Time-course gait analysis of hemiparkinsonian rats following 6-hydroxydopamine lesion



Contents lists available at Behavioural Brain Research Time-course gait analysis of hemiparkinsonian rats following6-hydroxydopamine lesion Tsung-Hsun Hsieh , Jia-Jin J. Chen , Li-Hsien Chen , Pei-Tzu Chiang , Hsiao-Yu Lee a Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwanb Institute of Basic Medical Sciences, National Cheng Kung University College of Medicine, Tainan, Taiwanc Department of Digital Media Design and Management, Far East University, No.49, Zhonghua Rd., Xinshi Dist., Tainan City, 74448, Taiwan Gait disturbances similar to those of human Parkinson's disease (PD) can be observed in animals after Received 20 August 2010 administration of neurotoxin 6-hydroxydopamine (6-OHDA) to induce unilateral nigrostriatal dopamine Received in revised form 10 March 2011 depletion. However, the relationship between gait disturbances and dopamine depletion following 6- Accepted 14 March 2011 OHDA infusion has not been determined. The present study investigated the longitudinal changes ofspatiotemporal gait patterns using a walkway system to acquire footprints and lateral limb images over a 6-week period following unilateral 6-OHDA injection into the medial forebrain bundle of rats. Our Parkinson's disease results indicated that hemiparkinsonian rats exhibited changes in gait patterns, as compared to normal controls, and pre-lesion levels, including a significantly decreased walking speed and step/stride length Gait patternDopaminergic neurons as well as an increased base of support and foot angle. The relative percentage of the gait cycle was alsoaltered, showing an increase in the stance to swing ratio, which was more evident in the affected hindlimb.
Time-course observations showed that these gait disturbances occurred as early as 4 days post-lesionand gradually increased up to 42 days post-injury. The extents of gait disturbances were compared withconventional apomorphine-induced turning behavior and akinesia bar tests, which were also apparentat 4 days post-lesion but remained relatively unchanged after 28 days. Our time-course gait analysis ofa unilateral 6-OHDA rodent model provides insight into the compensatory changes of motor functionsduring the 6-week development of a nigrostriatal lesion, which might be useful for future objectiveassessment of novel treatments for human PD subjects.
Crown Copyright 2011 Published by Elsevier B.V. All rights reserved.
1,2,3,6-tetrahydropyridine (MPTP) or by infusion of the neurotoxin6-hydroxydopamine (6-OHDA) in rats. Whereas MPTP injection Gait disturbances are commonly observed in subjects with causes acute and bilateral lesions in the nigrostriatal dopaminer- Parkinson's disease (PD) resulting from a degeneration of dopamin- gic system, unilateral injection of 6-OHDA into the rat SN, medial ergic (DA) neurons in the substantia nigra (SN) the forebrain bundle (MFB) or striatum (Str) has commonly been used hallmark changes of gait following PD include temporal asymme- to induce the changes of motor dysfunction observed in the hemi- try, which manifests as an inability to maintain internal gait rhythm parkinsonian rat model walking speed, increased cadence and increased dou- Several animal behavior tests have been devised to assess the ble stance time n addition, PD subjects exhibit abnormal spatial functional deficits and to quantify the behaviors that are simi- indices of gait patterns, which typically include short steps lar to human PD symptoms, including a rotation test for severity freezing gait decreased stride length gait abnor- of dopamine depletion bar test for akinesia a malities become more pronounced in the advanced stages of PD, stepping test for rigidity gait impairment is the car- inducing further disability or limitation of mobility. To understand dinal sign of PD in humans, gait analysis is used to quantify the the development of PD and to further explore effective thera- multifaceted and complex motor functions in PD animal models peutic strategies for improved management of gait disturbances, studies investigating PD gait disorders in rats often it is important to have relevant PD animal models, which can used footprints to monitor abnormalities in the spatial parameters be obtained by systemic administration of 1-methyl-4-phenyl- of gait. For example, the rodent hind paws were inked, and therodent was then allowed to walk on paper strips. Based on foot-print assessment, rats with a unilateral PD lesion were found to ∗ Corresponding author. Tel.: +886 6 597 9566x7652.
display a shuffling gait, motor asymmetries and short stride lengths E-mail addresses: , (H.-Y. Lee).
that resemble the key features of the human PD gait 0166-4328/$ – see front matter. Crown Copyright 2011 Published by Elsevier B.V. All rights reserved.
