Expression Changes Of Glutamate And Glutamate Receptor-1 In The Vestibular Nucleus Of Mouse Of Parkinson's Disease

ObjectiveParkinson's disease(PD) is a extrapyramidal disorder in the elderly characterized by tremor,muscle rigidity,bradykinesia and abnormal posture, affecting primarily the substantia nigra (SN) of the midbrain. In normal conditions, dopamine(DA) in the substantia nigra pars compacta (SNC) inhibits the excitatory effects of acetylcholine(Ach) in the striatum. The degeneration of dopaminergic neurons, declining levels of dopamine in the striatum, induced excessive output of excitation and the resultant boost in skeletal muscle's activity, which is believed to underlie the manifestations such as muscle stiffness, bradykinesia and postural instability in patients with PD. In recent years, in addition to studies of the association between DA and Ach, more attention was paid to the role of Glu in pathological mechanisms of PD. The excitatory neurotoxicity of Glu may be involved in the pathogenesis of PD.Vestibular system plays a key role in balance adjustment of body and postural reflexs. The previous clinical findings suggested that the disorder of balance and the diappearing of postural reflexs were connected with vestibular dysfunction in PD and apart from impairments of the SN and the striatum, the postural instability and the disorder of balance were linked with changes in vestibular function in this disease. A large number of experiments indicate that 1-methy-4-phenyl-1, 2, 3, 6-tetrahydropyri-dine(MPTP) can cross the blood-brain barrier and is thus selectively toxic to dopaminergic neurons in the SN to induce typical PD. Therefore, the present study using the MPTP model mice of PD and performing immunohistochemical technique, image analysis coupled with transmission electron microscopic technique to examine the expressions of Glu,GluR₁ and ultrastructures in the vestibular nuclei(VN), aims to provide with experimental data for further research in the pathogenesis of PD.Methods1. AnimalsWe used 30 nomal adult C57BL/6J mice (male, weighing 20 to 25 g, purchased from Experimental Animal Department of China Medical University) with food and water provided ad libitum. Protocols confomed to the China Medical University Guide for the Treatment of Laboratory Animals.2. PD mouse modelThese nomal adult C57BL/6J mice were divided randomly into the control group (n=6) and the experimental group(n=24). We treated the mice in the experimental group with 0.2ml MPTP (30mg/kg, i.p.), once a day, for 7days, and observed behavioral changes of these mice after administration. In the fourth day, the mice displayed trunk tremor, piloerection, limb stiffniss, tail hyperextension, standing instability, bradykinesia. The mice in the contol group were treated with nomal saline (i.p.).3.Nissl staining location for vestibular nuclei (VN)The perfused and fixed brain issue sections were dewaxed routinely, transferred into water, stained in carbol-thionine solution, colour-differentiated, dehydrated to be transparent in 80% alcohol, sealed with neutral gum and at last observed under a microscope.4.Immunohistochemical SABC detection of TH expression in thesubstantia nigra(SN), striatum and vestibular nuclei(VN)Mice in the control group and the experimental group were anesthetized, perfused and fixed respectively. Brains were removed, postfixed, transferred into 4% paraformaldehyde (PFA), and then cut into paraffin sections at 7μm thick. SN,striatum and VN were treated with TH immunohistochemical staining procedure respectively, then observed under a optical microscope, photographed and at last treated with image analysis and statistical analysis.5. Immunohistochemical detection of Glu and GluR₁ expression in thevestibular nuclei (VN)The mice in the control group and the experimental group were anesthetized , perfused and fixed repectively. Brains were removed, postfixed, then transferred into 4% PFA and made into paraffin sections at 7um, which were treated with Glu and GluR₁ immunohitochemical SABC procedure and subsequently were observed under a optical microscope. Micrographs were taken and then treated with image analysis and statistical analysis.6. Transmission Electron Microscopy (TEM) detection of the ultrastruc-tural changes in the substantia nigra(SN), the striatum and the vestibularnuclei (VN)The SN, the striatum and the VN of the mice in the control group and the experimental group were respectively fixed with 2.5% glutaraldehyde, postfixed with 1% osmium acid, embedded in Epon812 and cut into sections at very small thickness, which were stained with uranyl acetate and lead citrate. Then, the ultrastructures were observed under a transmission electron microscope.Results1. The immunohistochemical results(1) The immunohistochemical results of THAt a higher magnification, after incubation with specific TH antibody in the control group, we