Quantification of Retrograde Axonal Transport in the Rat Optic Nerve by Fluorogold Spectrometry 英文参考文献.docVIP

QuantificationofRetrogradeAxonalTransportintheRat OpticNervebyFluorogoldSpectrometry ChristianvanOterendorp*,StavrosSgouris,MichaelBach,GottfriedMartin,JuliaBiermann, JensF.Jordan,WolfA.Lagre`ze UniversityEyeHospitalFreiburg,Freiburg,Germany Abstract Purpose: Disturbed axonal transport is an important pathogenic factor in many neurodegenerative diseases, such as glaucoma, an eye disease characterised by progressive atrophy of the optic nerve. Quantification of retrograde axonal transportintheopticnerveusuallyrequireslabourintensivehistochemicaltechniquesorexpensiveequipmentforinvivo imaging. Here, we report on a robust alternative method using Fluorogold (FG) as tracer, which is spectrometrically quantifiedinretinaltissuelysate. Methods: To determine parameters reflecting the relative FG content of a sample FG was dissolved in retinal lysates at different concentrations and spectra were obtained. For validation in vivo FG was injected uni- or bilaterally into the superiorcolliculus(SC)ofSpragueDawleyrats.Theretinallysatewasanalysedafter3,5and7daystodeterminethetime courseofFGaccumulationintheretina(n=15).Insubsequentexperimentsaxonatransportwasimpairedbyopticnerve crush(n=3),laser-inducedocularhypertension(n=5)orcolchicinetreatmenttotheSC(n=10). Results: Spectrometry at 370nm excitation revealed two emission peaks at 430 and 610nm. We devised a formula to calculatetherelativeFGcontent(cFG),fromtheemissionspectrum.cFG isproportionaltotherealFGconcentrationasit correctsforvariationsofretinalproteinconcentrationinthelysate.AfterSCinjection,cFGmonotonouslyincreaseswithtime (p=0.002). Optic nerve axonal damage caused a significant decrease of cFG (crush p=0.029; hypertension p=0.025; colchicinep=0.006).Lysatesareamenabletosubsequentproteinanalysis. Conclusions: Spectrometrical FG detection in retinal lysates allows for quantitative assessment of retrograde axonal transport using standard laboratory equipment. It is faster than histochemical techniques and may also