T.-H. Hsieh et al. / Behavioural Brain Research 222 (2011) 1–9 ever, temporal data regarding the gait cycle in PD rats is insufficient mirror reflected the image of the rat's paws for convenient observation with a dig- due to the limitations of the inked footprint assessment system.
ital camera (EX-F1, Casio, Japan). For image capture, the camera was set to record The recent development of computer-assisted automatic gait anal- simultaneously a direct lateral view and a reflected underview of the walking track.
For lateral kinematical data acquisition, the rats were shaved and marked with red ysis, such as CatWalk, provides objective quantification of static on the skin of the lateral side of the bilateral hindlimbs before each test session. The and dynamic gait parameters from footprint analysis and has been marked landmarks included the lateral malleolus and the fifth metatarsal head as applied to bilateral 6-OHDA lesion rats Other simple video- identified by palpation while moving the joints. Use of colored landmarks provided based gait analysis systems have implemented a reflective mirror an easy way to determine the stance and swing phases of the gait cycle from heelcontact to toe off.
in a confined, transparent walking track for simultaneous recording Before the experiment, the rats were acclimated to the walkway by allowing of the plantar and sagittal views of the rat's hindlimbs; this allows them to walk freely on the track for 20 min before formal recording. The walking assessment of spatiotemporal and kinematics data in varied, freely task was repeated in both directions, thus permitting the recording of the move- ment of each hindlimb. The walking task was repeated until five or six satisfactory Although gait analysis in rats has been employed in vari- walks of at least 4 steps without pause were obtained. Only the hindlimb steppingpatterns were analyzed in our present video-based gait analysis system. The digital ous neuroscience studies the literature is scant regarding images obtained from each trial were processed with a threshold setting to detect the time-course changes of motor behaviors or locomotion func- the boundary of the soles, and critical points for derivation of paw indices were tions during the development of the 6-OHDA hemiparkinsonian determined using Matlab software (MatWorks, version 7.6., R2008a). After identifi- rat model. Understanding the relationships between development cation of sequential footprints, four spatial parameters, including step length, stridelength, base of support (BOS) and foot angle, and three temporal gait parameters, i.e., of motor disturbances and the degrees of DA cell loss might walking speed, stance/swing phase time and stance/swing ratio, were determined.
provide some insight into the quantitative assessment of novel Each gait parameter was averaged for at least 20 footsteps.
therapeutic strategies for PD. Thus, the aims of the present studywere to provide a detailed analysis of the time-course changes in gait spatiotemporal parameters and to observe the corresponding Impairment of the initiation of movement or akinesia has been commonly char- acterized by bar tests for immobility, stepping or cylinder tests bar dopamine loss in the rat's brain for 6 weeks following unilateral test was adopted in this study to observe the akinesia phenomenon of PD rats. During 6-OHDA injection.
the bar test, each rat was placed gently on a table. Each forepaw was placed alter-nately on a horizontal acrylic bar (0.7 cm diameter), which was suspended 9 cm 2. Materials and methods
above the table surface. The forepaw nearest the camera was recorded. The totaltime (in s) spent by each paw on the bar, i.e., the amount of time from the placing of the forepaw on the bar to the first complete removal of the paw from the bar, wasrecorded Animal studies were conducted on 41 adult male Wistar rats with a body weight range of 350–450 g and age range of 8–12 weeks at experimental onset. The ani- 2.3.3. Analysis of apomorphine-induced spontaneous rotation mals were separated into two groups for evaluating motor behaviors and DA cell A conventional behavioral assessment using apomorphine-induced rotation loss. Sixteen rats (eight normal control and eight lesioned rats) were assigned for was performed to quantify the unilateral nigrostriatal lesion-induced motor asym- time-course assessment of motor behaviors for six weeks. The other 25 rats were metry after ipsilateral 6-OHDA injection rotational tests were separated into five subgroups, which were sacrificed at pre-lesion and at 1, 7, 21 and performed with apomorphine (0.5 mg/kg in 0.1% ascorbic acid, i.p.; Sigma) injec- 42 days for evaluating the degree of DA neuron loss following the 6-OHDA lesion. All tion of PD rats. The rats were placed individually in a 30-cm-diameter round bowl rats were obtained from the Laboratory Animal Center, National Cheng Kung Uni- and assessed over a 60-min period stickers of two different colors were versity, Taiwan. The rats were housed at 25 ◦C with a 12/12 h light/dark cycle and pasted on the rat's back for easy identification of torso direction from the vec- continuous water and food. All experiments followed the Guide for the Care and Use tor change derived from the centers of the color circles. For precise calculation of of Laboratory Animals.
