| BACKGROUND:Parkinson’s disease(PD), is a progressively neurodegenerative disorder, resulting from the degeneration and loss of dopaminergic neurons in the substantia nigra of midbrain, typical symptoms include resting tremor, muscular rigidity, bradykinesia. About half of the advanced PD patients have the postural instability.gait disorder symptoms(PIGD), and it has a significant adverse impact on a person’s quality life. Although nucleus lesioning, dopamine replacement therapy, high-frequency stimulation at interior segment of globus pallidus, subthalamic nuclei, ventral nuclei of thalamus that can improve the typical symptoms, the effect on postural instability gait disorder is invalidation.Plaha P and his colleagues first reported that low frequency stimulation pedunculopontine nucleus(PPN), the equivalent of pedunculopontine tegmental nucleus in rats, can significantly improve postural instability gait disorder symptoms. Some research shows that PD patients’neuronal degeneration and apoptosis in pedunculopontine nucleus, residual neurons with irregular firing, compensate for the excessive discharges. These indicate that PPN is not only plays an important role in the pathogenesis of PD,but also may be involved in regulation of motor function Deep brain stimulation of pedunculopontine nucleus, has been received more and more attention, to treat PIGD symptoms in Parkinson’s disease. A lot of animal experiments and clinical trials have proven that low frequency stimulation at pedunculopontine nucleus (PPN) could significantly improve postural instability and gait disorder symptoms, but its mechanism remains unknown. Meanwhile the study of low frequency stimulation at pedunculopontine nucleus impact on animal behavior than clinical research lag, making more difficulty to clarify the mechanisms of this effect. It is therefore highly significant to been explored the effects of ethology with low frequency stimulation at pedunculopontine nucleus by animal experiments.Parkinson’s disease rats that received a unilateral injection of6-hydroxydopamine (6-OHDA) and at the same time the particular neuron nucleus have electrodes implanted, it was confirmed the ideal animal model for studying the effect of ethology on deep brain stimulation of the particular neuron nucleus. Currently, There are many behavioral approach(like BBB scale, foot printing, the staircase test, beam balance test) for detecting deviations of Parkinson’s disease rats,but, the behavior data from these experiments are subjective to some extent, the conclusion need for further research. Therefore, an objective way of detecting behavioral approach is extremely important. Catwalk and open field test, relatively ideal behavioral approach detection methods, which avoid artificial intervention were selected in this experiment. These techniques have been extensively applied in animal models of spinal cord injury, cerebral ischemia and Parkinson disease, which have been recognized more and more scholars.OBJECT: To evaluated6-OHDA-lesioned rats for Parkinson’s disease by low frequency stimulation at pedunculopontine nucleus detecting deviations in Catwalk and open field test. To provided experimental basis for clinical treatment of Parkinson’s disease.METHODS:1. Experimental group32adult male Sprague Dawley (SD) rats were divided into2groups according to randomly digital table:control group (6-OHDA-lesioned,8rats) and experimental group(6-OHDA-lesioned and electrode were implanted,24rats).2. Stereotactic operation and implanted electrodeRats were fixed on stereotaxic apparatus, under anesthesia of chloral hydrate through single intraperitoneal injection. Eyes of rats applying erythromycin ointment, avoiding corneal dehydrated during operation. Incisors rod below ear rod3.3mm, keeping bregma lie flush posterior. The skin is prepped with alcohol, and cut along the center of the head with1.5centimeter,3%of the hydrogen peroxide remove periosteum, fully exposed bregma. then according to Paxinos and Watson "rat brain stereotactic atlas" to determine the right forebrain medial longitudinal bundle (MFB)(coordinating from bregma:AP-1.8mm, ML-2.0mm, V-8.3mm). Microinjector was slowly pushed into a predetermined depth, and slow injection10ug/4ul6-OHDA solution (dissolved in physiological saline containing0.02%ascorbic acid),at the same time. the experiment group’s rats were implanted stimulating electrodes under the microscope, according to coordinates of the PPTg map (coordinating from bregma:AP7.9mm, ML2.1mm, V7.0mm), with dental cement fixation after implantation. The end of electrode outboard of skull was welded sockets, which can connect stimulate wires. The rats were given freely food and water after the first week of recovery. And the rats were given dietary control after recovery, with each daily10g, and drinking water is not limited to maintain body weight.3.Low frequency stimulation at pedunculopontine tegmental nucleus6-OHDA electrode rats had a Catwalk automatic gait and open field test detection with line shut down and belt line. Stimulation