IRBP deficiency permits precocious ocular development and myopia

Mouse husbandry

Mouse care and handling were approved by the Emory University Institutional Animal Care and Use Committee and adhered to standards recommended by the Association for Research in Vision and Ophthalmology (ARVO) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). C57BL/6J (WT) and congenic IRBP KO mice were used that had been backcrossed more than ten times to WT. The WT and IRBP KO mice colony is routinely screened for the Crb1^rd8/rd8 (rd8) mutation [40]. The WT and IRBP KO mice used in the current study showed the absence of the rd8 mutation. Mouse housing conditions and diet have been described [41]. The Emory University Division of Animal Resources housed and managed mice. Mice were housed at 23 °C on a 12 h:12 h light-dark Cycle and were given and standard mouse chow (Lab Diet 5001; PMI Nutrition Inc., LLC, Brentwood, MO) and water ad libitum. At 9:00 AM in the morning, we searched for new litters. New pups were defined as P1 if they were detected in the 9:00 AM census. The number of mice used per experiment is given in the Results and in each figure and table legend.

Slit-lamp examination

A slit lamp (Carl Zeiss, SL 130, Dublin, CA) was used to examine the mouse eyes. To test whether the optical media were clear in IRBP KO mice, we subjected the WT (n = 5) and IRBP KO (n = 6) mice at P21 to slit-lamp examinations. An examiner who was masked regarding the genotype of the mouse conducted the assessment. An average grading score (0–4) was given for the cornea and lens with a lower grade representing clear structures [42,43]. A Mann–Whitney test was used to assess the statistical significance between groups.

SD-OCT and fundoscopy

A Micron IV SD-OCT system and a fundus camera were used (Phoenix Research Labs, Pleasanton, CA) to analyze retinal morphology in the WT and IRBP KO mice at P15 (WT, n = 5; KO, n = 10), P30 (WT, n = 8; KO, n = 6), P45 (WT, n = 7; KO, n = 8), and P80 (WT, n = 5; KO, n = 7). Image-guided OCT images were obtained for the left and right eyes after a sharp and clear image of the fundus (with the optic nerve centered) was obtained. SD-OCT was a circular scan about 100 μm from the optic nerve head. Fifty scans were averaged. The retinal layers (indicated on the figure images) were identified according to published nomenclature [44,45]. Total retinal thickness and photoreceptor layer thickness were analyzed.

Keratometry

An automated keratometer was used to measure the corneal radius of curvature in the WT (n = 3) and IRBP KO (n = 5) mice at P53 to P55 [46]. The mean corneal radius of curvature (± SD) was recorded. An unpaired two-tailed Student t test with the Welch correction was used to assess statistical significance.

Whole-eye biometry

The WT and IRBP KO mice at P30 (WT, n = 8; KO, n = 6), P45 (WT, n = 7; KO, n = 8), P55 (WT, n = 3; KO, n = 5) and P80 (WT, n = 5; KO, n = 7) were subjected to in vivo whole-eye biometrical imaging using a Bioptigen R4310 deep imaging SD-OCT system (Leica Microsystems, Durham, NC). Post-imaging analysis included measurement of the following parameters: central corneal thickness (CCT), anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), retinal thickness (RT), and total ocular axial length. The mean thickness (± standard deviation, SD) was recorded. Two-way ANOVA with a Tukey test was used to assess statistical significance.

Metrics and metrology

Mice were euthanized with CO₂ asphyxiation and cervical dislocation before eye dissection, according to protocols approved by the Emory University IACUC and ARVO guidelines. After the mice were euthanized, each eye was removed with forceps, and the extraocular fat and muscles were removed. The optic nerve was cut to leave no stump. Eye weights were recorded with an A-160 analytical balance (Denver Instruments; Fisher Scientific, Pittsburgh, PA). Eye dimensions on three axes were obtained with a light-emitting diode (LED) noncontact micrometer (LS-7030; Keyence Inc., Schaumburg, IL) as described earlier [47] at P5 (WT, n = 10; KO, n = 9), P6 (WT, n = 8; KO, n = 10), P7 (WT, n = 4; KO, n = 4), P8 (WT, n = 6; KO, n = 6), P9 (WT, n = 6; KO, n = 8), and P10 (WT, n = 5; KO, n = 3).

