Gait Analysis In Three Different 6-hydroxydopamine Rat Models Of Parkinson’s Disease

BACKGROUND:Parkinson’s disease (PD), a pervasive motor disease, results from the depletion of dopamine (DA) in the nigrostriatal region of the Central Nervous System. As PD progresses, gait disturbance and postural instability become increasingly prominent. Thus, ameliorating gait disturbance is vital for PD patients to prevent unexpected injury, disability, and improve their qualities of life.The 6-hydroxydopamine (6-OHDA) induced rat model of PD has been extensively used as an animal model to mimic PD patients in basic science studies. The injection of 6-OHDA into different sites of the brain, i.e., the caudate putamen (CPU), the medial forebrain bundle (MFB) or the substantia nigra compact (SNC), causes different extent of the lesion in the corresponding brain area. Meanwhile, the lesion process in these three injection models can be quite different, leading to various kinds of performance of gait deficits. To quantify these deficits, experimenters use behavioral tests including the open field test, the rotarod test, the treadmill test, the cylinder test, and the ladder walking test. However, the accuracy of readouts can be affected by many factors. For example, timing of testing (day or night), training intention and frequencies, and testing environment are very important factors that affect the behavior performance. Animals behaviors tested in an involuntary state are hardly repetitive. Test indicators that are limited to monitor either static or dynamic state cannot adequately display both readouts simultaneously, and so on. Therefore, although great efforts have been made to measure the gait deficits in these 6-OHDA PD models, the validation and characterization of gait variability is still lacking.OBJECT:In this report we validated gait variability in three different rat PD models of unilateral 6-OHDA lesions using the CatWalk device. As the motor deficits are the direct consequence of DA loss in the nigrostriatal system, we evaluated the temporal expressions of tyrosine hydroxylase (TH) protein in the SNC and CPU of 6-OHDA treated rats and assessed the neuro-degeneration in the nigrostriatal regions by studying the correlation of TH expressions with changes of gait parameters.METHODS:1、Experimental groups:Adult male Sprague Dawley rats, weighing 290-310g at the time of surgery, were housed 2-3 per cage with ad libitum access to food and water during a 12 h light/dark cycle. The rats were randomly divided into three groups. Each group was comprised of 12 rats.2、Gait analysis:All rats were trained to cross the runway in a consistent manner at least six times a day for a week before any experimentation. A successful run is defined as an animal finishes running the tracks without any interruption or hesitation. Rats that failed the CatWalk training were excluded from the study. An average number of 5 replicate crossings made by each rat was recorded. Rats were subjected to computer-assisted Cat Walk from day 26 to day 30 after administration of 6-OHDA.3、Surgical procedure:All rats were anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg). Rats were placed in a stereotaxic frame (Stoelting) and 6-OHDA were injected into the right brain using a 10 ul Hamilton syringe fitted with a glass capillary. Partial lesion of the nigro-striatal pathway was obtained by injection of 3ul of 6-OHDA (3.5ug/ul free base dissolved in a solution of 0.2 mg/ml L-ascorbic acid in 0.9%w/v NaCl) (Sigma) (0.5 ul/min) in the CPU at the following coordinates (flat skull position):antero-posterior:+1.2mm, medio-lateral:-2.5mm, dorso-ventral:-5.0mm below dural surface, calculated relative to bregma according to the stereotaxic atlas of Paxinos and Watson (1986). Severe lesion of the nigro-striatal pathway was obtained by injection of the same dose of 6-OHDA in the MFB at the following coordinates:antero-posterior:-4.4mm, medio-lateral:-1.1mm, dorso-ventral:-7.8mm or above the SNC at the following coordinates: antero-posterior:-5.3mm, medio-lateral:-1.7mm, dorso-ventral:-7.2mm.4、Tissue processing:All animals were sacrificed 30 min after the last behavior test. After being anesthetized, rats were perfused through the ascending aorta with 250ml saline (0.9% w/v) at room temperature, followed by 250ml ice-cold paraformaldehyde (4% w/v in 0.1 M phosphate buffered saline). The brain was removed, post-fixed for 12h in 4%paraformaldehyde and cryoprotected overnight in sucrose (25% w/v in 0.1M phosphate buffered saline) before being sectioned on a freezing microtome (Leica). Coronal sections were collected in 6 series at a thickness of 20 um.5、Immunohistochemistry:Immunohistochemical staining was performed on sections using antibodies raised against TH (mouse,1:1000; sigma). Sections were