Quantitative assessment of gait
In this study, we developed a time-efficient and animal-friendly CatWalk test of motor functions in an MPTP-induced PD mouse model. Both static and dynamic parameters of gait variability were collected simultaneously. Static parameters of stance, print length, print width, print area, maximum contact intensity, and maximum contact area are based upon contacts of individual paw with the glass plate of walkway. Dynamic parameters are walk duration, variation of walk speed, swing speed, and base of support between limbs. The study showed that the CatWalk test was able to sensitively assess gait disorder and inter-limb coordination deficit in a mouse model of PD with bilateral lesion of DA neurons.
There are many behavioral tests in MPTP-induced PD mouse model. These have to be robust and sensitive, in order to detect functional changes, days and even weeks after challenge by a neuro-toxin. The widely employed rotarod test is a feasible means to analyze coordination disorders in a bilaterally lesioned PD mouse model, but it is insensitive. It was noted that the progress of pre-analytical training of animals may recruit DA in the striatum . The pole-, grid- and nest-building tests are sensitive to striatal DA level in MPTP-treated mice, but are heavily dependent on the skilled manipulation of fore-limbs of the animal being studied [17, 39, 40].
The open field test is the most commonly used method to evaluate motor deficits because of its ease and rapidity of use and rapidity in animals following MPTP insult [12, 41–45]. We conducted open field test to study motor deficits in mice three weeks after induction of sub-acute neural injury and applied the open field test as the control in the evaluation of the CatWalk test. Readouts of the open field test in the study agree with previous reports of significant motor deficits in mice a few days after MPTP injection [43, 44]. A significant upsurge of trajectories was also noted in mice one week post-MPTP administration compared to those read on day 4 and that of control mice on day 7. The scenario might be attributable to an increase of DA metabolism and fluctuation of catecholamines such as 5-hydroxytryptamine in the midbrains of mice induced by dopaminergic neurotic and MPTP and a booster of muscle strength to balance posture instability [46–49].
Although open field test is a friendly test, the time points to detect locomotor changes in animals are limited. Here, we could only apply the time points of the 4 day and three weeks after MPTP injury for the functional evaluation in the open field test. The stability of behavioral performance of animals in the open field is vulnerable to the toxic effects of MPTP on biogenic amines inside and outside the brain , administration regimens and dosages of MPTP [48, 50] and environments.
The computer-assisted CatWalk test paralleled the motor function assessment in the open field test by collecting static and dynamic parameters in non-stressed rodents to investigate gait variability and coordination changes simultaneously. The stride length, run duration, stance, step cycle, duty cycle, swing speed, and cadence can be used to assess gait changes. The base of support between limbs, initial dual stance, terminal dual stance and diagonal dual support, three-point support and four-point support reflects the posture alterations. The regularity index and step sequence evaluates inter-limb coordination. Animals therefore do not need to undergo any complex process of training. They are allowed to simply run freely along the glass walkway four to six times. Data from the study show that their abnormal gait , in terms of stride length, duty cycle, dual diagonal support, three-point support, four-point support, and base of support between limbs persisted up to three weeks post-MPTP administration. The parameters of inter-limb coordination, regularity index, and step pattern showed no significant difference in MPTP-treated mice, compared to those of control mice. The extent of MPTP-mediated nigrostriatal lesion might not elicit any observable dis-coordination of tetrapod . The observation may also be related to stronger postural adjustment in quadrupeds than in bipeds.
TH profiling at time of MPTP insult
MPTP-mediated dopaminergic cell loss occurred predominantly in the SN, whereas DA neurons in the ventral tegmental area were less affected [8, 51].
The remarkable reproducible neuroplasticity of the nigrostriatal dopaminergic neurons after MPTP injury has been reported in mice . The decrease in the expression of TH protein was noted to correlate with the loss of DA neurons in the SN due to acute MPTP injury . It is likely that the decline in striatal dopamine content was due to the decrease in the expression of TH protein as a consequence of MPTP lesion. The rescue of TH protein is one of the determinants for regaining striatal dopamine in surviving nigrostriatal dopaminergic neurons [40–42]. Readouts of western blotting in the study demonstrated that protein levels of TH in the SN and striatum appeared to rebound two weeks post-MPTP administration; however the expressions were still significantly lower than those derived from intact SN and striatum.