the number of rotations after apomorphine injection, the rotational behavior wasrecorded using a digital video camera, which was analyzed at 10-min intervals using 2.2. Chronic hemiparkinsonian rat model an image analysis program written in Matlab. The net number of rotations was cal-culated as the difference between the number of contralateral rotations and the For the 6-OHDA lesion, the rat was anaesthetized with intraperitoneal number of ipsilateral rotations with respect to the 6-OHDA injection side.
400 mg/kg chloral hydrate and placed into a stereotactic apparatus (Stoelting, IL,USA) to prevent head movement using a 45◦ non-puncture ear bar with the nose position at 3.3 mm below the interaural line. A 2-cm incision was made, and the areawas carefully cleared to expose the line of bregma. To cause destruction of the nigros- For evaluating DA neuron loss, tyrosine hydroxylase (TH) staining at five time triatal pathway, which results in near total depletion of dopamine in the ipsilateral points post-lesion was performed. The animals were deeply anaesthetized with Str and the SN ␮g/␮l of 6-OHDA (dissolved in 0.02% ascorbic saline, Sigma an overdose of pentobarbital and perfused transcardially with 0.9% saline and 4% Chemical Co., USA) was injected intra-cranially into the MFB (anterior–posterior: paraformaldehyde (PFA) in 0.1 M phosphate buffer solution (PBS). Brains were −4.3 mm from the bregma; lateral: 1.6 mm with respect to the midline and ven- removed and post-fixed for 3 days in the same fixative and dehydrated in 30% tral 8.2 mm from skull surface;) according to the stereotaxic brain atlas of Paxinos sucrose in 0.02 M PBS until the brain sank. The brains were cut into 30-␮m sec- and Watson was done on left side of the brain using a 26-gauge 10-␮l tions containing the Str and the SN on a cryostat (Thermo Shandon Ltd., UK). Every Hamilton microsyringe mounted vertically on the stereotactic frame. The syringe fourth section was selected from the region spanning from −5.20 mm to −5.80 mm was lowered through the burr hole, and the toxin was infused at a rate of 0.5 ␮l/min in the SN and from +1.70 mm to +2.30 mm in the Str with respect to the bregma with a syringe pump, giving a total volume of 4 ␮l. The needle was left in the brain free-floating sections were quenched for 10 min in 0.3% H2O2/PBS and for at least 5 min to prevent back filling along the injection tract rinsed in a 1:200 dilution of concentrated IHC Wash Solution with distilled water Rats with successful lesions were typically slower in their general activity and for 5 min. Rinsing was repeated 3 times. All sections were soaked in nonspecific had a tendency to turn toward the ipsilateral lesion side, but had a tendency to turn antibody binding solution that was blocked by Ready-To-Use IHC Blocking Solu- toward the contralateral side after apomorphine injection effectiveness of tion for 15 min. The sections were subsequently incubated with a 1:1000 dilution the MFB lesion was verified by an apomorphine-induced rotational test at 2 weeks of rabbit primary anti-TH (cat #AB125, Millipore) with Ready-To-Use IHC Antibody following the lesion f apomorphine-induced contralateral rotation behavior Diluent for 15–18 h at room temperature and then incubated in IgG anti-Rabbit did not occur, the rat was excluded from further statistical analysis.
IHC Antibody (Bethyl). Immunostaining was visualized by peroxidase reaction withstabilized, metal-enhanced diaminobenzidine for approximately 5–10 min with 2.3. Behavioral tests enhanced visualization by hematoxylin and, finally, bluing solution for 1–2 min. Sec-tions were mounted on chromalum-coated slides, dehydrated in ascending alcohol Three motor behavior tests (gait, bar and drug-induced rotation) were per- concentrations, cleared in xylene and coverslipped in DPX. The TH-positive neurons formed in same sequence on same day. For each test, there were at least 2 h of in the SN from both hemispheres were counted manually in each section loss resting time between each test.
rate of TH-positive cells in the lesion hemisphere was calculated and normalized asthe percentage of TH-positive neurons with respect to the unlesioned side.
