Abstract
Purpose: :
Many crystallins have been shown to be up-regulated in several retinal diseases. Studies suggest αA-crystallin may provide a protective role against oxidative stress and prevent photoreceptor apoptosis. This study was designed to evaluate the retinal up-regulation of crystallins in a murine experimental detachment model.
Methods: :
Retinal detachments (RD) were induced by subretinal injection of hyaluronic acid (HA, 10mg/ml) into left eyes of (22-24 week-old) C57BL/6 mice (n=28). The right eye was used as a control. Retinal samples were collected after 1, 2, and 4-5 weeks. Total proteins were extracted and 8-plex iTRAQ proteomic analysis was performed on 2 week (n=3), 4 week (n=3) and control (n=2) mice. Further protein analysis with Western Blot and immunohistochemistry was performed on αA-crystallin, the crystallin protein with the strongest up-regulation on proteomic analysis. α-Tubulin was used as a loading control.
Results: :
Mass spectrometry identified 11 total α-, β- or γ retinal crystallins with relative quantification which were up-regulated in detached retina. The up-regulation of αA-crystallin was the strongest, with no expression in the control retina. On Western blot, both 22kD and 20kD αA-crystallin isoforms were detected. During the first week post detachment, there was absent or low-level expression of the 22kD isoform averaging 1.26 relative intensity units (IU), while the average 20kD isoform density was measured at 12.83 IU. Both the 22kD and 20kD isoforms were substantially up-regulated at 2 weeks, averaging 24.33 IU and 50.16 IU, respectively. Five weeks post- detachment, αA-crystallin levels were reduced to 11.97 IU (22kD) and 17.39 IU (20kD). The 20kD isoform demonstrated consistently higher expression than the 22kD isoform over the 5 weeks period. We also detected low levels of αA-crystallin in some normal retinas (3/14), but expression was variable. As expected, the control protein, α-tubulin was expressed at a similar level at all time points. Immunohistochemistry demonstrated that αA-crystallin was up-regulated in the retina and localized to the photoreceptor layer.
Conclusions: :
Following experimental murine retinal detachment, retinal αA-crystallin expression significantly increases by 2 weeks and subsides by 5 weeks. The 20kD isoform was expressed at higher levels than the 22kD isoform. Since these proteins provide anti-apoptotic functions, we hypothesize that αA-crystallin may play an important protective role in patients with retinal detachment.
Keywords: crystallins • retinal detachment • proteomics