Funduscopy v2.13 1/Sep/2011 RIKENMPP_014_002 test 5 1 testSt test modification

Funduscopy refers to examination of the retina, blood vessels, and optic disc. It allows changes in the retina to be detected and is the only method for directly examining the blood vessels in the body. Funduscopy allows the extent of hypertension and arteriosclerosis to be estimated and can sometimes detect changes in the optic disc associated with certain brain diseases. The funduscopy test is based on observational assessment. To evaluate the fundus phenotypes for one mouse properly, 24 items relating to the clinical and pathological characterization of the ophthalmus are tested in this screening protocol (version 2.13).

These procedures are applicable to the mouse phenotyping platform at the RIKEN BRC. The tests should be performed by trained staff members who understand the safety and hygiene rules required by RIKEN. Any queries, comments, or suggestions, relating either to this SOP in general or to a specific problem encountered during a procedure, should be addressed to the Morphological Unit Leader. Any deviation from this protocol must be reported to the Morphological Unit Leader.

Testing is carried out in accordance with common animal handling procedures. Coveralls (in the rearing facility) or laboratory coats (in the laboratory facility) must be worn with gloves at all times in the work area. If animals are dropped on the floor, treat them in accordance with the safety and hygiene guidelines of the rearing facility at RIKEN. If something unexpected happens during the primary screening, it must be quickly reported to the Morphological Unit Leader and recorded.

Genesis hand funduscopy camera (Kowa Company, Nagoya) with a Volk 90D lens. An image filing system (VK-2; Kowa Company, Nagoya) should be on hand.

Mydrin P (Santen Pharmaceutical Co.): mydriatic eye drops containing 0.5% tropicamide and 0.5% phenylephrine

Apply Mydrin P diluted 5-7.5 times with physiological saline to the eye of the mouse. The eye drops quickly prepare the eyes for photographing and have a strong mydriatic effect. To prevent the whiskers from obstructing the photography, cut them down to the base. Check if the pupil has dilated 5-10 minutes later. An inadequately dilated pupil will result in a dark and unclear photograph. For such cases, apply the eye drops again. Turn on the power unit and the camera and adjust the illumination. When taking a photograph, set the illumination at 4-5 and the flash at 5-6, depending on the hair color of the mouse. Excessive flash will result in an image with too much reflection, whereas too little flash will result in a dark image with an inadequate level of detail. Adjust the lens position. Bring the camera to the lowest position. Lower the Volk Adjust the lens position. Bring the camera to the lowest position. Lower the Volk 90D lens fixed to the mount immediately below the camera. The view through the eye lens shows two circles formed by the reflection of the illumination from the camera on the spherical surface of the front lens. Make adjustments to bring the circles to the center of the camera viewer and fix the front lens at a horizontal position such that the two circles overlap to form one circle. As a consequence, the lens and the desk are approximately 7 cm apart. Adjust the camera position. Bring the camera up slowly while looking through the eye lens. Fix the camera position when the fringe of the front lens overlaps or moves a little inside the fringe of the viewer. When the camera and the front lens are too close, the image is out of focus; when they are too far apart, the image is small. Mice are photographed while keeping the device used to control the focal length range (called the focus switch) at +35D (maximum). Adjust the position of the mouse to take the photographs. Because the mouse is not anesthetized, it is important to restrain the mouse firmly. The mouse can be restrained by grasping the dorsal side (mainly from the shoulder to the neck), with the ring and pinkie fingers holding the tail. The other hand firmly holds the head by applying pressure above the nose, below the eyes, and on the jaw. Photographing is made easier by slightly pressing down below the eyelid to push the eyeball out of the socket. Place the head under the front lens and, while looking through the eye lens, move the head position so that the fundus stays inside the front lens. Move the mouse to a position in which the retina receives the largest amount of light without reflection. Press the foot pedal to release the shutter when the mouse is properly positioned; the cornea will be 1-2 cm below the front lens. Use both hands to keep the mouse oriented when photographing. Anesthetized mice can also be photographed. Care needs to be taken, however, because anesthesia may cause complications, such as the eyeballs turning turbid. Sometimes an unclean corneal surface causes blurred vision; rinse the surface with physiological saline. Take photographs of the broad area of the retina while turning the mouse eye to obtain alternative perspectives. Immediately repeat the procedure for the other eye as described in 9.6-9.8. Note any abnormal eye phenotypes. Record the grade, photographs, and comments for following 24 items related to eye characteristics. 0 = normal 1 = pale 2 = other 0 = normal 1 = optic disc coloboma 2 = other 0 = small 1 = normal 2 = large 0 = no tortuous or a little(normal) 1 = tortuous 0 = normal 1 = abnormal 0 = severely narrow 1 = slightly narrow 2 = normal 3 = dilated 0 = severely narrow 1 = slightly narrow 2 = normal 3 = dilated 0 = normal 1 = sheathed 2 = transparent 3 = other 0 = normal 1 = reflex 2 = sheathed 3 = ununiform 4 = other 0 = no branched or a little(normal) 1 = branched 0 = violet 1 = reddish violet 2 = pink 3 = whitish 4 = blue 5 = darkish blue 6 = other 0 = none 1 = temporal 2 = large portion of the retina 3 = whole retina 0 = none 1 = slightly mottled 2 = mottled 3 = spotted 4 = salt and pepper appearance 5 = small and large white spots 6 = large areas with hyperpigmentation and hypopigmentation 0 = none 1 = lepidic spots 2 = other 0 = none 1 = small number of isolated white spots (<= 10 spots) 2 = many small white spots (> 10 spots) 3 = white area(s) the size of an optic disc 4 = white area larger than an optic disc 5 = retinal fold 6 = other 0 = absent 1 = present 0 = absent 1 = present 0 = none 1 = conus 2 = mottled 3 = other 0 = full mydriasis 1 = moderate mydriasis (half size of fully dilated pupil) 2 = little or no mydriasis 0 = none 1 = corneal opacity 2 = intraocular 3 = other 0 = clear 1 = slightly cloudy 2 = moderately cloudy 3 = strongly cloudy (unobservable) 0 = absent 1 = present 0 = none 1 = cornea 2 = intraocular(lens or vitreous) 3 = other 0 = normal 1 = abnormal Make as many written notes and comments as possible.