2.3.1. Spatiotemporal analysis of gait patterns A walking track equipped with a video-based system was modified from pre- 2.5. Experimental design and statistical analysis vious studies for acquiring more spatiotemporal parameters of gait in this studywalking track apparatus consisted of a plexiglass chamber 80 (l) × 6 For motor behavioral testing, eight PD-lesioned and another eight normal (w) × 12 (h) cm with a mirror tilted at 45◦ underneath the walking track. The tilted animals were pre-tested (gait, bar and rotation tests) at least two days before



T.-H. Hsieh et al. / Behavioural Brain Research 222 (2011) 1–9 Fig. 1. Characteristics of stepping footprint during locomotion in (A) a pre-surgery and (B) a 42-day post-unilateral PD lesioned rat. Note the shorter step lengths of the
affected side (a) and the unaffected side (b) in the lesioned rat relative to the pre-surgery rat. The stride lengths (c) are also shorter, in contrast to the wider BOS (d) and larger
foot angle (e) relative to the pre-surgery level.
injection to establish baseline data. After induction of a unilateral 6-OHDA lesion, rection post hoc test when the main effect of time was significant. Also, a paired motor behavior test sessions were performed on the first and fourth day post- t-test was performed to investigate the differences between the ipsilateral (unaf- lesion, then at weekly intervals up to 6 weeks (i.e., 1, 7, 14, 21, 28, 35 and 42 fected) and contralateral (affected) sides over the time-course. For bar and rotation days post-lesion) under the same environmental conditions. For immunohisto- tests, a one-factor analysis (time) repeated-measures ANOVA was used to compare chemistry analysis, five out of 25 lesioned rats were sacrificed at each of five pre- and post-values at each time point followed by a Bonferroni correction post specific time points (i.e., pre-lesion and 1, 7, 21 and 42 days post-lesion) for hoc test in PD rats. For immunohistochemistry analysis, a one-way ANOVA was also TH staining.
performed to compare between groups followed by a Tukey's post hoc test. Data For statistical analysis of gait measurements, a two-way repeated measure anal- were analyzed using SPSS version 17.0 (SPSS Inc., USA) with the significance level ysis of variance (ANOVA) was used to test both group (PD versus normal) and time set at p < 0.05 for each assessment. All data were presented as the average ± standard factors. Multiple within-subject comparisons were taken with the Bonferroni cor- error of the mean (SEM).
Fig. 2. Time-course changes in the step length (A) and stride length (B) of the ipsilateral (left) and contralateral (right) hindlimbs were observed over 42 days in hemi-
parkinsonian rats. Asterisks represent significant differences as compared to the baseline data before surgery by using a paired t-test with a Bonferroni correction (*p < 0.05,
**p < 0.01, ***p < 0.001). Significant changes between two hindlimbs are represented with a square bracket (paired t-tests, #p < 0.05, ##p < 0.01, ###p < 0.001). Note that the
step length dropped significantly in the affected side (right) after 4 days post-lesion, and the stride length of the lesioned hemisphere (right) was particularly evident from
the earliest (day 4) until the last (day 42) time points. Data represent the mean (±SEM) length (mm) of the hindlimb during gait.


T.-H. Hsieh et al. / Behavioural Brain Research 222 (2011) 1–9 3. Results
and contralateral limb (F8,56 = 4.89; p < 0.001) groups (F1,7 = 175.80;p < 0.001 in ipsilateral limb and F1,7 = 224.93; p < 0.001 in contralat- 3.1. Spatiotemporal footprint analysis eral limb) and a significant time × group interaction (F8,56 = 5.41;p < 0.001 in ipsilateral limb and F8,56 = 7.09; p < 0.001 in contralat- Eight normal rats and eight PD rats completed gait analysis within 42 days post-lesion. a representative series of In the normal control group, no significant differences were footprint images captured from a pre-lesion rat (and a rat found in all gait parameters across the test time when compared to at 42 days post-lesion (The post-lesion footprints clearly pre-surgery baseline data (all p > 0.05). In PD rats, a post hoc analy- showed shorter steps, especially on the affected side, whereas the sis with a Bonferroni correction in the time effect showed that the pre-lesion animal exhibited a relatively consistent stride length.
