| BACKGROUND:Parkinson’s disease (PD) is a common, progressive and neurodegenerative disease of the central nervous system in the elderly, whose main pathological features is dopaminergic neuronsdegradation in the substantia nigra pars compacta (SNc) of the brain. Statistics show that incidence of PD exceeds 1% in the population over 65 years of age and affects severly the life of the elderly. The clinical manifestations of PD include mainly resting tremor, rigidity and bradykinesia. In advanced cases, postural instability and gait difficulty (PIGD) is also a common motor symptoms, which often disables the patients.The clinical therapy of PD include medication and surgery. The surgery has developed preceding the application of levodapa. The fomer has recently prevailed for drug refractory motor complication of advanced PD. In early stage, the surgery was through damaging stereotacticly central nucleus, centering in globus pallidus internus (GPi), ventral intermediate nucleus (Vim) and subthalamic nucleus (STN). Later, deep brain stimulation (DBS) becomes a atternative method, which is adjustable and reversible. Implanting electrodes in deep brain nuclei and stimulating the nuclei can regulate abnormal electrical activity that causes symptoms, hence improve the patient’s symptoms. So far, DBS has become the best surgery therapy for the advanced PD. Although Vim-DBS can effectively suppress tremor of PD, STN/GPi-DBS can effectively improve PD patients’ contralateral limb tremor, rigidity, bradykinesia, but all the treatments above have poor effect on PIGD. Recently, studies suggest that PIGD is related to dysfunction of PPN. The PPN has been recently proposed as a potential target for the treatment of PIGD in PD.Catwalk is an automated gait analysis system, which can detect objectively the dynamic and static gait parameters during animal moving. Catwalk method has been used for evaluating gait in series of animal model (such as pain model and spinal injuries)In spite of the increasing interest in PPN-DBS, however, it remains unclear that exact action mechanisms of PPN-DBS responsible for affecting PIGD in PD. We supposed that determining the effect of PPN-LFS on neurotransmitter levels in ventrolateral and Ventroanterior thalamus nuclei complex (VA/VL) is crucial for understanding the underlying mechanisms of PPN-LFS improving gait. The reasons are as follows:1. PPN mainly includes cholinergic, glutamatergic and GABAergic neurons. A significant loss of cholinergic neurons was observed within the PPN of PD paitients and PD animal model, which is related to PIGD.2. Basal forebrain mainly consist of cholinergic projection and receive cholinergic projection from PPN.3. Motor thalamus receive cholinergic projection from PPN and forebrain.4. Motor thalamus is connected to multi-aera of cortex, which plays an important role in integrating the information related to motor from basal ganglia, cerebellum and cortex.5. Locus coeruleus mainly consist of noradrenergic neuron, which exists degeneration in PD and PD animal model.Base on the mentioned above, first, it is helpful for understanding the behavioral effect of PPN-DBS that evaluating the PPN-DBS whether can improve gait defects in PD animal; secondly, the brain structure related to PIGD is connected to motor thalamus. The abnormal function of motor thalamus may be involved in PIGD. Hence, investigating the relation between neurotransmitter level motor thalamu and PPN-DBS may be crucial for understanding the underling mechanism of improving PIGD.OBJECT:Using the Catwalk automated gait analysis system, investigating gait in unilateral Parkinsonian rats and gait effect of PPN-DBS to furthering the understanding of behavioral effect of PPN-DBS in rodents. Evaluating the effect on neurotransmitter in motor thalamus (VL subregion in the current study) of PPN-DBS to further the understanding of underling mechanism of improving PIGD. The glutamic acid (Glu), y-aminobutyric acid (y-GABA), acetylcholine (ACh) and noradrenalin (NA) level were evaluated in the current study.METHODS:1. Experimental groups:Thirty-eight healthy adult male Sprague Dawley (SD) rats, weight control between 280g-320g, were randomly assigned to three groups. The group A, sham-lesioned group (n=10), receiveed stereotactic injection of saline in the right medial forebrain bundle (MFB); the group B, lesioned group (n=14), received stereotactic injection of 6-OHDA (4μl,3μg/μl) in the right MFB; the group C, the lesioned+electrode group (n=14) received injection of 6-OHDA in right MFB and an implantion of electrodes into the ipsilateral PPN simultaneously. All rats received implantion of microdialysis probe.2. Collecting gait baseline using catwalk:Prior to surgery, all rats were trained for a week to adapt for catwalk. That is the rat was placed in catwalk, and trained to run through the corridor spontaneously without stop (two times in the morning, two times in the night). Award the rats meet with the criteria that run through the corridor spontaneously without stop and more than 5 times. The trainning environment keep silent and dark. After trainning, detect the gait of all rats for gait baseline.3. Stereotactic surgery:Surgery 1, the group A rats were anaesthetized and placed into a stereotactic apparatus using a non-puncture ear bar with the nose position at 3.3 mm below the interaural line, received vehicle stereotactic injections (0.9% saline and 0.02% ascorbic acid,4μl) in MFB at the following coordinates relative to Bregma, AP-2.0mm, ML 2.0mm, DV -8.0mm (from the dura mater), according to the brain atlas of Paxinos and Watson (2008). The group B and C rats received injections of 6-OHDA (12ug/4ul,0.9% saline and 0.02% ascorbic acid).Surgery 2, after 2 weeks, the group B and C rats were anaesthetized again and placed into a stereotactic apparatus using a non-puncture ear bar with the nose position at 3.3 mm below the interaural line, received implantion of microdialysis probe in VL at the following coordinates relative