Ciliary Neurotrophic Factor-loaded PEG-PLGA Microbubbles Combined With Ultrosound Proteced Retinal Gangline Celles

PART Ι TO PREPARE THE CNTF-LOADED PEG-PLGAULTROSOUND CONTRAST AGENTS AND DETECTIONOF THERI PROPERTIESObjective：To prepare and observe the properties of CNTF-loaded PEG-PLGAultrasound contrast agents。Methods：1、To prepare and observe the properties of CNTF-loaded PEG-PLGAultrasound contrast agents,which is abbreviated as CNTF-PEG-PLGA，theCNTF-loaded PEG-PLGA ultrasound microbubbles were prepared usingthe technique of double emulsion.2、Morphology and drug distribution of CNTF-PEG-PLGA ultrasoundmicrobubbles were observed by light microscope and transmissionelectron microscope；Used Malvern laser particle size analysis the meansize and zeta potential of CNTF-PEG-PLGA microbubbles；The releasing behaviordetected by ELISA.Results:The CNTF-loaded PEG-PLGA ultrasound microbubbles were smoothand spherical with a mean size of312.5nm,the drug loading0.298ug/mgand encapsulation efficiency of62.35％．the releasing behavior of CNTF-PEG-PLGA nanoparticles in vitro was that after an obvious release ofabout35.59％in former24h，there were93.6％CNTF-PEG-PLGAreleased within28days．Conclusion:CNTF-PEG-PLGA ultrasound microbubbles were prepared by theemulsion solvent evaporation method with a smaller particle size、long-term sustained release characteristics、higher encapsulation efficiency anddrug loading. PART II CILIARY NEUROTROPHIC FACTOR-LOADEDPEG-PLGA MICROBUBBLES COMBINED WITHULTROSOUND PROTECED RETINAL GANGLINECELLES AFTER OPTIC NERVE INJURYObjective：To study the protection effects of CNTF-loaded PEG-PLGAmicrobubbles combined with ultrasound on retinal ganglion cells(RGCs)after optic nerve injury in rats.Methods：one hundred and forty five adult Sprague-Dawley(SD) rats weredivided into seven groups randomly:groupA (normal control group)、groupB (blank control group)、groupC(CNTF drug group)、groupD(ultrasound+CNTF drug group)、groupE(CNTF-PEG-PLGA microbubbles group)、groupF (blank-PEG-PLGA microbubbles group)、groupG (ultrasound+CNTF-PEG-PLGA microbubbles group).The1st，the2ndand the4thweekafter optic nerve injury, RGCs were retrograde labeled with the fluorescenttracer fluoro-gold(FG) to count RGCs number.The expressions of growthassociated protein43(GAP-43) were detected by immunohistochemistryon the3rdday,the1st，the2ndand the4thweek after optic nerve injury.Toobser-ve the retinal pathological morphology change at different times. Results:The results showed that after RGCs were retrograde labeled with theFG, The RGCs of group G、group E、group D and group C were signific-antly higher than group B at the1stweek,the2ndweek after optic nerveinjury (P <0.05)．At each time，the RGCs count of group G was higherthan group E、group D and group C (P<0.05),but less than group A(P<0.05)．The expression of growth associated protein43(GAP-43) wasweak，and mainly concentrated in the GCL on normal retina．The expre-ssion of group G was higher than other groups(P<0.05) on the1st,the2nd,and the4thweek after optic nerve injury．Normal retinal each layerarranged orderly and densely under the light microscope，the number ofRGC was more than injuer group.After optic nerve injury，the number ofRGCs count reduced on different degree，loose and vacuolization.Conclusion:The ciliary neurotrophic factor-loaded PEG-PLGA microbubblescombined with ultrasound can protect retinal ganglion cells against afteroptic nerve injury．...