observed that the immunolabeling was localized in the substantia nigra pars compacta(SNc), distributed in wedges, expressed in the membranes ,cytoplasm, dendrites of neurons and yellow-brown granular substances were ditributed unifomly in the cytoplasm. While in the experimental group, the immunolabeling in the SNc was distributed in wedges as well, but TH immunoreactive neurons were decreased significantly,dendrites were reduced relatively and the colour of cytoplasm was lighter.At a higher magnification, in the control group, we observed that TH immunolabelling in the striatum was localized in the fibers among neurons, ditributed in fascicles and a large number of fibers took on yellow-brown reticular substances which were evenly stained in the cross-section. While in the experimental group, TH immunolabeling was deceased significantly, fibers were reduced markedly and yellow-brown substances were in spotted, filamentous and linear shape.Under a optical microscope, TH immunolabelling was present only in the lateral vestibular nuclei (LVN) in mice. At a higher magnification, after incubation with specific TH antibody in the control group, we observed that the immunlabelling was localized in the membranes, cytoplasm and dendrites of neurons, yellow-brown granular substances were distributed uniformly in the cytoplasm, the cytoplasm was lighte blue. While in the experimentai group, TH immunoreactive neurons were deceased significantly in the LVN and the colour of cytoplasm was lighter.(2) The immunohistochemical results of GluAt a higher magnification, after incubation with specific Glu antibody in the control group, we observed that immunolabeling was localized in the somas, membranes and dendrites in the lateral vestibular nuclei (LVN), uniformly distributed yellow-brown granular substances in the cytoplasm, immunoreactive particles absent in the nuclei, light blue chromatin. Compared with the control, neurons of the LVN in the experimental group were inceased to some extent, dendrites were reduced, the immunoreactivity was inceased.(3) The immunohistochemical results of GluR₁At a higher magnification, in the control, GluR₁ immunolabeling was yellow-brown and localized in the somas, membranes, dendrites and axonal terminals of neurons in the LVN. Compared with the control, in the experimental group, GluR₁ immunoreactive neurons were significantly decreased, immunoreactive particles distributed scatteredly in the cytoplasm of neurons, the staining of cytoplasm was lighter, neuronal shape was not uniform and the border was hard to identified.2. The Transmission Electron Microscopic resultsThe transmission electron microscopic(TEM) results showed that in the control group, in the SN , the neuronal somas were large, the nuclei were in round, regular shape, the structures were intact, the chang chromatin was in large numbers. While in the experimental group, in the SNc, the ultrastructures of neurons displayed pathological changes: in the cytoplasm of neurons, we observed that the swelling degeneration of profuse mitochondria, ridge disappeaing, vacuolization,dilation of capsular cavities of partial RER, degranulation,shinking deformation of nuclei.In the control group, in the striatum, the boundary of nuclear envelope of neurons was clear, the structures were intact, rough endoplasmic reticulum and ribosome were in large numebers in the cytoplasm. While in the experimental group, in the striatum, we observed that dilation of capsular cavities of profuse RER, degranulation in the cytoplasm of neurons, shinking deformation of nuclei, under nuclear envelope, chromatin concentrating into masses, heterchromatin increasing.The ultrastructures of neurons in the LVN in PD mice displayed pathological changes: in the cytoplasm of neurons, we observed that swelling degeneration of profuse mitochondria, ridge disappearing, vacuolization; dilation of capsular cavities of partial RER, degranulation; shrinking deformation of nuclei; under nuclear envelope, chromatin concentrating into masses, heterchromatin increasing.3. Image analysis and statistical analysisThe results of image analysis showed that the expression of Glu in the LVN in PD mice was increased and was different significantly from that in the control group, as well as the expression of GluR₁(p<0.01), which was reduced. Conclusions1. The TH immunohistochemical changes of dopaminergic neurons in the substantianigra pars compacta (SNc) are consistent with dopaminergic fibers in the striatum in PD, which indicates that MPTP is toxic to the C57BL/6J mice and PD model has been developed successfully.2. The changes of dopaminergic neurons in the LVN in the PD mice implies that thenuclei may be involved in the pathogenesis of PD .3. The decease and incease of expressions of Glu and GluR₁ in the LVN in the PD mice have provided liable morphological basis for the vestibular dysfunction in PD.