Retinal uptake of intravitreally injected Hsc/Hsp70 and its effect on susceptibility to light damage.
OBJECTIVE
To evaluate the uptake by the rat retina of an intravitreally injected mixture of the constitutive and inducible forms of the 70 kD heat shock protein (Hsc/Hsp70) and test its potential to protect photoreceptors from light damage.


METHODS
Hsc/Hsp70 and actin (control protein) were labeled with fluorescein (referred to as fl-Hsc/Hsp70 and fl-actin). The labeled proteins were microinjected intravitreally into the normal or light damaged rat eye and each eye collected at three intervals after the injections. Retinal uptake of Hsc/Hsp70 or actin was studied in frozen sections using epifluorescence microscopy and in western blots of retinal homogenates using an anti-fluorescein antibody. Additionally, the cytoprotective effects of Hsc/Hsp70 were tested in rats that first were exposed to bright light (170 ft-c) for 24 h and then given an intravitreal injection of the protein immediately thereafter. Ten days later, photoreceptor damage was evaluated by measuring the area of the outer nuclear layer at fixed locations along the circumference of the retina.


RESULTS
The fluorescein-labeled proteins were detected in the retina one h after administration and were retained there for more than 6 h. They were diffusely distributed, primarily in the nerve fiber layer, ganglion cell layer, and plexiform layers. Fl-Hsc/Hsp70 was also found in the outer nuclear layer (ONL) at 6 h after injection. At 24 h post-injection, the proteins were undetectable by epifluorescence microscopy of retinal sections, but could still be detected in western blots of retinal homogenates. The pattern of protein uptake was similar in light-damaged retinas. Ten days after light damage, the retinas in those eyes that received injections of Hsc/Hsp70 had greater ONL areas compared to either the light-damaged retinas of uninjected eyes or those that had received actin. The difference was statistically significant (p<0.05).


CONCLUSIONS
Intravitreally injected Hsc/Hsp70 is taken up by retinal cells and, when administered after an acute injury like light damage, increased the number of surviving photoreceptors.
Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1010, USA.
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D000818:Animals; D015153:Blotting, Western; D019793:Fluorescein; D019084:Fluorescent Antibody Technique, Indirect; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000493:pharmacokinetics*; D007267:Injections; D008027:Light / Q000009:adverse effects; D008297:Male; D008856:Microscopy, Fluorescence; D009412:Nerve Fibers / Q000378:metabolism; D010786:Photoreceptor Cells / Q000378:metabolism; D011833:Radiation Injuries, Experimental / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000378:metabolism* / Q000528:radiation effects; D012162:Retinal Degeneration / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control*; D012165:Retinal Ganglion Cells / Q000378:metabolism; D014822:Vitreous Body
D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494373:Hspa8 protein, rat; D019793:Fluorescein