step and stride lengths in the affected side gradually decreased and Furthermore, the ventral view of the footprints showed that post- reached a significant difference at 4 days post-lesion (p < 0.05) fol- lesion rats walked with a wider BOS than the pre-lesion rats. The lowed by a progressive increase of severity up to the sixth week of bilateral stride length in the post-lesion animals was markedly observation (p < 0.01). Furthermore, the difference in step length of shorter than that of the pre-lesion animals, as exemplified in the affected side revealed a decreasing time-course trend, with the Compared to pre-lesion measurements, post-lesion data unaffected side showing a slower trend than the affected side.
showed significant differences in the spatial gait parameters of the Regarding the BOS, there was a significant time × group interac- affected (contralateral) side of the lesioned rats. tion (F8,56 = 3.53, p = 0.00). The main effect for time was significant the time-course changes of bilateral step length and stride length. A (F8,56 = 3.45, p = 0.003). A significant group difference was also two-factor ANOVA on the step length over the 42 days showed a sig- observed (F1,7 = 59.48, p < 0.001). shows that post-lesion rats nificant time × group interaction in the ipsilateral limb (F8,56 = 3.30, showed a significantly larger BOS at 7 days post-lesion compared to p = 0.004) and the contralateral limb (F8,56 = 8.24, p < 0.001) as well pre-lesion levels (p < 0.001), with a gradual but persistent increase as significant effects of time (F8,56 = 2.44, p = 0.02 in ipsilateral limb; up to the end of the measurement period (p < 0.001). shows F8,56 = 3.93, p = 0.001 in contralateral limb) and group (F1,7 = 159.97, the time-course change of the foot angle. Much more external rota- p < 0.001 in ipsilateral limb; F1,7 = 452.6, p < 0.001 in contralateral tion was exhibited in both the ipsilateral and contralateral sides limb). For stride length, a two-factor ANOVA showed signifi- one day after the lesion in comparison with the pre-lesion state.
cant effects of time in the ipsilateral limb (F8,56 = 5.77; p < 0.001) Interestingly, the foot angle in the unaffected side reached a sig- Fig. 3. Time-course changes of the BOS (A) and foot angle (B) of the hindlimbs after 6-OHDA injection through 42 days. Asterisks represent significant differences in the
foot angle between measured values and pre-surgery data (*p < 0.05, ***p < 0.001) by using Bonferroni correction post hoc tests. The bracket indicates significant differences
between the hindlimbs of the two sides using a paired t-test (#p < 0.05, ##p < 0.01).


T.-H. Hsieh et al. / Behavioural Brain Research 222 (2011) 1–9 Fig. 4. (A) The time-course changes in the duration of stance and swing phase within 42 days of observation. Note the gradually increased trend in both phases, which was
maintained at relatively high levels until 21 days after PD lesion. (B) The percentage of stance phase in the full gait cycle also shows a gradual increase either in the affected
side (black circle) or the unaffected side (white circle) following 6-OHDA injection. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to pre-operative values with a Bonferroni
correction.
nificant difference at the second time point (4 days post-lesion) (F8,56 = 6.51, p < 0.001), as reflected in the main effect for test days. A and remained substantially stable throughout the remaining time significant group effect was also observed on the changes of walk- points. In contrast, the foot angle of the affected side exhibited a ing speed (F1,7 = 224.07, p < 0.001). The time × group interaction continuous increase over the full observation period.
was found to be significantly different (F8,56 = 320.69, p < 0.001).
