The Research On Gait Regulation Of Deep Brain Stimulation In The Pedunculopontine Tegmental Nucleus In Parkinsonian Rats

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. The clinical manifestations of PD is motility disorders, such as limb resting tremor, rigidity, bradykinesia and so on. Gait disorder is also a common motor symptoms in advanced PD patients. The patients’ walking speed getting slower and slower, upper limb swing reducing and festination gait seriously affect the quality of life of patients.Deep brain stimulation (DBS), which implanted electrodes in deep brain nuclei by stereotactic surgery, can regulate abnormal electrical activity that causes symptoms by stimulate neural nuclei and improve the patient’s symptoms to achieve a therapeutic effect. DBS is the best surgical treatment for advanced PD, especially for the drug refractory PD patients.There are three main commonly targets for DBS in the PD:thalamotomy ventral intermediate nucleus (Vim), the globus pallidus medial part (GPi) and the subthalamic nucleus (STN). Targets choice primarily depends on the main symptoms to be treated, and should be selected flexibility in accordance with the patient’s clinical symptoms. In generally, Vim-DBS can effectively suppress tremor of PD, but has poor effect on muscle rigidity and bradykinesia. GPi is the most commonly damaged target and GPi-DBS treatment can effectively improve PD patients’ contralateral limb tremor, rigidity, bradykinesia, but is not good for axis symptom, such as gait disorder. STN, adjusting both GPi and SNr in the indirect channel on the movement loop, is the most commonly preferred target for DBS treatment of PD patients. STN-DBS is effective for muscle rigidity, bradykinesia, tremor and can significantly reduce the PD patients levodopa dosage, but also poorly improves axial symptoms such as postural instability and gait disorders. In recent years, Clinical studies suggest that the pedunculopontine nuclear (PPN)-DBS can be effective in improving motor symptoms of advanced PD patients, in particular, gait and posture disorders. Studies have shown that PPN neurons degenerated and discharge irregularly and compensatory abnormal excessive discharge in the PD state; low frequency electrical stimulation of the PPN can significantly improve PD patients with postural instability and gait disorders. This suggests that PPN involved in regulating motor function. The PPN has become a new target for DBS treatment of PD patients with gait disorders and postural instability.The technology of using6-hydroxydopamine (6-OHDA), a neurotoxin, to make Parkinson’s disease model in rats and to study the pathophysiology of PD and to assess the effect of various treatment methods is matured and widely used. There are a lot of behavioral methods to verify the PD rat model assessment and treatment effects, such as the treadmill test, the cylinder test (forelimb asymmetry test), turn-bar test, balance beam walking test, ladder walking test, footprint analysis and so on. But some of these methods confined forelimb function test, some restricted on the forced movement, and some can only test a single dynamic parameters or static parameters, and artificial records are affected by subjectivity. Compared with other behavioral testing methods, Catwalk automated gait analysis system can detect the rats four legs’ claw dynamic and static parameters as well as four-leggs’ claw coordination and detect the spontaneous motor behavior of rats, and computer system acquires and offline analysis data to avoid the subjectivity impact of artificial record. Therefore, more and more scholars are concerned about the use Catwalk automated gait analysis system to evaluate the behavior of the various experimental animal models to learn the characteristics and treatment effects assessment.Pedunculopontine tegmental nucleus(PPTg) in rats is equivalent of the PPN in human. What will change in Parkinsonian rats’ gait? Can PPTg-DBS also improve Parkinsonian rats’ gait? This study is to use Catwalk automated gait analysis system to assess the effect of PPTg-DBS on Parkinsonian rats’gait and further provides the basic experimental evidence for the mechanism of PPN-DBS in improving the PD gait.OBJECT:Using the Catwalk automated gait analysis system to access gait changes of unilateral Parkinsonian rats and to investigate the rescue effect in gait of the rat Parkinson model after deep brain stimulation (DBS) in the pedunculopontine tegmental nucleus (PPTg) and further to provide the basic experimental evidence for the mechanism of PPN-DBS in improving the gait in PD.METHODS:1、Experimental groups:Twenty-seven healthy adult male Sprague Dawley (SD) rats, weight control between280g～320g, were randomly assigned to three groups. The sham-operation group (n=6) received stereotactic injection of saline in the right medial forebrain bundle (MFB). The6-OHDA group (n=6) received to be unilateral Parkinson’s disease (PD) rat model. The6-OHDA+electrode group (n=15) received ipsilateral injection of6-OHDA and simultaneously an electrode implanted into the ipsilateral PPTg. The CatWalk system was employed to investigate changes in gait parameters of all the three groups four weeks after surgery and furthermore to assess gait function before and after the PPTg-DBS in the electrode-implanted group.2、The rats’ Catwalk adaptive training:All rats should be trained to be adaptive to Catwalk for one week before stereotactic surgery. Put rats into the Catwalk channel, training its spontaneous continuous non-stop through the