to Bregma, AP -2.28mm, ML 2.4mm, DV-5.4mm. Determine the PPN coordinates relative to Bregma, AP-7.9mm. The group C rats received a slow implantation of electrodes. At last the implanted microwire electrodes and microdialysis probe were embedded in dental acrylic with the anchoring screws to form a headstage on each rat’s head. After the surgery, the rats recovered for 1 week.4. Detect the gait parameterPrior to detecting, train the rats for 5 days using catwalk method, strengthen the memory of rats. The following 2 days, detect the gait parameter of all the rats, 4times/day. The rats must run through the corridor no less than 5 time. The group C rats receive electric stimulation for 15min, then were placed in the runway for 5 min and receive electric stimulation simultaneously.5. Electric stimulation procedureThe threshold for stimulation-induced behavioral side-effects, such as twisting to one side or erecting the whiskers, was determined at a frequency of 25 Hz and pulse width 80μs in a stair step procedure (from 20μA, in 5μA steps). Stimulation started with a current of 20μA below the individual threshold (25Hz,80μs). The rats with electrodes were test for gait under the conditon that with stimulation and without respectively.6. Microdialysis procedureProbe activition:(1) The probe was fixed on the bracket and was infused with artificial cerebrospinal fluid (ACSF) to emptying air bubbles, when connecting the tube,the tube linked to outlet end should not be too long (about 5 cm), otherwise the semipermeable membrane was easy to expand under the great pressure.(2) Place the probe, joint pipe and adaptor in the ethyl alcohol (70%). The probe was infused with alcohol for at least 1 h at the rate of 1μl/min. The flow velocity is less than 10μl/min. Then the probe was infused with ACSF for at least least 1 h at the rate of 1μl/min.Sample collection:Collect samples once every 30 min. Samples collected in the first 2h were discarded. The following 2 samples were regarded as baseline. Then collect additional 6 samples. At last, the samples have been placed in refrigerator (-80 degrees Celsius) until to detect.Determination of recovery rates:The probe was placed in ASCF (including standard substance) which concentration is set to 0.5nm, lnm,5nm, 10nm and 20nm respectively. Then the probe was infused with ASCF (no standard substance) at the rate of 2μl/min. At every concentration level,4 sample was collected. Every sample volume was 40μl. The recovery rates=concentration of collecting dialysis fluid/concentration of ASCF×100%.7. Liquid chromatography:Liquid chromatography for Glu andy-GABA:Hypersil ODS chromatography column (4.0mm×125 mm, size 5Lm); mobile phase (A):10mM Na2HPO4-NaH2PO4, PH7.2, phosphate buffer (PB), containing 0.5% tetrahydrofuran (THF); mobile phase (B):PB-methanol-acetonitrile (volume ratio 50:35:15); detection wavelength:0 min, excitation wavelength of 340 nm, the emission wavelength of 450 nm, after 20.5 min, the wavelength switch to the excitation wavelength of 260 nm, the emission wavelength of 305nm.8. Liquid chromatography and liquid mass:Mass spectrometry conditions for ACh:m/z 146.1, collision energy 12eV, fragmentor 72V, electrospray ion source, positive ion MRM mode; mass spectrometry conditions for NA:m/z 170, collision energy 4eV, fragmentor 40V, electrospray ion source, positive ion MRM mode.Chromatography conditions:C18 chromatography column (2.1mm×50 mm, size 1.8μm); mobile phase (A):acetonitrile; mobile phase (B):5mM ammonium acetate+0.1% formic acid; gradient elution conditions:0～2 min,100%B,2～2.4 min, 100%～20%B,2.4～2.6 min,20%～100% B,2.7～5.0 min,100%B; flow rate 0.3 mL/min.9. Histochemical:At the end of the experiments, each animal was subjected to the same mesthesia as described for the surgery session. Carry out electrical stimulation for 50s (30μA), so that the electrode tip tract was leaved in the brain tissue. Open the :hest of the rats, perfuse saline irrigation, then perfuse fixed 4% paraformaldehyde solution, and the brain was placed in 4% paraformaldehyde to be fixed overnight. Then the brain then was placed in a 25% sucrose solution for dehydration. After rozen section, Sections with the PPN or VL were stained with Cresylviolet to reveal he stimulation sites or probe sites; the sections containing the SNc were processed for immunohistochemical staining for tyrosine hydroxylase.10. Statistical methods:SPSS 17.0 statistical software was used to analyze data. Measurement data are given as"the mean ± standard deviation". And p<0.05 was considered statistically significant. The data concerning gait among the multiple group was evaluated using one-way analysis of variance (ANOVA). The data between the two condition of PPN-DBS and without DBS in the same group was evaluated using paired t-test.RESULTS:1. Gait analysis result:Compared to the sham-lesioned rats, the 6-OHDA-lesioned rats displayed a significantly increased base of support (BOS), and Duty cycle, but a remarkable reduction in Stride length, Maximum contact aera and Intensity (left forelimbs). Compared to pre-DBS, PPN-DBS improved sigficantly BOS and Max aera, however had no significant effect on the other gait parameters.2. neurontransmitter result:Compared to the sham-lesioned rats, the 6-OHDA-lesioned rats displayed a significant increase both of glutamic acid (Glu) and γ-aminobutyric acid (y-GABA) concentration in VL, a apparent decrease of acetylcholine (ACh), no changes of NA. PPN-DBS improved sinificantly Glu level in VL in lesioned rats, and improved unsignicantly ACh concention, but did not affect NA and GABA level.CONCLUSION:The current study showed PPN-low frequency stimulation (LFS) partially improved gait deficiency in PD rats, supporting the proposal that PPN-LFS is useful for treating gait deficits of PD, and suggested PPN-LFS rebalance Glu and ACh levels in the PPN-VL pathway, which yield a possible insight into the potential mechanisms underlying PIGD in PD. |
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