Step Sequence Is a Critical Gait Parameter of Unilateral 6-OHDA Parkinson's Rat Models

Abstract

Parkinson's disease is a progressive neurological disorder, marked by the loss of dopaminergic neurons in the nigrostriatal pathway that leads to abnormal gait, rigidity, slowness of movement, and tremor. The ability to recapitulate and measure the neurological sequelae in rodent models of Parkinson's disease is important for studying and evaluating potential therapeutics. Individual variability in lesion severity and injury progression are key factors in the 6-hydroxydopamine model that require normalization when evaluating therapeutic effects. The gait parameters that were found to be affected by 6-hydroxydopamine lesioning of the nigrostriatal pathway in rats may be used to study novel transgenic models of Parkinson's disease as well as to test novel therapeutics. Previously, studies have used a video-based system to analyze gait abnormalities in the 6-hydroxydopamine model of Parkinson's disease, but these studies did not account for individual variability on reported gait parameters. By analyzing the ratio of parameters from the injured to uninjured sides and correcting for speed in related parameters, hindpaw step cycle parameters, hindpaw print area, and step sequence are significantly altered in different ways for each type of lesion, when compared to saline-injected controls. These findings enable new metrics for evaluating therapeutic efficacy of drug-, gene-, or cell-based therapies in rat models of Parkinson's disease.

Figure 1.

Experimental overview. (A) Timeline of behavioral assays (CatWalk and methamphetamine-induced rotations) in relation to induction of lesion. (B) Schematic of targeted sites for unilateral 6-hydroxydopamine (6-OHDA) lesion to create the intrastriatal (intra-STR; two striatal injections) and medial forebrain bundle (MFB; single injection) model of Parkinson's disease.

Figure 2.

Loss of dopamine neurons impairs locomotion. (A) Representative images of tyrosine hydroxylase (TH) loss in the striatum (above) and substantia nigra (below) of each lesion group compared to sham. (B) Semiquantification optical density measurements of TH immunoreactivity relative to contralateral hemisphere in the striatum and midbrain. (C) Net ipsilateral turns (ipsilateral minus contralateral turns) following methamphetamine administration. (D) Average speeds of rats tracked throughout the study. Intra-STR, group with injections of 6-OHDA in the striatum; MFB, medial forebrain bundle/group with injections of 6-OHDA in the medial forebrain bundle.

Figure 3.

The 6-OHDA lesion alters proportion of step cycle spent in stand phase. (A) Left: Image of left front paw print taken from the CatWalk software. Middle: Ratio of hindpaw (left or lesioned side/right or unlesioned side) print area is different between types of lesion, indicating a change in limb loading. Right: Print area does not correlate with speed of crossing the apparatus. (B) Schematic of step cycle made up of the stand (paw contact with glass) and swing (paw not in contact with glass) phases. Step cycle as a whole did not change in both the lesion and sham groups. 6-OHDA, 6-hydroxydopamine; Intra-STR, group with injections of 6-OHDA in the striatum; MFB, medial forebrain bundle/group with injections of 6-OHDA in the medial forebrain bundle; LF, left front paw.

Figure 4.

The 6-OHDA lesions alter step sequence preference. (A) Schematic demonstrates the order of each paw in the six-step sequence pattern classifications. (B) Overall step sequence: the proportions of each step sequence type out of total step patterns identified. (C) The cruciate A step sequence is more common after lesion, permitting quantification of parkinsonian gait in an animal model. LF, left front paw; LH, left hind paw; RF, right front paw; RH, right hind paw; 6-OHDA, 6-hydroxydopamine; Intra-STR, group with injections of 6-OHDA in the striatum; MFB, medial forebrain bundle/group with injections of 6-OHDA in the medial forebrain bundle.

Figure 5.

Correlation of step sequence and TH immunoreactivity in the midbrain. To examine the relationship between TH staining and changes in gait, optical density of TH staining in the midbrain was correlated to (A) the percentage of steps taken in the alternate sequences and (B) in the cruciate sequences on week 6. Although negative and positive correlations are shown with TH staining/alternate step sequences and TH staining/cruciate step sequences, respectively, a floor effect (0% of alternate sequence in MFB lesion) and a ceiling effect (100% steps taken as alternate in sham lesion) negate statistical analysis. TH, tyrosine hydroxylase; 6-OHDA, 6-hydroxydopamine; Intra-STR, group with injections of 6-OHDA in the striatum; MFB, medial forebrain bundle/group with injections of 6-OHDA in the medial forebrain bundle.