A new version of the Funduscopy (version 2.13) was added. This version involves 24 items (D01, D02, D03, V01, V02, V03, V04, V05, V06, V07, R01, R02, R03, R04, R05, R06, R07, R08, O01, O02, O03, O04, O05, and O06) relating to the clinical and pathological characterization of the ophthalmus. In this version, the title of one item and the term of criteria of several items were improved from the Ver. 2.12. The term of criteria for "R03: Appearance of a pigmentation pattern suggesting retinal degeneration in the whole retina" was changed from "1 = slightly rude", 2 = rude" to "slightly mottled", "2 = mottled". The choices for R03 include seven possibilities: "0 = none", "1 = slightly mottled", "2 = mottled", "3= spotted", "4 = salt and pepper appearance", "5 = small and large white spots" and "6 = large areas with hyperpigmentation and hypopigmentation". The term of criteria for "R08: Change around the optic disc" was changed from "2 = rude" to "2 = mottled". The choices for R08 include four possibilities: "0 = none", "1 = conus", "2 = mottled", "3= other". The title of "R04" was changed from "Pattern (not retinal degeneration) on the whole retina" to "Appearance of a pattern (not retinal degeneration) on the whole retina". The term of criteria for "R05: Appearance of white spots on a portion of the retina" was changed from "2 = many white spots (> 10 spots)","3 = white spots the size of an optic disc" to "2 = "many small white spots (> 10 spots),"3 = white area(s) the size of an optic disc". The choices for R05 include seven possibilities:" 0 = none","1 = small number of isolated white spots (<= 10 spots)","2 = many small white spots (> 10 spots)","3 = white area(s) the size of an optic disc","4 = white area larger than an optic disc"," 5 = retinal fold" and "6 = other". v.2.12 This protocol (version 2.12) was described as the SOP. v.2.11 This protocol (version 2.11) was described as the SOP. v.2.10 This protocol (version 2.10) was described as the SOP. v.2.01 This protocol (version 2.01) was described as the SOP. v.2.00 This protocol (version 2.00) was described as the SOP. v.1