Histology and immunohistochemistry

Routine paraffin-embedded histology with hematoxylin and eosin (H&E) staining, plastic sectioning, staining with toluidine blue, and morphometric measurements were conducted as previously described [22,48]. We detected TH-positive cells in the inner nuclear layer (INL) by performing immunohistochemistry with primary anti-TH antibodies raised in rabbits (Pel-Freez Biologicals; Little Rock, AR). The secondary antibodies against the primary antibody were donkey anti-rabbit Alexa Fluor 488 from Life Technologies, Inc. (Carlsbad, CA). The ages and numbers of the mice were P23 (WT, n = 6; KO, n = 6), P26 (WT, n = 6; KO, n = 5), P30 (WT, n = 5; KO, n = 7), and P36 (WT, n = 10; KO, n = 13). Briefly, the cornea was perforated with a 27 gauge beveled needle, and the eye was fixed for 10 min at room temperature in freshly prepared 4% paraformaldehyde in Dulbecco’s PBS (1X ; 2.66 mM KCL, 1.47 mM, KH₂PO₄,137.93 mM NaCl,8.05 and mM Na₂HPO₄-7H₂O,pH 7.4; without calcium or magnesium). An eyecup lacking the lens was dissected with iridectomy scissors. The eyecup was filled with 2 mg/ml hyaluronidase (Type IV-S H3884; Sigma-Aldrich, St. Louis, MO) and incubated for 30 min at 23 °C. The eyecup was frozen at −80 °C and thawed at 23 °C to further permeabilize the retina. The retina was removed from the underlying RPE and was flatmounted on a glass slide with the photoreceptor cells face down inside a silicone gasket [48]. Retinas were blocked in 0.25% (V/V) Triton X-100 in PBS and 2% (V/V) normal donkey serum (NDS) overnight at 4 °C, rinsed three times in PBS for 5 min each and then incubated at 4 °C for 3 days in the rabbit anti-rat TH primary antibody at 1:250 dilution in PBS with 0.25% (V/V) Triton X-100 and 2% NDS. The retinas were washed three times in PBS, 5 min each, and incubated in the secondary antibody at 1:500 dilution in PBS with 0.25% (V/V) Triton X-100 in darkness at 4 °C overnight. Retinas were mounted in Vectashield Hardset (Vector Labs Inc., Burlingame, CA), dried for 1 to 2 days at 4 °C in the dark, and imaged with a Nikon C1 confocal microscope (Nikon Instruments, Melville, NY) as described [49,50]. The focal plane was adjusted to image and count TH-positive cells in the INL (dopaminergic amacrine cell bodies).

DA and DOPAC measurements

For the DA and DOPAC analyses conducted in the neural retina, the eyes were harvested, and the neural retina was dissected from the eye as previously described [51] at P7 (WT, n = 7; KO, n = 10), P10 (WT, n = 9; KO, n = 9), P12 (WT, n = 8; KO, n = 9), P23 (WT, n = 9; KO, n = 8), P26 (WT, n = 10; KO, n = 10); P36 (WT, n = 9; KO, n = 12), and P60 (WT, n = 9; KO, n = 9). The retinas were homogenized in 0.2 N perchloric acid containing 0.01% sodium metabisulfite and centrifuged; the supernatant fractions were analyzed with high-performance liquid chromatography (HPLC; Agilent, Santa Clara, CA) eluted with an isocratic mobile phase (5% (V/V) acetonitrile, 0.32 mM sodium octylsulfate, 50 mM sodium phosphate, 0.1 mM EDTA, pH 2.7 to 3.2) on a 5 μm Ultrasphere 250 × 4.6 mm C18 column (catalog number 235,329; Hichrom, Ltd., Reading, UK) with reverse phase chromatography and an electrochemical detection system [30,52,53].