channel. Each rat had a training in the morning and afternoon, with each run across the channel back and forth three times to five times and the entire experimental training in the darkroom. Training eligibility criteria:the rats need continuous non-stop through the channel, at least three times. After qualified training, we acquire Catwalk data for a baseline value.3、Making model and implantating the stimulation electrode:Sham-operation group rats were anesthetized and were fixed in stereotactic coordinates, then according to Paxinos and Watson "rat brain stereotactic atlas" to determine the right forebrain medial longitudinal bundle (MFB)(coordinating from bregma:AP1.8mm, ML2.0mm, V8.3mm). Microinjector was slowly pushed into a predetermined depth, and slow injection4ul saline containing0.02%ascorbic acid.6-OHDA group were injected10ug/4ul6-OHDA solution (dissolved in physiological saline containing0.02%ascorbic acid), with other steps being the same.Beside6-OHDA injection,6-OHDA+electrode 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 daily12-14g, and drinking water is not limited to maintain body weight. 4、Catwalk data collection:Catwalk data acquisition time:all the rats were collected as a baseline after qualified Catwalk training; four weeks after stereotactic surgery.6-OHDA+electrode rats had Catwalk data acquisition before and after PPTg-DBS.5、PPTg-DBS:6-OHDA+electrode rats had a Catwalk automatic gait detection with line shut down and belt line. Stimulation parameters:frequency of25Hz, pulse width80us, amplitude2-6V. Stimulation amplitude of each rat is80%of the individual stimulation threshold. Each rat stimulation from2V,0.1V to increase, 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.6%Histochemical:After all of the detection, rats were generally anesthetized, and the stimulation is turned on through the stimulating electrode, giving a strong 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 placed in4%paraformaldehyde immersion to be fixed overnight. After that, the brain then was placed in a25%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.7、Statistical Methods:Using the SPSS19.0statistical software to analyze data and measurement data was performed with "the mean±standard deviation". Comparing with the numbers between the two groups were performed with two independent samples t-test. The comparison before and after the stereotactic surgery and the PPTg-DBS were performed with paired samples t-test.And the difference of P<0.05was considered statistically significant.RESULTS:1、For Sham-operated rats, comparing stereotactic postoperative with preoperative (4weeks), four claws’ stride, swing speed, support phase time, the time ratio of the maximum contact area, maximum contact area, the average pressure of these parameters were no significant difference (P>0.05). And the reaction of four claws’ coordination parameters:support base, the normal step sequence mode, walking support mode index parameters of the law were also not statistically significant (P>0.05).2、6-OHDA group compared with the sham group:the four paws’stride, swing speed is reduced, supporting increased relative to time and time ratio of the maximum contact area, and the differences were statistically significant (P<0.05). However, both the two parameters, maximum contact area and mean pressure, only in the ipsilateral (left) front and rear paws reduce statistically significant, while the contralateral (right) front and rear paws of these two parameters, the difference was not statistically significant (P>0.05). The support base of the front paws showed no significant difference (P>0.05), but the hind claws of the support base became bigger, statistically significant difference (P<0.05). Both the parameters, walking support mode and the regularity index, which fleet four claws’coordination of the normal step sequence patterns, were not statistically significant (P>0.05).3、6-OHDA+electrode group compared with6-OHDA:four claws’ stride, swing speed, support phase time, time of maximum contact area ratio, the maximum contact area, mean pressure differences of these parameters was not statistically significant (P>0.05); Both the two parameters, which reflect four claws coordination were no significant difference (P>0.05); However, normal step sequence mode, walking support mode the difference was statistically significant difference (P <0.05).4、Comparing before PPTg-DBS with after PPTg-DBS in the6-OHDA+the electrodes group rats, four claws’s stride, swing faster, support phase time, reduced time ratio of the maximum contact area, the difference was statistically significant (P <0.05); Ipsilateral (left) before and after the claw maximum contact area and mean pressure increase, there is a statistically significant difference (P<0.05) and contralateral (right side) of these two parameters change is not statistically significant (P>0.05). Meanwhile, the base of support of the hind limbs restored (P <0.01); However, the type of normal step sequence patterns accounted PPTg-DBS walking support mode and regular index no significant difference (P>0.05).CONCLUSION:1、Catwalk automated gait analysis system allows automated and objective quantification of a large number of dynamic and static gait parameters and interlimb coordination.2、Established unilateral6-OHDA damaged PD rat model for the study of PPTg-DBS improving the gait disorder.3、The PPTg-DBS can effectively improve gait function of unilateral PD rat model.4、Implantation of PPTg electrodes may change the regular step sequence and support formula.