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Figure 3. C57BL/6 mouse with a hemispherical plastic diffuser attached in front of the right eye. The plastic collar was attached to prevent the mouse from removing the diffuser (from Ref. 22).
no effect on corneal curvature in C57BL/6 mice was found. After 37 days in continuous white light with about 500 lux ambient illuminance, corneal radius was 1.42 ± 0.04 mm (n = 25 eyes), versus 1.40 ± 0.05 mm (n = 20 eyes) in animals kept under regular 12/12 h light/dark cycle. There were significant differences in refraction (+3.1 ± 3.6, n = 40, versus +6.4 ± 4.3, n = 51, p < 0.001), but these small changes were in the opposite direction as in chickens.8
Finally, lid suture was also used to induce deprivation myopia2,33 in 20 days or four weeks, respectively. Non-visual effects on eye growth cannot be excluded, such as increased mechanical pressure on the globe, which might cause a rebound effect after lid re-opening, changes in the metabolic conditions due to reduced oxygen supply or elevated ocular temperature.
Techniques to measure the induced refractive errors and changes in eye growth
Refractive state
In a number of studies, refractive states were measured by white light streak retinoscopy (e.g. Refs. 2, 7, 33, 34). In streak retinoscopy, a slightly
311 The Mouse Model of Myopia
defocused light streak is projected onto the eye from the retinoscope, which is held at about an arm’s length from the mouse. A small fraction of this light is reflected from the back of the eye, the fundus, and is visible in the pupil. The movement of the reflection in the pupil must be compared to the movement of the light streak seen on the fur surrounding the eye, while the streak retinoscope is tilted up and down.
If the reflection in the pupil appears stationary with no clear direction of movement, the “reversal point” is reached and the eye can be assumed to be in focus with the observer. Otherwise, differently powered trial lenses are held in front of the eye until the reversal point is reached, and the lens power provides the information about refractive state. The procedure works well in animals with large pupils, but it is very difficult to judge the direction of movement of the light bar in small pupils (1 mm in diameter, or even smaller, if the pupil constricts due to the white light emitted by the retinoscope). In a trial carried out by a certified optometrist, no correlation was found between streak retinoscopy and infrared photoretinoscopy in22 alert, non-cyclopleged black mice.8 Streak retinoscopy also provided generally more hyperopic readings than infrared photoretinoscopy (see below). High hyperopia was also found in other studies using streak retinoscopy (+20 D34; +13.5 D2; +15 D33; and >+10 D21 — see Fig. 1B). An interesting case involves albino mice (as used by Barathi et al.).21 In these mice, the iris is scarcely pigmented and light penetrates easily. Therefore, these animals are, in fact, mainly refracted through the iris, mimicking a large pupil — finally limited only by the diameter of the cornea. The movement of the retinoscopic reflection can therefore be judged much more easily than in (non-cyclopleged) black mice. Given that light scatter in the iris should further degrade the retinal image, it is interesting that myopia could still be induced by negative lenses in front of the eye.
A perhaps powerful technique for refracting small vertebrates is infrared photoretinoscopy. This technique is video-based, uses infrared light, and has been successfully applied in a variety of vertebrate eyes (e.g. barn owls; toads and tadpoles; frogs and salamanders; water snakes).29,35–37 Since infrared light is used, the animal is not disturbed by the measurement and the pupil does not constrict. To measure a mouse, it is sufficient to slightly restrain it by holding its tail while it rests on a small platform and turning down the room light since the pupil of the mouse is very responsive to light.38 An infrared sensitive video camera is positioned at about 60 cm distance. Attached to the camera lens is an arrangement of
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infrared light emitting diodes (IR LED; see Fig. 4A). A small fraction of this light enters the pupil, is diffusely reflected from the fundus of the eye, and returns to the camera. Because the IR LEDs are positioned directly below the camera aperture, they produce a brightly illuminated pupil — like the “red eye effect” seen with flash cameras. Furthermore, the brightness distribution in the vertical pupil meridian displays a gradient, with more light in the bottom in the case of a myopic eye (a screen dump of the refraction software is shown in Fig. 4B), and more light in the top of the pupil in a hyperopic eye. The brightness distributions in the pupils of mice are not smooth, but bumpy, indicating that the optics has considerable aberrations; furthermore, they are affected by the first Purkinje image. Figure 4B shows the measured brightness profile, together with a linear regression line fit through the pixel brightness values. Refractions can be determined from the slopes of these regression lines. The only unknown variable is then the conversion factor from the slope of the brightness profile in the pupil into refraction. However, this factor can be determined by placing trial lenses of known optical power in front of the mouse eye, inducing known refractive errors, and recording the slopes.22
Figure 4. (A) USB2 monochrome video camera, 50 mm lens with focal length extender and 10 mm extension ring, and custom-built photoretinoscope. The camera and the infrared LEDs of the photoretinoscope can be run through the USB port of the laptop, making additional power supplies unnecessary. (B) Screen dump of the software, developed in Visual C++, designed to measure refraction and pupil size with 62 Hz sampling rate. In addition, light-induced pupil responses can be recorded, which are elicited by a green LED attached to retinoscope (not shown in the version in (A)) and flashed through the USB port.
313 The Mouse Model of Myopia
The temporal sampling rate of this technique is currently 62 Hz, a typical frame rate for USB2 cameras (Fig. 4A). As soon as the mouse eye appears in the video frame, the image processing software detects the pupil — which is a simple task because it is brightly illuminated over a dark background — and fits a linear regression through the pixel brightness values in the vertical pupil meridian.
Even though the measurements are easy to perform, some limitations have to be considered:1 because of the excavation of the optic disc (nicely visible in the frozen section of the mouse eye presented by Remtulla and Hallett7), the eye is more myopic (or less hyperopic), close to the pupillary axis and appears considerably more hyperopic in the periphery due to the thickness of the retina.22 Therefore, for consistent refractions, it is important to align the eye with the camera axis.22 It was observed that mice sometimes change their refractive state for a few seconds and become a few diopters more myopic. The mechanism behind this optical change has not yet been systematically studied but it is clear that it occurs without visual stimulation and does not represent accommodation. It was also observed under cycloplegia with Tropicamide,22 and Woolf 39 was unable to find a ciliary muscle for accommodation in the mouse eye. Also, Smith et al.40 stated that the ciliary muscle in the mouse eye is weak and lacks accommodation. An alternative explanation for this change in refraction is that it is produced by activity of the retractor bulbi muscles,41,42 which can pull the globe back into the orbit, causing a temporary change in intraocular pressure, which, in turn, could affect the refractive state. Therefore, it is important to observe mice for several seconds to ensure that their eyes are in a relaxed condition.22 It was found that mice were measured more hyperopic when they had larger pupils (about 0.9 D more hyperopia per 0.1 mm increase in pupil size).22 This effect could result from negative spherical aberration (more hypopic refractions in the pupil periphery). On the other hand, positive spherical aberration was found in mouse eyes by Hartmann-Shack aberrometry,10 and it is more likely that the increasing hyperopia results from non-linearities in the video system. Larger pupils return more light, proportional to the pupil area, and pixel values are not perfectly log-linear to the absolute brightness. A more extensive calibration with different camera aperture sizes would be necessary to control this factor. The standard deviation typically obtained in the same eyes in repeated measurements was about 2.7 D22 — much less than the optical and behavioral depth of focus (see below).