Tropicamide solution

Prior to treatment initiation and prior to sacrifice (2 months of treatment) for biochemical analyses, animals were subjected to ERG analyses to evaluate the changes in the electrical activity of the retina as we have done previously [2 (link),21 (link)]. After dark adaptation overnight, ERG responses were recorded from both eyes using platinum wire corneal electrodes, forehead reference electrode, and ground electrode in the tail. Pupils were fully dilated using 1% tropicamide solution (Alcon, Ft. Worth, TX). Methylcellulose (Celluvise; Allergan, Irvine, CA) drops were applied as well to maintain a good electrical connection and body temperature was maintained at 37°C by a water-based heating pad. ERG waveforms were recorded with a bandwidth of 0.3-500Hz and sampled at 2kHz by a digital acquisition system and were analyzed using a custom-built program, which allowed a measurement of a-wave, b-wave and oscillatory potential from all animals (MatLab, Mathworks, Natick, MA). Statistics were done on the mean ±SD amplitudes of the a- and b- wave of each treatment group.

ERG analyses were carried out to evaluate the changes in the electrical activity of the retina between the right and left eye of control, diabetic, diabetic+CMV, and diabetic+IGFBP-3 NB animals [4] (link), [20] (link). Briefly, rats were dark-adapted overnight and ERG responses were recorded from both eyes together using platinum wire corneal electrodes, forehead reference electrode, and ground electrode in the tail. Pupils were dilated using 1% tropicamide solution (Alcon). Methylcellulose (Celluvise; Allergan, Irvine, CA) drops were applied as well to maintain a good electrical connection and body temperature was maintained at 37°C by a water-based heating pad. ERG waveforms were recorded with a bandwidth of 0.3–500 Hz and samples at 2 kHz by a digital acquisition system and were analyzed a custom-built program (MatLab). Statistics was done on the mean ±SD amplitudes of the a- and b- wave of each treatment group, including oscillatory potentials. Comparisons were made between the right (OD) and left eyes (OS).
Intraocular pressure (IOP) was measured monthly using a tonometer (TonoLab, Colonial Medical Supply, Franconia, NH). Briefly, the tip of the probe of the tonometer was placed at the cornea of the eye. During measurements, the tip of the probe hit the cornea six times and gave the IOP reading of that eye.

OCT was performed at the indicated times using the Micron III intraocular imaging system (Phoenix Research Labs, Pleasanton, CA, USA). Before the procedure, eyes were dilated with 1% tropicamide solution (Alcon, Fort Worth, TX, USA) and lubricated with hypromellose ophthalmic demulcent solution (Gonak) (Akorn, Lake Forest, IL, USA). Mice were then placed on a custom heated stage that moves freely to position the mouse eye for imaging. Several horizontal and vertical images were taken per lesion to allow calculation of CNV lesion volume. Three-dimensional quantification of CNV lesion volumes was performed using an ellipsoid quantification method as previously described24 (link). Fluorescein angiography was performed 14 days post laser by intraperitoneal injection of 50 μL of 25% fluorescein sodium (Fisher Scientific, Pittsburgh, PA, USA). Fundus images were taken using the Micron III system and Streampix software.

OCT was performed at the indicated times using the Micron III intraocular imaging system (Phoenix Research Labs, Pleasanton, CA, USA). Before the procedure, eyes were dilated with 1% tropicamide solution (Alcon, Fort Worth, TX, USA) and lubricated with hypromellose ophthalmic demulcent solution (Gonak) (Akorn, Lake Forest, IL, USA). Mice were then placed on a custom heated stage that moves freely to position the mouse eye for imaging. Several horizontal and vertical images were taken per lesion. In addition, gross retinal/choroidal structure, and vascular patterns were examined for possible adverse effects of the test compound or vehicle.