In this study we measured locomotor deficits in rat models of PD, HD and stroke using a novel automated gait analysis test, the catwalk method, and compared the results to deficits recorded using established behavioral motor tests. To the best of our knowledge, the present study is the first to evaluate these rat models of neurological disorders using an automated gait analysis concurrently measuring static and dynamic gait parameters.
The quantitative gait analysis used in the present study, the catwalk method, provides both static gait parameters as well as time-based information and a pressure parameter, in contrast to a conventional gait analysis test, such as the analysis of footprints obtained by allowing rodents to walk with inked paws along a sheet of paper, which provides information about static gait [15
]. The 6-OHDA rat model for PD recapitulates one of the main features of PD, namely the loss of midbrain dopaminergic neurons. The loss of dopaminergic innervation of the caudate putamen leading to reduced activity of the basal ganglia thalamocortical motor circuit and motor deficits has been well characterized both in the rat 6-OHDA model as well as in the human disease [2
]. One of the most sensitive rodent behavioral tests available to assess midbrain dopaminergic cell loss is the amphetamine induced rotation test; however a pharmacological challenge is necessary to induce the behavioral readout [18
]. It has proved challenging in the field to develop behavioral readouts for dopaminergic cell loss without pharmacological challenge [2
]. The 'cylinder' test (paw use asymmetry test) can be used to observe contralateral-ipsilateral motor imbalances in the forelimbs in unilaterally 6-OHDA lesioned animals without pharmacological challenge [19
], as is confirmed in our experimental groups where the degree of impairment correlates to the degree of lesion (Fig. ). This contralateral-ipsilateral imbalance is also observed in the gait analysis, as the intensity of the lesioned forepaw is significantly lower compared to the non-lesioned paw, indicating that the 6-OHDA lesioned animals preferably use the non-lesioned paws to support their body weight. The amplitude of the intensity imbalance was consistent with the severity and type of lesion. Indeed, strongest measures were observed for the group showing the largest lesion, the MFB lesion group. In this group, strong paw intensity deficits were seen at 1 week post-lesion. On the contrary, striatal injections which yielded lower levels of dopaminergic fiber loss showed lower pawprint intensity deficits compared to the MFB group. A recent study has similarly analyzed 6-OHDA lesioned rats using the Catwalk gait analysis [20
], including experimental groups with mild bilateral 6-OHDA lesions in the striatum combined with subthalamic nucleus stimulations. Because Vlamings et al. [20
] performed bilateral lesions and our major observation is a contralateral-ipsilateral imbalance in pawprint intensity, it is difficult to draw parallels between both studies. We nevertheless observed that the contralateral-ipsilateral imbalances were bigger in the forepaws compared to the hindpaws, similar to differences of effects of forepaw vs hindpaw as observed by Vlamings et al. [20
Interestingly, despite the availability of multiple parameters from the catwalk analysis, the only parameter which consistently yielded results corresponding to a unilateral motor deficit was the intensity parameter measured for the forepaws. This parameter is highly analogous to the paw usage parameter monitored in the cylinder test and we can therefore conclude that information obtained from motor behavior assessment of hemiparkinsonian rats using the catwalk test is comparable to that obtained by the cylinder test.
We have also monitored the onset and early progression of HD-like symptoms related to all motor systems affected in HD in a transgenic rat model of HD. In line with previous studies in this tgHD rat and in a knock-in mouse model of HD [14
], gait analysis revealed a hyperkinetic profile early on in the pre-symptomatic stage. Whereas previous studies used the accelerod or open field monitoring [14
], we have demonstrated by the aid of the Catwalk that the hyperkinetic profile in 2-month-old transgenic rats is more specifically reflected by increased swing speed and decreased paw contact of quasi all limbs, and lasts until the age of 3 months; the latter in accordance with a previous report [14
]. 2-month-old transgenic rats also showed increased locomotor speed (Additional file 1
), in line with previously described negative correlation between locomotor speed and stance duration [22
]. This early hyperkinetic profile was not observed on the rotarod test used as a reference test in this study, allowing us to conclude that the catwalk method is more sensitive in measuring behavioural deficits in these animals than the rotarod test (Additional file 2A
]. Whether alternative behavioural tests, of which some perform better than the rotartod, e.g. the balance beam tasks, will also detect this hyperkinetic profile needs to be further studied ([24
Consistent with observations in humans, in which even years before the onset of overt motor symptoms such as chorea, subtle motor deficits are present (i.e alterations in finger-tapping rhythm and rate) [27
], we also noticed in 3- and 4-month-old tgHD rats a deviant gait pattern, in which they placed the right forepaw more to the inside than WTs (Fig. ). No significant differences in paw angle were observed between the time points of both groups.
Aberrations in paw placement have so far only been described in symptomatic mice transgenic for HD and in 3-NP-treated mice [15
]. Specifically, R6/2 transgenic mice at 13-14 weeks of age exhibited a gait that lacked a normal, uniform, alternating right-left step pattern with inside paw placement of all limbs, although within 3-NP-treated mice, the paw placement angle of the hind limbs was more open [15
]. The unilateral onset of this paw angle placement phenotype in the tgHD rats may be explained by reports suggesting that asymmetric striatal degeneration due to ventricle enlargement is not uncommon [6
]. Whether the paw angle measured using the catwalk is indeed an early marker for motor deficits needs to be clarified in further longitudinal designs.
Thirdly, we used the Catwalk method to quantify disturbances in gait in rats with a unilateral photothrombotic lesion in the parietal sensorimotor cortex compared to sham operated animals. As for the tg HD animals, the rotarod test which we used as a reference test proved to be insensitive since only a fraction of the lesioned rats fell off the machine (Additional file 2B
). As expected by the anatomofunctional location of the infarct, the rats showed a deficit in usage of the forepaw contralateral to the lesion 48 h after surgery as seen with the cylinder test. However, this test is not suitable for the detection of impairments in the hindpaws. In contrast, the gait analysis with the Catwalk method demonstrated a significant difference in the usage of the contralateral vs. ipsilateral hindlimbs. This was expressed by the 'intensity', 'print area', 'print width' and 'max area' parameters (Fig. ). All these parameters showed that the lesioned animals put a higher pressure on the non-affected ipsilateral hindpaw than the contralateral hindpaw. This imbalance is likely due to a compensation of the animal to spare the affected paw and enforce the paw with non-injured motoric excitation. These results confirm data where cortical ablation lesions impaired the performances of the rats on a beam walking task caused by hindlimb deficits [30
]. Our data therefore clearly demonstrate the advantage of using automated gait analysis for the study of cortically lesioned rats.
Although the present study has focused on gait analysis in rats, extension to mouse models is in principle possible. For example, robust effects on stride length during continuous locomotion following pyramidotomy of the cortical tract in adult mice have been observed [32
]. Also, unsteady gait with strongly reduced paw print area for both fore- and hindpaws and reduced base of support for the hindpaws have been measured in a mouse model for Refsum disease [34
]. Further research will be required to delineate gait deficits in mouse models of PD, HD or stroke.