Eye Examination
Material and Methods
Eye Dom, S. Nusinowitz, Visual Physiology Laboratory,UCLA, USA
Photic Stimulator, FLC-33A, Astro-Med, Inc., Grass Product Group, Warwick, RI
Amplifier, AC, CP511, Astro-Med, Inc., Grass Product Group, Warwick, RI
Red Light Bulb, 25W, type Mexico, Philips Corporation, USA
Red transparent paper
Kodak Wratten Gelatin Filter # 47A
Headlamp, Petzel, Inc. USA
I/O board, National Instruments, Lab PCI-1200
Heating Pad, Baxter K Mod 100, Baxter Laboratory Equipment
Electrodes, F-E2, platinum sub-dermal needle electrodes
Astro-Med, Inc., Grass Product Group, Warwick, RI
Gold wire (99.9%), 0.508 mm (Jewelry Store)
Gold loop electrodes for recording and reference; they are made
by S. Nusinowitz, Visual Physiology Laboratory,UCLA, USA and
consist of modified platinum electrodes to which a gold wire loop has been attached.
Atropine Sulphate 1%, Alcon Laboratories,USA
Cyclomydril, Alcon Laboratories,USA
Ketaset Hydrochloride, 100mg/ml, Fort Dodge Animal Health
Xylazine, 20mg/ml, Phoenix Pharmaceutical, MO
Goniosol, Ciba Ophthalmics, USA
Custom software for data analysis, S. Nusinowitz, Visual Physiology Laboratory,UCLA, USA
Working Solution
0.6ml Ketaset
1ml Xylazine
3.4ml saline
Primary Screens
Slit Lamp Routine exams of cornea and iris are performed with a slit lamp attached to a microscope (SL-7E, Topocon Corporation, Japan) and camera (Coolpix 4500, Nikon Corporation, Japan). Using a 40x objective, the appearance of the cornea, (e.g. clarity, size, surface texture, vascularization etc.), pupil, (e.g. size, shape, constriction, reflected luminescence), and iris (e.g. presence of atrophy and synechia) are determined, and abnormalities will be documented with photographic images of the afflicted area.
Indirect Ophthalmoscopy In preparation for indirect ophthalmoscopy the eyes are dilated with 1% atropine (Atropine Sulphate, 1 drop, Alcon Laboratories, USA). Subsequently, the lens is examined for cataract formation and pseudoexfoliation with an indirect ophthalmoscope (Heine Omega 180, Germany), and using 60 or 90 Diopter double aspheric lenses (Volk Incorporation, USA), the fundus is examined for signs of retinal degeneration (e.g. retinal vessel constriction, retinal pigment epithelial disturbance, drusen or deposits, structural or optic nervehead anomalies, vitreal abnormalities, and abnormal vascularization).
Phenodeviants for any one of the above anomalies are tested for heritability.
Secondary Screen
Electroretinogram (ERG)
In preparation for the ERG, mice are kept in a dark room for at least 2 hours prior to recording, and light sources in the testing room are turned off or covered with red transparent plastic; a red light bulb (25W, Philips, Corporation) is used to illuminate the room.
Mice are weighed, and 1 drop of Atropine and 1 drop of Cyclomydril (ophthalmic solutions) are applied to induce mydriasis, and Ketaset/Xylazin /saline solution (0.1ml/20g body weight) is injected (i.p.) to induce anesthesia. Once the pupil dilating and anesthetic agents have taken affect, the mouse is placed on a heating pad (38ºC) and the electrodes are attached: tail (ground), mouth (reference) and cornea (recording), using one drop of Goniosol to moisten the cornea.
The rod test is performed with a short-wavelength Wratten filter (# 47A) using stimulation intensities of 1, 4, 8 (lumen sec/ft2, flash duration 10¼sec, and 0.2HZ frequency).
The subsequent cone test is performed with the Dom light on, and the animals are allowed a 10-minute adaptation time; cones are then tested with white flashes at flash intensities of 16, 8, or 4, at 1.0 Hz.
Future development
Primary screens
We plan to develop additional screening methods for functional deficits or for eye alterations not detected by a conventional exam, including a) gonioscopy to examine anterior chamber angle structures; b) non-invasive measures of retinal ganglion-cell and optic nerve function, such as pattern ERG, visual-evoked potential, and a contralateral pupillary response assay for evaluating the visual system from the retina through the primary visual cortex; and c) non-invasive intraocular pressure measurements. Once fully developed, these non-invasive functional measures may be used for primary screening or as a first line of secondary analysis.
Secondary screens
Longitudinal screens on all heritable eye mutants of interest
This level of study includes further electroretinograms, photo documentation, fluorescein angiography, intraocular pressure measurements and histology. Secondary screens will be performed on mutants to provide further characterization of eye diseases.