OPL grading

Paraffin and plastic sections of WT (n = 16) and IRBP KO (n = 8) mouse retinas at P5 and P6 were ranked by three observers who were masked regarding the genotype of the mouse. Grade 1 included a distinct and continuous outer plexiform layer (OPL). Grade 2 represented a less distinctive OPL, and grade 3 had little to no evidence of an OPL. A Mann–Whitney test was used to assess statistical significance between groups.

Goggling

A diffuser lens covering the right eye was connected to a head pedestal to induce visual form deprivation in mice from P28 to P56, as previously described [54]. The diffusers were plano contact lenses made from polymethyl methacrylate (PMMA; X-Cel Contacts, Duluth, GA) painted with partially translucent white paint (Top Speed Fast Dry Nail Enamel, 0,429,920; Revlon, New York, NY). The goggles were counterbalanced to ensure even weight distribution. The contralateral (left) eyes were not goggled. An infrared automated photorefractor [55] was used as previously described [22,54,56]. A two-way repeated-measures ANOVA with the Holm-Sidak test was used to assess statistical significance between the groups.

Visual acuity

Optokinetic tracking responses (OKTs) were obtained with an optomotor system (Cerebral Mechanics Inc., Lethbridge, Canada). Visual acuity was measured at 100% contrast as previously described [57-59]. Three experienced observers evaluated mice from different litters at P26 (KO, n = 6), P30 (WT, n = 8; KO, n = 2), P36 (KO, n = 2), P45 (WT, n = 7; KO, n = 1), P95–P96 (WT, n = 6; KO, n = 7), and P265 (KO, n = 6).

Statistical analysis

Data from each group are summarized as the mean ± standard deviation with the sample size indicated in each figure, legend, and table. In each sample group, mice from different litters were included. Analysis was performed with an unpaired two-tailed Student t test with the Welch correction, ANOVA with Tukey’s test, or other tests as indicated in the text or figure legend. GraphPad Prism 6.0 for Mac OS X (GraphPad Software, Inc., La Jolla, CA) was used.

Optical clarity, fundus imaging, keratometry, and SD-OCT

One explanation for the KO effects on axial elongation is that the media were turbid, cloudy, and obscured form, leading to eye elongation by form deprivation myopia as routinely occurs in retinal degenerations that are associated with posterior subcapsular cataracts [37,38]. Thus, an early experiment was to test the clarity of the optical media in the two mouse strains (WT and IRBP KO) at four ages: one (P15) just after eye opening, one after the burst of photoreceptor cell death had occurred in the IRBP KO mice (P30), and two later time points (P45 and P80) in the living mice. About 50% of the IRBP KO mice (n = 10) at P15 had a slightly cloudy optical path. In the WT mice, only one of five mice had a cloudy axis at P15. However, the optical media (the cornea, anterior chamber, lens and vitreous) all were equally clear and transparent in both strains and at all other ages as established by sharp and distinctive layering within the retina of the same mice with noninvasive SD-OCT (Micron IV) and by equally crisp fundus photographs (Figure 1A–H). To test whether the curvature of the cornea is altered in IRBP KO mice, we measured the corneal radius of curvature in the WT and IRBP KO mice at P55. We observed no significant difference between the corneal curvature of the WT mice and that of the IRBP KO mice (mean = 1.413±0.012 mm for the KO mice, n = 8; for WT mice, 1.392±0.015 D, n = 3; p = 0.3301, unpaired two-tailed Student t test with the Welch correction).

We next examined the eye with a slit lamp to evaluate the clarity in the cornea and lens. We did not detect any obvious opacity or haze at P21 in either eye in five KO mice and five WT mice. The slit-lamp exam revealed no significant difference in the average grading score for the IRBP KO and WT mice (the Mann–Whitney test, cornea, p = 0.318 and lens, p = 0.0541). These results did not support the hypothesis that cloudiness, corneal defects, lens defects, or back scattering of light prevented form detection in the IRBP KO mouse eye, which alone would have explained profound myopia in the IRBP KO mouse.