The study data were in line with those reported previously by Ookubo et al. . The TH protein level fell to the lowest extent after three weeks. The stable lesion in PD mice can serve as a proxy for Parkinsonism in mice, but not for the assessment of MPTP neurotoxicity. The up-regulated expression of TH protein expression two weeks after MPTP injury might have a robust compensatory increase resulted by surviving nigrostriatal dopaminergic neurons against the neurotoxicity of the MPTP [54, 55]. The study data suggest that the open field test is insensitive to the extent of DA lesion except on day 4 and three weeks post-MPTP challenge.
Translational investigation of gait variability in MPTP-treated mice
The study provided a thorough analysis of the very fine locomotor aspects in a mouse model of sub-acute PD mediated by MPTP. The CatWalk test revealed detailed gait changes in mice at one week and three weeks post-MPTP challenge. That stride length decreased markedly in MPTP-treated mice may be the result of an increase in muscle rigidity and hypokinesia [40, 56, 57]. Results of the study were in accordance with previous studies [23, 27, 34, 58] and reports on PD patients [59–61], suggesting that stride length serves as a successful translational parameter for PD.
Bradykinesia is one of the major hallmarks of PD [59, 62]. Run duration, speed variation, swing speed, and step cycle are parameters related to velocity in mobility analysis. Using the CatWalk analysis system, we identified a significant increase in walking duration accompanied with a great variation in the walking speed in MPTP-treated mice. The greater variation in walking speed, which is similar to the fractal-like gait in PD, suggests possible marked inconsistencies in the timing of gait and inadequate postural adjustments [63–65]. The step cycle was increased in the MPTP-treated mice of the study due mainly to the longer contact between the paws and the glass plate. Results of the study were in line with a previous gait assessment in a bilateral 6-OHDA rat model . Gait cadence decreased significantly in MPTP-treated mice. However, an increase in step frequency was noted in PD [66, 67]. Cadence varies directly with stride length and inversely with the step cycle. The decrease in stride length noted in MPTP-treated mice of the study might not counter-balance the increase in the step cycle. As a consequence, the cadence decreased in MPTP-treated mice.
Support, either temporal or spatial, is another aspect related to gait in PD. The significant increases of initial dual stance, terminal dual stance, three-point support, and four-point support and the substantial decrease in diagonal dual support in the study suggested an increased duration of the postural phase in the MPTP-treated mice. The study data agree with reports on a longer double limb support time in PD, which may stem from the long time taken to prepare for the generation of propulsive forces [3, 68]. Results are in line with delays of freezing of gait or gait hesitation in PD [69–71]. The increased duration of the postural phase and the decrease in propulsive forces during the postural and movement phases resulted in a shorter step length and a slower step velocity. The spatial parameters of base of support between hind limbs increased substantially in MPTP-treated mice but there was no obvious difference in base of support between fore limbs. This might be related to hind limb rather than fore limb compensation, at least in part, playing a role in the gait instability of MPTP-treated mice in the study. On the other hand, the study data contrast with the findings of Westin et al. in which the base of support between fore limbs decreased significantly after 6-OHDA injury. This might be attributed to the difference in injured cerebral areas of animals in the two studies .
Interlimb coordination parameters of regularity index and step pattern were also studied. There are three categories in the step patterns: cruciate (Ca, Cb), alternate (Aa, Ab), and rotary (Ra, Rb). The ‘Ab’ alternate pattern is the most common step cycle, constituting 80% to 95% of the total step cycles in intact rodents, the other patterns are very rare in intact animals. The Ab pattern remains dominant in the Parkinson’s disease animal model . In this study, there was a slight but not significant decrease in the incidence of this step pattern in mice after MPTP injury. This result is supported by Chuang , and may be related to stronger postural adjustment in quadrupeds than in bipeds.
Correlation between the gait parameters and TH levels in the SN
Various strategies have been employed to verify the correlation between the motor deficits and the impairment of the nigrostriatal system. Here, we used correlation analysis of the motor parameters and TH protein levels in the SN. Compared with the travel distance measured in the open field test, parameters of the run duration, stride length, duty cycle, swing speed, base of support between hind limbs, three-point support, and four-point support in the CatWalk test were much more strongly correlated with the SN TH protein level. This is one of the first studies to apply correlation analysis to gait measures and TH protein levels in the SN to further confirm gait and posture deficits in the classic sub-acute MPTP PD model.