Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

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FRANK SCHAEFFEL

Like a number of small mammals, mice are not predominantly “visual animals.” Duke-Elder (1958, p. 639) wrote, “Mice are nocturnal in type and obviously depend visually on the appreciation of differences in luminosity and movement, rather than on the very imperfect pattern vision of which their eyes are capable.” Had researchers not advanced beyond these assumptions, mice might never have been proposed as a model to study the mechanisms of myopia. As it happens, the visual control of eye growth in the mouse appears to be marginal. On the other hand, the findings from numerous knockout models and microarrays and extensive knowledge of the physiology, biochemistry, and genetics of the mouse cannot be ignored. Furthermore, spontaneous ocular mutations such as the albino locus and the p locus caught the attention of eye researchers long before knockout animals were known. These two eye color mutations represent the first gene loci that were ever mapped in vertebrates, in this case to chromosome 7. For these reasons, attempts to establish the mouse as a new model for myopia studies may be worthwhile.

How good can spatial resolution be in a mouse eye?

The eye of an adult mouse has an axial length of little more than 3 mm (Remtulla and Hallett, 1985; Schmucker and Schaeffel, 2004a). At this size, the mouse eye is comparable to the eyes of other mammals of similar weight, as illustrated in an impressive figure by A. Hughes (1977, p. 654). Hughes showed eye length versus body weight for many different vertebrates. If eye weight is scaled to body weight, the mouse eye is five times larger than the human eye. Thus, the mouse eye cannot be considered vestigial.

To focus an image of the environment onto the retina of a mouse eye, the refractive power of cornea and lens must be more than 500 diopters (D) in air (a third of power is lost because the backside of the eye’s optics is, as in all vertebrate eyes, filled with water-like fluid). Because of this high power, a refractive error of a few diopters—most disturbing for humans, who have only about 60 D of total power in the eye—is not so relevant for the mouse. For the same reason, small imperfections in the optics of the mouse eye are less important than in the human eye.

Even if optically optimal, small vertebrate eyes generally have lower visual acuity (Kirschfeld, 1984). This is because the image projected onto the retina is proportionally smaller. To sample the fine details in a tiny image, the sampling units—the photoreceptors—should also be proportionally smaller. However, this is not possible, for physical reasons: when photoreceptor diameters approach the range of the wavelength of light, “optical crosstalk” between receptors occurs since light energy “leaks out” (Kirschfeld and Snyder, 1976). This phenomenon limits further increase in sampling density. Probably for this reason, vertebrate photoreceptors are not thinner than about 1 μm—two times the wavelength of visible light.

In a human eye, 1° of the visual field maps on 0.29 mm linear distance on the retina. In a 28-day-old mouse, the image magnification is only a tenth (0.03 mm/deg). Accordingly, even with the best possible optics, a mouse eye can achieve only a tenth of the human eye’s spatial resolution. As shown in chapter 7 in this volume, the spatial resolution of the mouse eye is even lower, by almost another factor of 10, in part as a result of optical aberrations but mainly because of the large photoreceptor diameters (>2–3 μm; Carter-Dawson and LaVail, 1979) and neuronal spatial processing (de la Cera et al., 2006). Given this poor visual acuity, it seems likely that emmetropization (the developmental matching of axial eye length to the focal length of the optics) may not be as precise as in some birds or primates that have high visual acuity and are, in part, not limited by ocular optical aberrations but rather by physics—the diffraction of light.

Schematic eye modeling

The first schematic eye for the adult C57B1/6J mouse was presented by Remtulla and Hallett in 1985. It was developed based on frozen sections of 14 eyes of 20to 23-week-old animals. Schmucker and Schaeffel (2004a) developed a paraxial schematic eye model for C57BL/6J mice at different ages, also based on frozen sections (figure 5.1). Although frozen sections have limited resolution because of the distortions that can occur during freezing and sectioning, it is possible to average measurements from several eyes and to

Figure 5.1 Frozen sections of mouse eyes at age 26 and 44 days. (Replotted from Schmucker and Schaeffel, 2004a.) The overproportional growth of the lens reduces the depth of the vitreous chamber with age. Such large lenses make it unlikely that accommodation is present in the eye, because these lenses cannot be

fit the biometrical data from different ages by polynomials. The averaging procedure reduces the impact of measurement variability, and a few general statements can be made about the optics of the mouse eye:

1. In line with an observation by Zhou and Williams (1999), the eyes grow linearly over the first 100 days, with no signs of saturation. Axial length increases from 3.0 mm at 22 days of age, by 4.4 μm/day.

2.Interestingly, because the lens increases in thickness from 1.74 mm at 22 days of age by 5.5 μm/day, the vitreous

chamber actually declines in depth from 0.83 mm at 22 days, by 3.2 μm/day.