With regard to temporal gait parameters after unilateral For post hoc comparisons, the time-course changes dopaminergic lesion, the data in show that the precise dura- of walking speed. A significant difference clearly appears at day 4 tion of swing and stance phase can be clearly observed by high post-lesion (p = 0.011). Interestingly, walking speed showed a con- speed video recording. A significant increase in the stance phase tinuously decreasing trend over the time-course of observation, time could be seen at 14 days post-lesion in the affected hindlimb, although the most dramatic change in walking speed occurred by but no apparent change was seen in the swing phase time until 21 the first post-lesion test. From day 28 to 42, the decrease in walking days post-lesion (p < 0.05). Furthermore, shows that there speed seemed to reach a plateau. The average walking speed was was a significant difference in the percentage of stance phase as 30.8 ± 2.3 cm/s at the pre-lesion state, which was quite different compared to the pre-lesion level in the affected side by 28 days than the final speed (days 28–42) of approximately 8.0 ± 0.7 cm/s post-lesion (p < 0.05). The time-course measurements also indi- (p = 0.004).
cated progressive increase in the percentage of the stance phasein the affected hindlimb, from 69% at pre-surgery to 78% at 42 days post-lesion, as shown in The walking speed of the post-lesion animals was also severely shows the time-course changes of post-lesion paw decreased. A significant difference was observed across time immobility according to the bar test for both limbs. One-way



T.-H. Hsieh et al. / Behavioural Brain Research 222 (2011) 1–9 Fig. 7. Time-measurement changes in the rotational response to apomorphine. Bars
Fig. 5. Time-course changes of walking speed in normal and PD lesion rats while
represent the mean (±SEM) number of net contralateral rotations performed by the rats performed the walkway locomotion test during the 42 days. Note the gradual animals as the total number of full body rotations in 60 min. **p < 0.01, ***p < 0.001 decrease in the walking speed, which started to reach significance at day 4 after 6- as the significant difference compared with the pre-lesion stage with the Bonferroni OHDA injection. Levels of significance: *p < 0.05; **p < 0.01 as compared to pre-values correction post hoc test.
with the Bonferroni correction. Data are expressed as the means ± SEM.
repeated measures of ANOVA revealed a significant effect of time sistent asymmetry in turning behavior difference (p = 0.001). The in the ipsilateral limb (F time-course measurement showed a gradual increase in the rota- 8,56 = 18.27, p < 0.001) and contralateral tion number until 21 days post-lesion, after which a plateau was 8,56 = 10.65, p < 0.001). Following 6-OHDA lesion, both limbs exhibited similar trends of increasing immobility. Compared with reached and the values remained basically unchanged.
the pre-lesion level, the bar test scores showed a statistically signif-icant increase (p = 0.012) at approximately 4 days after the PD lesion 3.4. Histological tests in the affected forelimb, but the unaffected side did not reach statis-tical significance until 7 days post-lesion (p = 0.026). The behavior The results of the TH-immunohistochemistry in the Str and the asymmetry in the forelimbs became evident at day 4 post-lesion SN at pre-lesion and at 1, 7, 21 and 42 days post-lesion are shown and remained statistically significant from day 4 until the end of in At the Str and SN regions, infusion of 6-OHDA induced observation (paired t-tests, t = 3.07, p < 0.02).
a progressive loss of TH-immunoreactive cells. A mild lesion wasalready apparent at 1 day post-injection in the ipsilateral Str and 3.3. Apomorphine-induced rotation behavior SN. A decrease in TH-immunoreactive density on the side of theinfusion was markedly apparent at 1 week post-lesion, a trend depicts the time-course changes in rotation behavior which progressed throughout the course of observation. Six weeks for the test animals from pre-lesion until the end of the test after unilateral 6-OHDA infusion, tyrosine hydroxylase immunore- period. Post-lesion animals revealed significant time-dependent activity in the Str and SN was less detectable on the side of the contralateral rotation to the side of infusion following apomorphine infusion. The quantification of DA neuron loss in the SN at each time point is presented in n our longitudinal analysis, the aver- 8,56 = 21.65, p < 0.001). At the first day after unilat- eral 6-OHDA administration, the rats displayed occasional rotation, age TH cell loss in the SN region was 11.30 ± 1.10%, 67.13 ± 5.28%, but the behavior did not reach a level of significance (p = 0.416).
88.66 ± 4.68% and 95.43 ± 2.97% at 1, 7, 21 and 42 days post- However, at 4 days post-lesion, the injured animals revealed con- lesion, respectively. All lesioned groups had significant differencesin the time factor (one-way ANOVA: F4,20 = 58.29, p < 0.001). TheTukey's post hoc analysis indicated that the survival rates of theTH-immunoreactive neurons reached a significant level after 7 dayspost-lesion (p < 0.001).
