Abstract
Abstract: :
Purpose: We have previously demonstrated selective uptake of fluorophore conjugated Tat permeation peptides by rodent inner retinal neurons, predominantly retinal ganglion cells (RGCs), following intraocular injection. As such, these peptides may be useful for translocating therapeutic or imaging substrates into RGCs. To evaluate retinal uptake in vivo, we performed fluorescent retinal imaging following their intraocular injection in rats. To determine their possible intra–axonal transport, we also examined the temporal course of RGC axonal labeling in the nerve fiber layer, optic nerve head, and optic nerve following injections. Methods: The permeation peptide used was based on the wildtype HIV–1 Tat sequence (Tat 47–60), but contained only D–amino acids and an ornithine substitution for glutamine, as this enhanced uptake in in vitro models. Alexa Fluor–594 or fluorescein maleimide were thiol–conjugated to the C–terminal cysteine to enable visualization of cellular uptake. Rats were anesthetized and 5 µl of a 250–500 µM solution was injected into the vitreous. Retinal uptake was imaged with the ADIS–9000 digital retinal imaging system at 1–7 days. Animals were euthanized, the eyes enucleated, the tissue fixed in 4% paraformaldehyde and then either embedded in paraffin or prepared as flat mounts. Immuno–histochemistry was performed to confirm cell types exhibiting uptake. Results: In vivo fluorescent imaging showed diffuse punctate retinal staining at all time points examined, with more intense signal near the injection site. This same pattern was seen in fixed retinal flat mounts and ocular sections. Paraffin sections confirmed selective fluorescent labeling of both RGCs and a subset of inner nuclear layer cells. Labeling of the retinal nerve fiber layer continued into the anterior optic nerve head and spread posteriorly along the optic nerve with increasing time post–injection. Conclusions: Modified Tat permeation peptide constructs conjugated to flurophores are taken up rather selectively by RGCs following intraocular injection in the rat. The pattern of uptake is similar with both in vivo imaging and in tissue sections, arguing against fixation related artifact. The temporal pattern of labeling in the optic nerve suggests transport along RGC axons following uptake. Since various moieties can be conjugated to these permeation peptides, they may be useful for delivery of therapeutic agents to RGCs and/or for delivery of constructs to enable in vivo imaging of RGC physiology.
Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • ganglion cells • imaging/image analysis: non-clinical