3.A recent study using video photokeratometry in Egr-1 −/− mice and their wild-type littermates (Schippert et al.,

2007) showed that the corneal radius of curvature increases from 1.35 mm to 1.53 mm from day 22 to 100.

4.To make a mouse eye more myopic by 1 D, axial length must be increased by 5.41 μm at the age of 22 days and by 6.49 μm at the age of 100 days.

5.Retinal image magnification increases from 0.032 mm/ deg at 22 days to 0.034 mm/deg at 100 days.

6.As in other animals (e.g., chicken: Schaeffel et al., 1986; barn owl: Schaeffel and Wagner, 1996), the retinal image brightness increases with age. Image brightness is proportional to the inverse squared f-number, the ratio of anterior focal length to pupil size. Typical for humans are f-numbers around 5 (for a 3.3-mm pupil), but for the mouse, the f-number is 1.02 already at day 22. This produces a 25 times brighter retinal image than in humans. Until the age of 100 days, the f-number in mouse declines even further,

easily deformed. Note the relative thickness of the retina, which should give rise to a large “small eye artifact” in retinoscopic measurements (Glickstein and Millodot, 1970). Scale bars denote millimeters.

to about 0.92, providing another 20% more brightness. Even owls do not have such a low f-number (1.13; Schaeffel and Wagner, 1996). The mouse probably has one of the brightest retinal images among vertebrates.

7. The thickness of the retina, relative to axial length (which increases from 0.178 mm at 22 days of age, by 0.6 μm/day), should give rise to a large difference in the position of the photoreceptor layer and of the light reflecting layer(s) in retinoscopy—resulting in large amounts of apparently measured hyperopia (the “small eye artifact”; Glickstein and Millodot, 1970). From the schematic eye, the small eye artifact was calculated to more than 30 D (Schmucker and Schaeffel, 2004a). Since infrared photorefraction and even streak retinoscopy did not show such large amounts of hyperopia, either mice must be myopic (but see the discussion of behavioral depth-of-field measurements in the next section) or the light-reflecting layer(s) is (are) not at the vitreoretinal interface but rather deeper in the retina.

New techniques to measure optical parameters in very small eyes like the mouse’s

To study the mechanisms of visual control of eye growth and myopia in a mouse, optical parameters of the eye must be measured in vivo. Standard optometric techniques generally fail, owing to the tiny size of the eye.

Infrared Photoretinoscopy First, refractive state must be determined. In a number of previous studies, this was done by white light streak retinoscopy (e.g., Drager, 1975;

Remtulla and Hallett, 1985; Beuerman et al., 2003; Tejedor and de la Villa, 2003). In streak retinoscopy, a slightly defocused light streak is projected onto the eye from the retinoscope, which is held at about arm’s length distance from the subject, 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 is compared to the movement of the light streak on the fur surrounding the eye as the streak retinoscope is titled up and down. If the reflection in the pupil appears stationary with no clear direction of movement, the “reversal point” is reached, where the eye is assumed to be in focus with the observer. Otherwise, trial lenses of different power are held in front of the eye until the reversal point is reached, and the lens power then 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 the movement of the light bar in small pupils (1 mm in diameter, or 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 could be found between streak retinoscopy and infrared photoretinoscopy in 52 alert, noncyclopleged mouse eyes (refraction measured with streak retinoscopy = 0.276 × refraction measured with infrared [IR] photoretinoscopy + 15.988, R2 = 0.047; Schmucker, 2004). The range of refractions measured with IR photoretinoscopy was from +2 and +12 D, but the values obtained with streak retinoscopy ranged between +12 and +22 D. As can be seen, streak retinoscopy also provides considerably more hyperopic readings than IR photoretinoscopy. High hyperopia was also found in other studies using streak retinoscopy (+20 D: Drager, 1975; +13.5 D: Tejedor and de la Villa, 2003; +15 D: Beuerman et al., 2003).

A more powerful technique for refracting small vertebrates may be IR photoretinoscopy. This technique is videobased, uses IR light, and has been successfully applied in a variety of vertebrate eyes (e.g., toads and tadpoles: Mathis et al., 1988; frogs and salamanders: Schaeffel et al., 1994; water snakes: Schaeffel and Mathis, 1991; barn owls: Schaeffel and Wagner, 1996). Because IR 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. An IR-sensitive video camera is positioned at about 60 cm distance. Attached to the camera lens is an arrangement of IR light–emitting diodes (IR LEDs; figure 5.2A, white arrow at top right). 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 top in

