The Effects On Macromolecules Transfer And Heat Shock Protein 70 Expression Caused By Lens Zonule Rupture

Purpose: 1. Zonular fibers are a specific form of extracellular matrix composed mainly of fibrillin what believed to be secreted by non-pigmented ciliary epithelium. The variation of fibrillin-1 distribution in lens capsule after zonule rupture was investigated in the research. 2. Little is known about the mechanism of translocation of macromolecules into lens epithelium and fiber cells. The study was to observe whether the rupture of zonule affected translocation of macromolecules into the lens and to determine the role of zonule in transferring these macromolecules. 3. HSPs (heat shock proteins) may play an important role in the maintenance of supramolecular organization of the lens proteins. The zonule rupture model was made to determine whether zonule dehiscence affected HSP-70 expression in lens. Methods: Guinea pigs were applied as experimental animal and microsurgeries were taken to tear the inferior part of zonule, range about 120°. 1. The eyes were removed from guinea pigs at 1, 4, 8 and 12 weeks post-operatively and cryo-sections were prepared. Immunofluorescence technique was used to observe fibrillin-1 variation in lens capsular. 2. HRP (horseradish peroxidase, 40kD) was coupled with the fluorochrome FITC and encapsulated with liposome (BioPORTER). pEGFP-N1 plasmid (expressing green fluorescent protein, 4.7kD) with encapsulation in commercially available liposome (DOSPER) was prepared. The eyes were enucleated 1, 4 and 8 weeks after zonule rupture and anterior segments comprising lenses were incubated in medium containing one of these components. Cryo-sections were made and translocation of fluorescent macromolecule from the medium into the lens and green fluorescent protein expression in the epithelium were observed by fluorescent microscopy. 3. Genea pig's lenses were obtained 1 and 8 weeks after zonule rupture. Lenses were dissected to separate epithelium and cortex. The proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose paper. Western blot analysis was performed withspecific HSP-70 antibody. The difference of HSP-70 expression in zonule dehiscence eye and controls was identified. Those images were digitalized and measured by image analysis system and the data was statistically analyzed by t-test respectively.Results: 1. 8 and 12 weeks after surgery, immunofluorescence indicates that fibrillin-1 in capsule of zonule dehiscence side is less than which in the control side (p<0.01). 2. FITC labeled HRP was found in the epithelium and cortex in equatorial region. At 8 weeks post-operatively the fluorescent value of FITC-HRP on zonule dehiscence side is significantly lower than the control side (p<0.05). pEGFP-N1 was able to enter equatorial region of intact lens and expressed the green fluorescent protein. The green fluorescent protein expression is weaker both on zonule dehiscence side and the control side and difference is not significantly (p>0.05). 3. At 1week post-operatively, HSP-70 expression in lens capsule of surgical eye was stronger than which in the controls (p<0.05). At 8 weeks post-operatively, HSP-70 expression in cataract capsule seemed weaker than the controls. Conclusions: 1. After zonule rupture, decrease of the zonular components in lens capsule can be detected in a short time (about 8 weeks) and ingredients of the lens capsule alternated. It is possible that the alternation affects the permeability and function of the capsule. 2. The macromolecules were transferred into the lens mainly at the equatorial region where zonule attaches to the lens. Translocation of macromolecules may be affected by zonule rupture in the late stage and the alternation of zonular components in lens capsule seems one of the possible reasons. 3. HSP-70 expression level in surgical eye elevated in the early stage may due to the stress induced by surgery. However the decrease of HSP-70 expression in the late stage after surgery accounts for the increased density in some animals' lens.