rinsed three times in potassium-phosphate buffer (KPBS) between each incubation period. The sections were quenched for 10 min in 3% H2O2/10% methanol. One hour of pre-incubation with 5% normal goat serum was followed by incubation overnight with the primary antibody in 2% serum at 4℃ and incubation with 1:200 dilution of biotinylated goat anti-mouse antibody, followed with avidin-biotin-peroxidase complex, and visualized using 3,3-diaminobenzidine (DAB) as a chromogen.6、Cell counting and optical densitometry analysis:Assessment of the total number of TH+ neurons in the SNC was made according to the optical fractionator principle, using the Image-Pro Plus (Media Cybernetics, Inc., USA). Every 6th section covering the entire extent of the SNC was included in the counting procedure. A coefficient of error of<0.10 was accepted. CPU TH+ fibre density was measured by densitometry at four coronal levels (+1.2, 0.8,0.00 and -0.4mm). The measured values were corrected for non-specific background staining by subtracting values obtained from the cortex. The data are expressed as a percentage of the corresponding area from the intact side.7、Statistical Methods:All the data was expressed as mean+standard error of mean (SEM). The comparison before and after surgery was performed with a paired-samples t-test. One-way ANOVA analysis followed by Bonferroni post hoc test was used to determine inter-group and intra-group differences. The correlations of the TH expression levels in the SNC and CPU with the motor parameters in all tests were evaluated by Pearson’s product-moment correlation coefficient. A p value<0.05 was considered as to be statistically significant. All of the data was analyzed using the SPSS 17.0 software (SPSS Inc, Chicago, USA).RESULTS:1、Degeneration of striatal DA fibres was assessed by optical densitometry of TH immunostained forebrain sections. Similar to the stereological cell counts, ANOVA analysis revealed a severe loss of terminals when 6-OHDA was injected in the MFB and SNC (7.7±3.5% for the MFB group and 12.7±8.1% for the SNC group; P<0.05 compared to intact side), while CPU 6-OHDA injection induced a moderate loss of CPU fibres (35.4±14.6% for the CPU group; P<0.05 compared to intact side). Degeneration of striatal DA fibres was assessed by optical densitometry of TH immunostained forebrain sections. Similar to the stereological cell counts, ANOVA analysis revealed a severe loss of terminals when 6-OHDA was injected in the MFB and SNC(7.7±3.5% for the MFB group and 12.7±8.1% for the SNC group; P<0.05 compared to intact side), while CPU 6-OHDA injection induced a moderate loss of CPU fibres (35.4±14.6% for the CPU group; P<0.05 compared to intact side). The lesion induced by injection of 6-OHDA in the MFB and SNC was significantly more remarkable than that in the CPU(P<0.05), while no difference was observed between 6-OHDA injection in the MFB and SNC (P>0.05).2、Gait analysis parameters collected by the Cat Walk system were first analyzed to compare to the pre-test. The max contact area, mean intensity, stride length and swing speed decreased, whereas the stance, step cycle, duty cycle, terminal dual stance and bases of support increased in varying degrees in the three groups.3、On the other hand, all parameters in the MFB group took a more profound deterioration than the CPU group. Similar to the MFB group, there was also significant difference in all parameters between the SNC and CPU groups except the duty cycle. However, readouts of gait parameters of mean intensity, stride length, swing speed, step cycle, duty cycle and terminal dual stance of the MFB and SNC group were significantly different (all P<0.05).4、Significant contralateral (’lesioned’) vs. ipsilateral (’nonlesioned’) differences were observed in the max contact area, mean intensity and terminal dual stance of the MFB and SNC groups (all P<0.05). There was significant difference of max contact area between the contralateral and the ipsilateral fore paw (contralateral< ipsilateral, P<0.05) and terminal dual stance (contralateral> ipsilateral, P<0.01) in the CPU group. Besides, other parameters were similar between the contralateral and the ipsilateral side.5、Significantly positive correlations exist between TH levels and values of swing speed, stride length of all limbs, max contact area, and mean intensity in the left limbs. Substantially negative correlations between TH levels and values of stance, terminal dual support, step cycle, duty cycle in all limbs, and base of support were noted.CONCLUSION:1、The gait readouts in the MFB and SNC 6-OHD A rats are more profound than the CPU.2、Many gait parameters show a close correlation with the protein levels of TH.3、Catwalk system can provide reliable and objective criteria to stratify gait changes arising from 6-OHDA rats.