Discussion Of Toll-like Receptor 4 And Its NF-κB Signal Transduction Pathway At The Rats Retinal Injury And Mechanism With Chronic IOP Elevation

Discussion of TOLL-like Receptor 4 and Its NF-κB Signal Transduction Pathway at the Inj ury and Mechanism with Chronic RatS Retinal IOP ElevationIntroductionAt present glaucoma has been listed as in the world the second blinding ophthalmopathy,but its pathogenesis also not very clear,in the world still did not have one kind of optic nerve protection medicine to be able to invest through the United States Food and Drug Surveillance Administrative bureau's verification authorization into the clinical use.At present urgent needs to protect to glaucoma optic nerve conducts the deep research,but the nerve immunity protection is proposes most newly prevents RGCs to carry on one which of strategies the nature loses.Worldwide, glaucoma damage the optic nerve and optic nerve immunological mechanism of immunological protection by means of increasing concern to researchers,the study abroad,as a result of autoimmune disorder regulated,resulting in the retina and optic nerve changes in certain components and with antigens,thereby triggering the autoimmune response,resulting in damage to the retina and optic nerve.The immune system in the pathogenesis of glaucoma role is twofold,both have neural protective effects of nerve damage.A better understanding of the immune system in glaucoma the role of optic nerve injury,will help us in the prevention and treatment of glaucoma to create a better control strategies.For glaucoma patients,how to make a protective immune and auto-immune disease caused by neurodegenerative injury equilibrium will determine the fate of ganglion cells.CNS glial cells through its own and the effects of CNS-infiltrating cells to immune defense functions of the exercise,TLR4 is a receptor of innate immunity,TLRs expression of microglia and their activation status are closely related to the combination of TLRs and PAMPs in small Glial cells provide a stimulus in the pathogenic immune response and the links between the key mechanisms of exogenous pathogens combined with the TLRs to activate innate immune responses to promote the acquired immune response.Glaucoma and optic nerve injury in TLR4 specific mechanism is unknown.The topic to be from the level of gene and protein research TLR4 and its NF-kB signal transduction activation of microglial cells in the role and significance of the use of immunohistochemical method of TLR4 and microglial cells in the normal rat retina and chronic ocular hypertension on the expression of retinal location and relevance,and to explore the TLR4 and NF-κB signaling pathway in the optic nerve injury in the mechanisms.For an in-depth TLR4 revealed microglia cells and functional relationships,to find effective antagonists of TLR or TLR signaling pathway blocked in order to achieve the best possible state of microglial cell activation, for glaucoma optic nerve damage and provide a theoretical basis for immunotherapy.Materials and methods1.Rat chronic high intraocular pressure model preparation:In the cauterize 3 venae sclerales technique unites the silk crack mildew element the application to establish the big mouse chronic high intraocular pressure model.Before TONO a PENII pen type eye pressure meter photomicrometrology,after the technique,30min,2h,1d,3d,7d,14d,28d,56d intraocular pressure.2.Under light microscope retina morphology observation:HE dyes,and surveys in the retina the membrane to the outside membrane distance is retina thickness.3.After model building 2h,1d,3d,7d,14d,28d,56d take 5 high intraocular pressure groups,the sham-operation group and the blank control group big mouse nose side half retina separately,dyes in the retina shop piece upward immunity histochemistry,in the laser altogether focuses under the microscope to observe retina TLR4,the small spongiocyte's expression situation.4.Law examines in each group of retina organization using RT a PCR TLR4,the small spongiocyte's gene mRNA expression and Western a Blot law examines in each group of retina organization TLR4,the small spongiocyte's protein expression,the statistical result indicated by the mean value gentleman standard deviation that during many group of mean value's comparisons with oneway a ANOVA analysis,the surgery group and the blank control group mean value's comparison with pair the T-test analysis,uses the SPSS 13.O software to carry on the statistical analysis. 5.Small spongiocyte activation model establishment:Cell model which the scratch establishment's small spongiocyte activates on the small spongiocyte's culture medium,law examines in each group of small spongiocyte using RT-PCR TLR4,P50, IL-6,the TNF-αgene mRNA expression and Western a Blot law examines in each group of retina organization TLR4,P50,IL-6,the TNF-αprotein expression,the statistical result indicated by the mean value gentleman standard deviation,during many group of mean value's comparisons with oneway a AN OVA analysis,the scratch group and the control group mean value's comparison with pair the T-test analysis,uses the SPSS 13.0 software to carry on the statistical analysis.Results1.Rat model of chronic high intraocular pressure Preparation:The burning branded three episcleral veins on intraoperative application of mitomycin United set up rat model of chronic high intraocular pressure.TONO type 1 PENll pen tonometer measuring preoperative and postoperative 30min,2h,1d,3d,7d,14d,28d,56d of intraocular pressure.2.Light microscopy observation of retinal morphology:HE staining,and measurement of retinal internal limiting membrane to the outside world that the retinal thickness from the film.3.Model set up after 2h,12h,24h,1w,4w,8wrespectively five high-tension group, sham-operated group and the normal rat retina at the nasal half of the retina chip up Shop immunohistochemical staining,laser at retina was observed under confocal microscope TLR4,a small expression of glial cell line.4.The application of RT-PCR assay in retinal tissue of each group TLR4, microglial cells express the gene mRNA and Western First Blot assay in retinal tissue of each group TLR4,microglial cells express the protein,are both results standard deviation of the number of people said that many groups were compared between the number one ANOVA using oneway analysis,drug group and the number of non-treatment group comparisons were paired t test analysis with all SPSS13.0software used for statistical analysis.5.Microglial cell activation model:RT-PCR Detection of microglial cells in each group of TLR4,P50,IL-6,TNF-a gene mRNA expression and Western First Blot assay in retinal tissue of each group NF-kB,P50,IL-6,TNF-a protein expression results are in both the standard deviation of the number of people said that many groups were compared between the number one ANOVA using oneway analysis of the treatment group and non-treatment group mean comparison between the analysis of paired t tests were used for statistical analysis software SPSS1 3.0.Conclusion1.Normal rat retinal pigment epithelium organizations shows that a small amount of TLR4 expression,and can be seen static microglia cells.2.TLR4 chronic high intraocular pressure in rats at different time points of the retina expresses its chronic high intraocular pressure in the rat optic nerve degeneration in the role of immune regulation.3.The activation of microglial cells and TLR4 expression in space and time relevant.4.TLR4 and its signaling pathway NF-κB microglial cells in the activation process plays an important role,TLR4 and the activation of microglial cells positive feedback regulation.