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E. M. Barnett, Q. Chang, X. Zhang, B. Elangovan, D. Piwnica-Worms; Visualization of Effector Caspase Activity in Rat Models of Retinal Ganglion Cell Apoptosis Using an Activatable Fluorescent Permeation Peptide. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3272.
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The eye is an attractive model for studying molecular imaging probes because of its accessibility and the ease of optical imaging. There is particular interest in in vivo imaging of apoptosis-related enzyme activity in retinal ganglion cells (RGCs), which undergo apoptosis in glaucoma. RGC uptake of fluorescent probes can be enhanced by permeation peptides such as HIV-1 Tat, as previously demonstrated (Invest Ophthal & Vis Sci 2005 47:2589) . An activatable fluorescent peptide probe incorporating an effector caspase cleavage site has been developed and validated in vitro (J Med Chem 2005 48:5404). This probe was evaluated for the imaging of apoptosis associated enzyme activity in RGCs in animal models of RGC degeneration.
RGC apoptosis was induced in rats either through unilateral optic nerve transection, the induction of uniocular elevated intraocular pressure, or phamacologically using intravitreal injection of staurosporine. The activatable peptide probe is a small peptidomimetic consisting of an all D-amino acid modified Tat permeation peptide, an L-amino acid effector caspase recognition sequence, a quencher, and a fluorophore. Upon cleavage of the caspase recognition sequence and subsequent loss of fluorescent quenching, fluorescence from the retained intracellular fluorophore is detectable via fluorescence imaging. Retinal imaging was performed using a fluorescence macroscope both in vivo and ex vivo (after removal of the cornea and lens) following intravitreal injection of the activatable probe. Retinae were also examined as flat mounts and paraffin sections.
Fluorescent retinal cells were detected diffusely throughout the retina in eyes with induced RGC apoptosis using in vivo and ex vivo imaging. Subsequent examination of ocular sections revealed the labeled cells to be predominantly RGCs. Control eyes showed either no fluorescent cells or a small focus of labeled cells in the retinal periphery at the injection site. Immunohistochemistry for activated caspase-3 showed correspondence with probe labeling in ocular sections.
The use of a molecular imaging probe to detect apoptosis related enzyme activity in vivo in the retina shows promise and has applications in the study of animal models of glaucoma and potentially in the diagnosis and management of human glaucoma.
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