Abstract
Purpose: :
To report a model for easy assessment of pressure-dependent damage of retinal ganglion cells (RGCs) utilizing B6.Cg-TgN(Thy1-CFP)23Jrs transgenic mouse strain (CFP mouse), in which the Thy1 promoter linked to cyan fluorescent protein (CFP) reporter.
Methods: :
In 24 CFP mice, one of both eyes was chosen for laser-induced ocular hypertension eye (treated eye) and the other remained untouched to serve as control. After anesthesia with an i.p. injection of a xylazine/ketamine mixture and pupil dilation by instillation of 5 µl of 0.5% tropicamide, laser photocoagulation (532 nm wavelength, 200 mW power, 0.05 second duration, 200 µm spot size) was performed at whole circumference of the limbus (50-56 shots), after flattening the anterior chamber by aspiration of aqueous humor. Intraocular pressure (IOP) was measured every week, by a microneedle method in both eyes. When the IOP in the treated eye decreased to 120% or less of that in the control eye, animals were sacrificed, both eyes enucleated, fixed and the retina was flat-mounted. The number of RGCs was determined by counting RGCs expressing CFP using fluorescence micrographs in 12 separate areas of 40,000 µm2 each 600, 1200 and 1800 µm being apart from the optic disc in each 4 retinal quadrant to obtain the average density of RGCs/mm2 for each retina sample. Based on the line plot of time (x) and IOP (y) in both eyes, the area under curve ( IOP(y) dx) was calculated as a surrogate value of the pressure-insult and ( IOP(y) dx) was correlated with the RGC density.
Results: :
In 21 of 24 treated eyes, 20% or more IOP increase compared to that in the control eye was obtained at 1 week and IOP was maintained by 20 % or higher in 19, 7, and 4 eyes at 2, 3 and 4 weeks respectively. The pressure insult and the RGC density showed significant negative correlation (y=-0.045x+97.31, r=0.51, p=0.015).
Conclusions: :
The use of CFP mice enabled easy evaluation of pressure-dependent RGC damage without retrograde labeling of RGC. This model should be useful in evaluating relationship between the IOP and RGC damage and effects of various treatments on it.
Keywords: ganglion cells • intraocular pressure • transgenics/knock-outs