| BackgroundFragile X syndrome (FXS) is caused by chromosomal abnormality, is one of the most common forms of inherited mental retardation,and the case rate is just second only to down syndrome . Fragile X syndrome (FXS) results from a trinucleotide repeat (CGG) expansion in the 5'untranslated region of the gene FMR1 and subsequent hypermethylation which lead to transcriptional silencing of FMR1 and loss of its encoded protein, the fragile X mental retardation protein (FMRP). The main symptoms of FXS include facial abnormality, macro-orchidism, mental retardation and high susceptibility to epilepsy.It is reported that epilepsy occurs in 20 to 25% of individuals with FXS and paroxysmal EEG abnormality presents in about 50% of prepubescent boys with FXS. It has been found that FMRP is a brain specific mRNA binding protein that suppresses translation of target mRNAs. However, the exact mechanism remains elusive how the reduced FMRP causes the clinical manifestations.FMR1 knockout mouse has been served as a valuable tool in elucidating the mechanisms of FXS. The model mouse has shown the multifaceted phenotypes consistent with those in human, especially hyperactivity and susceptibility to auditory seizure. Our previous study showed that ABR threshold has significantly difference between immature KO mice and immature WT mice, the ABR threshold in immature FMR1 KO mouse were higher than immature WT mice. ABR threshold has no significantly difference between mature KO mice and mature WT mice and the ABR threshold in FMR1 KO mouse had age-dependence .Our previous study also showed that FMR1 KO mice were more susceptible and more serious than wild type control mice during the patterns of hyperthermal-indused convulsion. FMR1 KO mouse has been generally accepted as an Serviceable epilepsy or seizures model. It was reported that limbic epileptogenesis markedly increased of mossy fibre sprouting in FMR1 KO mice.FMR1 knockout mouse, a single gene disease model, also provides a unique opportunity to study the molecular mechanism of epilepsy in hereditary mental retardation diseases.Γ-aminobutyri acid(GABA), a major inhibitory neurotransmitter in the central nervous system, plays an important role in the regulation of the excitability of neurons. Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for the coversion of glutamic acid intoγ-aminobutyri acid. Recent year's researches have confirmed that GAD is closely related to epilepsy. It has been found that mutation of GABAA receptor gene may be predisposing factors of childhood absence epilepsy (CAE) and febrile seizures. Some study has shown that the GABA-immunoreactivity neurons and the expression of GABAARα5 subunit decreased after post-epilepticus.The studies indicate that the amount of PV positive neurons and the GABAA receptors are significantly decreased in FMR1 KO mice when compared to the WT mice. In hippocampal slices from FMR1 KO mice, the prolonged epileptiform discharges was recorded by blocking GABAergic transmission with the GABAA receptor antagonist bicuculline. Therefore, we speculate that whether or not the change of the cochlea morphology may be the reason of the AGS in FMR1 KO mice, we also speculate that the decreased number and function of interneurons might lead to the increase of seizures susceptibility in FMR1 KO mice; and the GABA receptors might be one of important roles in abnormal excitability of interneurons.There are no intimate report about this in internal and abroad. PurposeThis research use the HE dyeing to observe different weeks FMR1 KO mice and WT mice cochlea morphology;Using the way of immunohistochemistry to compare with the changes of GAD, GABAα1 receptor expression between FMR1 KO mice and WT mice cochlea and auditory cortex , to investigat the GAD,GABAα1 receptor expression whether or not influenced by FMRP, and to approach the reason and mechanism of audiogenic seizure ,to find some treat evidences for FXS.Methods1. Experimental animalsMale FMR1 KO mice of the FVB strain (The DutchBelgian Fragile X Consortium) and their WT littermates were used. The genotype of mice was identified before the experiment.2. Genotype appraisementFmr1 knockout mice were identified using the PCR technical and Western blotting.3. Animail guoupsExperimented animals were divided into 4 groups according to their genotype and age: KO group (3w, 4w, 6w, 8w) and WT group (3w, 4w, 6w, 8w), each group has 10 FVB mouses.All of them are used to immunohistochemistry and cochlea HE dyeing.4. Image analysis and statisticsThe numbers of immunostaining signal of the GAD and GABAα1 receptor protein in cochlear and auditory cortex were determined by Image J 13.7 system. All the data were calculated and analysis with statistic software SPSS 16.0. The accepted level of significance was 0.05.Results1. All the mice at different ages were KO mice through genes and Western blotting type identification. 2. There are no difference between FMR1 KO mice and WT mice cochlea morphology by HE dyeing.Compare FMR1 KO mice with the WT mice cochlea in different weeks, we first find that FMR1 KO mice's cochlea structure is complete,there are no hemorrhage and exudate between scala tympani, scala vestibuli and scala media.The basal membrane, Corti organ , ligament of cochlea,SGC and psalterial cord are normal.3. Increased expression of GAD and decreased number of GABAα1 receptor in cochlear and auditory cortex in FMR-1KO miceNumber of GAD in cochlea: 3W FMR1 KO mice 59.67±18.02, WT mice 36.60±8.56 (P=0.000); 4W FMR1 KO mice 69.71±23.41, WT mice 36.06±7.96(P=0.000); 6W FMR1 KO mice 50.14±12.85, WT mice 34.05±11.8 (P=0.000); 8W FMR1 KO mice 35.26±9.07, WT mice 31.39±7.20 (P=0.081).Number of GAD in auditory cortex: 3W FMR1 KO mice 75.40±13.83, WT mice 40.64±7.99 (P=0.000); 4W FMR1 KO mice 67.00±13.21, WT mice 52.45±10.22(P=0.015); 6W FMR1 KO mice 62.4±8.84, WT mice 48.3±12.42 (P=0.004); 8W FMR1 KO mice 57.50±5.93, WT mice 48.00±10.544(P=0.118).Number of GABAα1 receptor in cochlear: 3W FMR1 KO mice 38.49±6.43, WT mice 49.71±9.07(P=0.003); 4W FMR1KO mice 45.71±8.56, WT mice72.48±27.27(P=0.000); 6W FMR1 KO mice 43.82±6.57, WT mice 53.19±15.89 (P=0.003); 8W FMR1 KO mice 39.50±9.47, WT mice 44.28±9.97 (P=0.111).Number of GABAα1 receptor in auditory cortex: 3W FMR1 KO mice 35.17±4.26, WT mice53.38±7.27 (P=0.000);4W FMR1 KO mice 28.00±2.55, WT mice 47.5±8.23(P=0.001); 6W FMR1 KO mice 40.20±8.26 , WT mice 74.8±12.16 (P=0.000); 8W FMR1 KO mice 52.00±5.29, WT mice 54.8±4.66(P=0.462).Conclusion1. There are no difference between FMR1 KO mice and WT mice cochlea morphology by HE dyeing.2. Increased expression of GAD and decreased number of GABAα1 receptor in cochlear and auditory cortex in 3W, 4W, 6W FMR1 KO mice and there is no distinct between FMR1 KO mice and WT mice in 8W.3. The expression of GAD and GABAα1 receptor in cochlea and cortex auditory in Fmr1 knockout (KO) mouse are characterized by age-dependence, they may be the reasons of the Audiogenic seizure in Fmr1 knockout (KO) mouse. Key words Fragile X syndrome; Epilepsy; Audiogenic seizures; GAD; GABAα1... |
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