of vehicle (6-OHDA-SA-8w) as the control. The intensity and max area (A and B) were reduced in the left paws and the swing speed (C) of all paws decreased after 6-OHDA injection. After implantation of dopaminergic neurons, the intensity, max area, and swing speed of all paws improved. The BOS between the hind paws increased after injection of 6-OHDA and partially recovered after dopaminergic neuron implantation (D). 6-OHDA-lesioning and dopaminergic neuron implantation did not lead to a significant change in step patterns and inter-limb phase dispersions (E, F, G and H). Rotational behavior in response to methamphetamine was tested at pre-transplantation (pre-TP) and at 4 and 8 weeks post-grafting. A significant decrease in absolute numbers of drug-induced turning was seen in the transplantation group compared with control animals at eight weeks (I). *P < 0.05; **P < 0.005.

Figure 2

The percentage of ES cells differentiates into TH-positive cells in vitro culture. (A) GFAP (red) and tyrosine hydroxylase (green) immunostaining after exposure FGF8b and Shh. At this stage almost no GFAP-positive glia cells were detected in the differentiating ES cells on day 10. (B) TH and DAT staining are two markers of dopamiergic neurons. (C) The efficiencies of the dopaminergic neuron induction: 18.7 ± 4.3% (Shh/FGF8b-treated ES cells) v.s. 2.4 ± 1.1% (mock-treated ES cells). The TH-positive cells were manually counted in total cells. The scale bar is 15 μm.

Figure 3

The number of TH-positive cells was evaluated at four weeks after 6-OHDA lesioning. Normal TH immunoreactivity was detected in the substantia nigra (A) and striatum (C) of all the sham animals. Depletion of TH immunoreactivity was noted in the substantia nigra (B) and striatum (D) at the side treated with 6-OHDA. Immunohistochemical staining was done at 8 weeks after implantation of ES-derived dopaminergic neurons into 6-OHDA lesioned striatum. The M2 mouse-specific antibody (E and F, red) and the nuclear marker DAPI (F, blue) were detected in numerous cells at the implantation site, suggesting that the grafts survived. TH-positive neurons were found within the graft (G, green). All TH-positive profiles coexpressed the M2 mouse-specific antibody (H, yellow), and one cell revealed M2 staining only (arrowhead). The scale bar: A, B: 200 μm; C to H: 50 μm.