The present study investigated time-course changes of motor behaviors, including gait spatiotemporal patterns, conventionalbar and rotational behavioral and TH-immunohistochemistry pat-terns of DA neuron loss for 6 weeks following unilateral 6-OHDAlesion in rats. As compared to the pre-lesion state, animals withthe development of unilateral dopamine depletion exhibited grad-ual reduction in step/stride length and walking speed but anincrease in BOS and foot angle. The gait cycle of stance and swingphases were also affected. Additional behavioral tests, includingapomorphine-induced rotation and the akinesia bar test furtheridentified asymmetric motor behavior during the 6 weeks of grad-ual DA neuron loss following 6-OHDA injury.
Fig. 6. Time-course changes of akinesia observed from the bar test scores of 6-
OHDA lesioned rats. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from the
The time-course observation of gait patterns and behavior pre-lesion time point with the Bonferroni correction post hoc test.
tests helped confirm behavioral compensation and quantify the T.-H. Hsieh et al. / Behavioural Brain Research 222 (2011) 1–9 Fig. 8. Representative microphotographs demonstrating TH-immunoreactive fibers in the SN (A) and Str (B) from animals sacrificed at pre-surgery as well as at 1, 7, 21 and
42 days post-6-OHDA lesion. At pre-lesion, neurons in the bilateral hemisphere (darkly stained TH-positive cells), are present throughout the SN. Note that obvious reduction
of TH-immunoreactive neurons in the lesioned hemisphere (left side) can be observed after 7 days of 6-OHDA lesion.
relative dopaminergic cell depletion at the measurement time variation after day 4, whereas the affected side showed a progres- points, which provided a clearer picture of the development of sively increasing trend until day 28, followed by a plateau for the induced PD from pre-lesion to full symptom manifestation. After remainder of the test (Similarly, the rotational response the unilateral injection of 6-OHDA, gradual impairment in gait (progressively increased up to day 21 and reached a rela- performance reached a plateau at around 28 days post-lesion tively stable plateau. According to our time-course measurement (Similar observations could be found in the bar test of TH-immunoreactive neurons in the SN, the DA loss increased and induced rotational behavior test. Across the 6 weeks of bar slowly from 89% at 21 days to 95% at 42 days post-lesion tests, the immobility duration of the unaffected side showed little indicating that the depletion of DA neurons also reached a plateauat about the same time points of 21 or 28 days. Thus, rats withinfusion of 6-OHDA in the MFB displayed a gradual development ofdifficulties in locomotion, which was well correlated to progressiveloss of nigrostriatal DA neurons.
According to our detailed spatial gait analysis, our seven-day post-lesion data revealed significant modification of step/stridelength and foot angle. At the same time point, the majority of DAdepletion (about 67%) was observed from our TH staining. Inter-estingly, these results concurred with those of previous studiesdemonstrating that approximately 75% of DA cell loss in rats 68% of DA cell loss in PD patients pronounced locomo-tion deficits. Also, the asymmetric gait patterns became evident at4 days post-lesion. The asymmetry in locomotion can be attributedto the significant reduction in step length, stride length and footangle in the affected hindlimb of hemiparkinsonian rats. However,a smaller step length, stride length and foot angle than those of nor-mal rats were also found in the unaffected hindlimb. Although theSN of the intact hemisphere did not experience dopamine deple-tion, the level of gait performance of the unaffected hindlimb also Fig. 9. Time-dependent histogram representing the percentage of neuron survival
showed mild impairment, which has also been reported in a previ- rates determined by quantification of TH-immunoreactive neurons of the SN in the ous study using electromyographic (EMG) recordings lesioned hemisphere as compared to the SN of the intact hemisphere. Values are work also indicated that the intact hindlimb plays an important expressed as the mean ± SEM. Note that TH-immunoreactive neuron loss becomesapparent after 7 days post-lesion as compared to the pre-lesion value (***p < 0.001, compensatory role during locomotion, carrying more weight to Bonferroni correction post hoc test).