the case of a hyperopic eye (figure 5.2A) and more light in the bottom in a myopic eye (figure 5.2B). The measured brightness profiles are shown to the right of the pupils in figures 5.2A and 5.2B, together with a linear regression line fit through the pixel brightness values (arrows in the figures). The refractions can be determined from the slopes of these regression lines. The only unknown variable then is the conversion factor from slope of the brightness profile in the pupil into refraction. 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 (Schaeffel et al., 2004). The temporal sampling rate of this technique is determined by the video frame rate (analogue cameras: 25 Hz [PAL] or 30 Hz [NTSC], and 30 Hz or more with firewire cameras). 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 Hallett, 1985), 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 (Schaeffel et al., 2004). Therefore, for consistent refractions, it is important to align the eye with the camera axis. (2) 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 most likely is not accommodation. It was also observed under cycloplegia with tropicamide (Schaeffel et al., 2004), and Woolf (1956) was unable to find a ciliary muscle for accommodation in the mouse eye. Also, Smith et al. (2002) state that the ciliary muscle in the mouse eye is weak and accommodation lacking. An alternative explanation for this change in refraction is that it is produced by the activity of the retractor bulbi muscles (Lorente de No, 1933; Pachter et al., 1976), which can pull the globe back into the orbit, causing a temporary change in intraocular pressure that, in turn, could affect refractive state. Therefore, it is important to observe mice for several seconds to ensure their eyes are in relaxed condition. (3) Mice were measured more hyperopic when they had larger pupils (about 0.9 D more hyperopia per 0.1 mm increase in pupil size; Schaeffel et al., 2004). This effect could result from negative spherical aberration (more hyperopic refractions in the pupil periphery). On the other hand, positive spherical aberration was found in mouse eyes by Hart- mann-Shack aberrometry (de la Cera et al., 2006), and it is more likely that the increasing hyperopia results from non-

Figure 5.2 Infrared photorefraction in alert mice. A, Hyperopic eye. B, Myopic eye. The IR LED arrangement (A, inset, white arrow) causes brightly illuminated pupils. The brightness gradient in the vertical pupil meridian can be fit by linear regression (white lines indicated by arrow to right of pupil). Calibration with trial lenses showed that the slopes of the regression lines are linearly related

linearities in the video system. Larger pupils return more light, proportional to 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 for this factor. The standard deviation typically obtained in the same eyes on repeated mea-

to refractive error of the eye. The brightness distributions in the pupils (see 3D brightness profile of the pupil, left, and brightness profile, right) are quite heterogeneous in eyes with poor optics, such as in the mouse, but smooth in eyes with high optical quality, such as in humans (not shown). (Replotted from Schaeffel et al., 2004.)

surement was about 2.7 D (Schaeffel et al., 2004)—much less than the optical depth of focus.

Photokeratometry The corneal surface is the optically most powerful surface of the eye—in most vertebrates it generates more than 60% of the total optical power.

Therefore, it is important to measure its radius of curvature. This can be done by IR photokeratometry in alert mice. Mice are placed on a platform (figure 5.3A, white arrow) and slightly restrained by holding their tail. The platform is positioned about 15 cm from a metal ring with a diameter of 300 mm that carries eight IR LEDs (figure 5.3A). The reflections of the IR LEDs on the corneal surface, the first Purkinje images, are also arranged in a circular pattern (figure 5.3B). In a digital video image of the eye, the reflections can be detected by an image-processing program and fit by a circle in real time (at 25–60 Hz, depending on the hardware platform). The diameter of the fitted circle is proportional to the curvature of the cornea—small circles for steep corneas with small radius of curvature and larger circles for flat corneas. Although the equations to calculate corneal curvature from camera distance, distance of the IR LEDs of the keratometer, distance of the keratometer to the mouse,

and camera magnification have been worked out (Howland and Sayles, 1985), it is easier just to measure a few ball bearings with a known radius of curvature (figure 5.3C) and extrapolate to the corneal radius of curvature of the animal under study. The standard deviation of this procedure is about 1%, with the major source of variability the depth of focus of the video camera. The video camera depth of field ultimatively determines how precisely the distance of the mouse to the keratometer and the camera are controlled.

Low-Coherence Interferometry Perhaps the most important variable in myopia studies is axial length. Human myopia is axial in most cases (Curtin, 1985), which means that the eye is growing too long in the anteroposterior axis compared to an eye of an emmetropic control group. In a few cases myopia develops because the refractive power of the lens increases (e.g., when diabetes or cataract develops),

measured radius [mm]

C

calibration with ball bearings

4

3

2

1

ball bearing radius of curvature [mm]

Figure 5.3 Infrared photokeratometry. A, The alert mouse is positioned on a wooden platform and is slightly restrained by the tester holding its tail (white arrow). The block is slowly moved back and forth until the reflections from eight IR LEDs are visible in a highly magnified video image of the eye (B). Once the first Purkinje images are in focus, the software detects their centers and fits a

circle through them. This occurs at a video frequency between 25 and 60 Hz, depending on the hardware. The high-speed measurements also provide the current standard deviations of the radii. The diameter of this circle is proportional to the curvature of the cornea. C, The calibration factor can be determined by comparing the measured radius to the radius of ball bearings.