parameters:frequency of25Hz, single pulse, square wave,dutation80us, amplitude2-8V. Stimulation amplitude of each rat is80%of the individual stimulation threshold. Each rat stimulation from2V,0.1V to increase, each stimulation epoche lasting less than10s with at least1min in between, until the stimulus side effects (such as the rat body to reverse to one side or the head jitter) and this is the amplitude of the individual stimulation threshold.4. Behavioral examinationCatwalk gait and open field test data were acquired at1week before stereotactic surgery and3weeks after operation in the control and experimental group rats; the behavioral data of rats in the experiment group were recorded again4weeks after operation when they were under ongoing LFS-PPTg(1) Open field test:The apparatus used is:diameter80cm, height50cm, plastic alloy synthesized by the reaction of a cylindrical tank mines (Netherlands Noldus company). The rats were placed in the central portion of the reaction tank, keeping quiet surroundings, moderate light. After the rats were adapted to the environment10min,then record the traveled distance, trajectory and speed.(2) Catwalk gait acquisition:Catwalk gait analysis system is an automated computer-assisted gait analysis technology, which mainly consists of the built-in fluorescent glass, high-frequency camera components. When the rats through the glass, the reflection fluorescence from the bottom of the shape of footprints were below the camera to capture, transmit to the computer for offline analysis. Start recording in rats after one week adaptation training data, the recording standard rat continuous, uninterrupted by the channel, and each time by at least10steps, each rat was recorded at least three times.5. Immunohistochemical and Nissle stainingOne day after behavioral testing, rats were generally anesthetized, and the stimulation is turned on through the stimulating electrode, giving a high voltage to leave the electrode tip tract in the brain tissue in order to determine the position of the tip of the stimulating electrode. Open the chest of the rats, perfusing saline irrigation, then perfusing fixed4%paraformaldehyde solution, and the brain was removed and placed in4%paraformaldehyde immersion to be fixed overnight. After that, the brain then was placed in a30%sucrose solution dehydration until it was completely sinked. Fixing and dehydrating the brain tissue to frozen sections, then taking the substantia nigra and pedunculopontine tegmental nuclear slices to TH immunohistochemical staining and Nissl staining to determine the damaged substantia nigra dopaminergic neurons and whether electrode was implanted in the right position.6. Statistical analysisSPSS17.0was used to for the statistical analysis of the data. The measurement data was showed as mean±SD (x±s), the difference was consider statistically significant when P<0.05.Results1. Results of Catwalk gaitRun speed variation and Couplings (right front→left front, right front→left hind, right hind→left front, right hind→left hind) of all rats postoperative have increased significantly compare to preoperative, while Cadence decreased significantly. Run speed variation and Couplings of the6-OHDA electrode group rats by low frequency stimulation at pedunculopontine tegmental nucleus (LFS-PPTg) have a significant reduced in the postoperative, cadence increasing, and the difference had statistical significance (P<0.05). 2. Results of Open Field testMotional distance of all rats postoperative have decreased significantly compare to preoperative, and the difference had statistical significance (P<0.05). The6-OHDA electrode group rats by low frequency stimulation at pedunculopontine tegmental nucleus (LFS-PPTg) have increased significantly in the postoperative,, and the difference had statistical significance (P<0.05).3. The behavior of experimental group and control groupResults of Catwalk gait and Open Field test in the experimental group and control group before the operation had no statistical difference(P>0.05), also the difference postoperative had no significant difference(P>0.05).ConclusionsExercise capacity and other indicators of Parkinson’s disease rats, reflected gait of PIGD in clinic, through low frequency stimulation at pedunculopontine tegmental nucleus had significantly improved. Combined with hemiparkinson’s disease rats, electrodes were implanted at pedunculopontine nucleus, Catwalk gait and Open Field test that adequately reflect changes of symptoms of Parkinson’s and exercise capacity shall be preferable. This study on mechanisms of low frequency stimulation at pedunculopontine nucleus provided behavioral foundations for further research. This kind of behavior research with pathophysiology and simultaneously recorded electrophysiological activity, may provide a new theoretical basis. While it promoted clinical application of low frequency stimulation at pedunculopontine nucleus, relieving the confusion in facing of mental and emotional disturbance. |
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