support the unaffected side during propulsion a T.-H. Hsieh et al. / Behavioural Brain Research 222 (2011) 1–9 significant increase in the BOS and foot angle of both hindlimbs was of gait initiation, akinesia and locomotion. Deep brain stimulation found in the PD rats. The progressive decrease in walking speed can of the PPN has been proposed as a new therapeutic means for gait explain a compensatory increase in the BOS and foot angle, which restoration in animal models or PD patients the seems to be necessary to increase balance and stability during loco- loss of neurons and the changes of neuronal activity in the PPN following 6-OHDA lesion in rodent studies still reveal certain dis- With regard to gait temporal information, our results showed crepancies the mechanisms of impaired that dopamine deficiency induced significant changes in stance and gait development from animal models may provide a basis for swing duration of the gait cycle after 7 days post-lesion. In addition implementing new therapeutic approaches for functional recovery to the elongation of swing and stance time as compared to pre- of PD subjects, such as repetitive transcranial magnetic stimula- lesion measurements, the gait cycle showed a gradual increase in tion (rTMS), a non-invasive brain stimulation technique, which the percentage of stance phase (The extension of both swing may modulate brain activity and improve motor performance in and stance duration indicated both the slowness of hindlimb move- ment and a strongly reduced walking speed slow steppingpattern in the affected hindlimb might reflect akinesia or hypoki- nesia induced by dopamine depletion, which was supported by theforelimb akinesia observed during the bar test. The extended dura- The present study characterized the time-course development tion of hindlimb swing and stance during locomotion indicated that of rodent gait impairment and the extent of cell loss in the SN the PD rats had difficulty in initiating steps with the affected limb from the immediate post-lesion state to the stable plateau state due to the 6-OHDA injury observation of an ele- following 6-OHDA injection. The unilateral 6-OHDA rat model vated percentage of stance phase and a decreased percentage of study has generated interesting findings regarding the pre-lesion to swing phase has been demonstrated previously in both human full-symptom time-course development of gait impairment, rota- and animal studies can be explained by a delayed onset tional response, bar test behavior and TH-immunohistochemistry.
of the swing phase due to akinesia or muscle rigidity.
The time-course changes in gait impairment started as soon as Compared to dynamic gait analysis, the bar test is useful for eval- day four post-lesion and progressively increased to a peak level uating the motor asymmetry and akinesia of the forelimbs under around four weeks post-lesion; they then persisted at a plateau static conditions increase in the immobilized duration state over the remaining 6 weeks of the test period. The rotational observed in our study confirms the well-known phenomenon that response to apomorphine and the bar test for akinesia also pro- an established nigrostriatal lesion causes akinesia/bradykinesia in vided similar developmental curves. The video-based methodology the bar test with the values of pre-surgery and and the experimental data provide new insight into the progres- unaffected forelimb, we observed that the unilateral 6-OHDA lesion sive changes affecting rodent gait pattern during the development caused akinesia in bilateral limbs but was especially severe in the of nigrostriatal lesions and suggest that the hemiparkinsonian rat contralateral side. Regarding animal behavior, the rats tended to model can be used as an animal model for human Parkinson's dis- avoid the use of the affected forepaws after unilateral DA lesions.
ease. Future researchers may use the presented methodology and Clearly, the forelimbs play an important role in supporting the body data for enhanced understanding of the general mechanisms of PD weight during walking in four-legged animals avoiding and in the development of novel treatment protocols for functional the use of a forelimb would also contribute to the abnormal gait recovery from PD.
patterns, which could be confirmed from our time-course obser-vation of bar test data showing gradual aggravation similar to ourgait data.
Researchers often start to perform therapeutic examination or manipulation after confirmation of the PD rodent model from rota- The authors would like to thank the National Science Council and tional behavior tested at 2–3 weeks post-lesion the National Health Research Institutes of Taiwan for financially data showed that the rotational response presented at day 4 and supporting this work under contract numbers NSC 96-2628-E-269- progressively increased to day 21 post-lesion. These results agree 001-MY3 and NHRI-EX98-9535EI. We also would like thank the with previous findings that the rotational response to apomorphine anonymous reviewers for their constructive comments.
was present at about 3 days post-lesion that sig-nificant dopaminergic cell loss occurred at early time points. In Appendix A. Supplementary data
addition, previous work reported that the minimal dopaminergiccell loss required to elicit a rotational response was about 40–50% Supplementary data associated with this article can be found, in for SN was very close to our TH-immunoreactive the online version, at .
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