d placed on a heating pad just before in vivo imaging. Pupils were dilated with tropicamide (5 mg/mL) drops. Dilation in the lateral tail vein was achieved by placing the animal below a heat lamp. The animals had been positioned on the Micron III stage and Hypromellose coupling fluid applied to the eye. The camera and eye position was adjusted ensuring correct alignment and concentrate around the optic nerve head plane applying normal colour fundus photography ahead of adjusting towards the acceptable filter set for Fluorescein Angiography. 0.1 mL/kg, 10% fluorescein sodium was then administered by way of intraperitoneal injection or intravenous injection via the lateral tail vein. The fluorescein bolus was delivered over 3 seconds; simultaneously with the very first image capture. Pictures have been captured employing the Streampix Computer software (Phoenix Investigation Laboratories) as XGA resolution (1024×768 pixels) 24bit RGB sequential tiff files together with the Micron III light supply at maximum 19569717 intensity and acquire setting of +4 db. Pictures have been taken at 7 day intervals for three weeks just after laser therapy. FFA images with fluorescein administration by intraperitoneal injection had been taken 1 frame/second for 120 seconds, and 1 frame/5 seconds thereafter as much as ten minutes post injection. For intravenously administered fluorescein, pictures were taken at 30 frames per second for 120 seconds. All FFA image analysis was performed with open-source application, ImageJ (National Institute of Mental Wellness, Bethesda).
For SC-1 region measurement, pictures had been imported into ImageJ, exactly where trained graders blinded to the experimental treatment, determined lesion location and intensity. RGB Tiff files had been imported into ImageJ and individual colour channels separated. Red and Blue Channels had been removed and all region and intensity values had been measured from the Green Channel. Beneath digital magnification (800%) and making use of the `freehand selection tool’ the maximal border of each and every hyperfluorescent CNV lesion or the hypofluorescent laser burn was determined along with the region recorded in pixels (Fig 1). Unavoidable magnification of fundus photos to varying extent is introduced by the lens program exerting pressure around the corneal surface. As such every single CNV or CR burn region was normalised against the averaged optic nerve head region (determined in an identical manner to lesion area measurements). The normalised CNV area was then plotted against time soon after laser treatment. The fluorescence intensity (average grey level) inside the maximum border of each CNV lesion and CR burn was calculated employing ImageJ, areas where substantial vessels overlapped 
the hyperfluorescent region was excluded (Fig 1). Background fluorescence intensity was measured by defining an annulus region around the CNV lesion, from which, six representative regions avoiding retinal vessels have been identified and an average background fluorescence intensity worth calculated. The inner border of the annulus was defined as the outer limit of the CNV lesion whilst the outer border in the annulus was defined employing twice the radius of the inner border. The net fluorescence intensity above background was thus calculated by subtracting the calculated background value from the CNV hyperfluorescence intensity. Net lesion fluorescence from every sequential fundus image was calculated and plotted against time just after fluorescein injection via intravenous and intraperitoneal administration. The time corresponding to peak fluorescence intensity was identified and this worth was made use of for all subsequent intensity calcul