SD-OCT images from individual living mice verified previous histological work [22] on enucleated eyes that identified substantial outer nuclear layer (ONL) layer loss after P15 and before P30, P45, and P80 (Figure 1; statistics given in Table 1, all p<0.0001) in the IRBP KO mouse eye. At P15, the ONL was 13% less thick in the IRBP KO mouse eye than in the WT mouse eye. At P30, 34.4% of the ONL thickness was lost in the IRBP KO mouse eye compared to the WT mouse eye at the same age. At P45–P80, similar thinning in ONL thickness was observed in the IRBP KO mouse eye compared to the WT mouse eye (35.9% at P45), and 38.6% loss of the ONL in the IRBP KO mouse eye at P80 compared to the WT mouse eye. Total retinal thickness was lost in the IRBP KO mouse eye relative to the WT mouse eye following the same pattern (Table 1) with almost all of the differences between the KO and WT mouse eyes accounted for by the profound loss of ONL thickness. A noteworthy observation was an 11% increase in photoreceptor thickness in the WT mouse eye from P15 to P30 (88.1±1.42 to 97.8±3.53 μm, respectively, which was statistically significant at p<0.01; Tukey’s test). This thickness increase agrees with our previous observation [22] of a similar increase in photoreceptor nuclei counts across the ONL between P15 and P30 in histological cross sections.

Fundus imaging (Figure 1) detected no mottling at P15 in either the IRBP KO or WT mice. However, from P30 to P80, there was readily observed mottling at the layer of the outer segments and the RPE in the IRBP KO mice, but no such mottling was observed in the WT mice at P30–P80. These images highlight the subtle character of the changes in the eye caused by the absence of IRBP until after P15 and the profound ONL loss in a tight time window from P23 to P30 as previously observed in histological sections [22]. Mottling of the IRBP KO fundus coincided temporally with ONL loss; however, any causal relationship of mottling and ONL loss is not understood.

Table 2 illustrates axial measurements shown with SD-OCT (Bioptigen, model 4310) from living mice. We obtained the total optic axis length (anterior cornea to the RPE, an internal diameter measure), CCT, ACD, lens thickness, vitreous depth, and retina thickness. The total of these measurements summed to about 50 μm thinner than the measurement from the LED noncontact micrometer for external eye size [47]. This difference in thickness was contributed by the choroid and the sclera. The chorioscleral thickness, estimated from the difference, was the same as the measurement of the chorioscleral thickness from histological sections. These measurements were all consistent internally and with previous histological and LED micrometer measurements [22], which validated the SD-OCT measurements in live mice.

The vitreous chamber was substantially deeper (about 60% more) in the IRBP KO mice than in the WT mice at all four ages (P30, P45, P55, and P80; Table 2). This difference accounted for all the excessive eye elongation in the IRBP KO mouse over the WT mouse. None of the other biometric parameters were significantly different in the IRBP KO and WT mice. The lens of the IRBP KO mouse eye was the same thickness as in the WT mouse eye at equivalent ages. These lens thickness measurements with SD-OCT in live mice are the same as those we previously reported with an LED micrometer in unfixed harvested tissue [22] and with measurements of histological H&E-stained sections of the lens after formalin fixation or freeze-substitution and paraffin embedding (data not shown).

Onset of aberrant eye elongation in IRBP KO mice

We previously found changes in the size and axial length in IRBP KO mice as early as P10 [22]. To refine the onset of KO eye elongation, we studied IRBP KO and WT eyes at 1-day increments from P5 to P10 (Figure 2 and Figure 3). It was clear that the eye weight of the IRBP KO mice began to deviate by P7 (Figure 2), but not at earlier ages, P5 or P6. By P8 and older, the IRBP KO mice had heavier eyes than the WT mice (Figure 2), according to two-way ANOVA with the Sidak multiple comparisons test (p = 0.0021). As shown in Figure 3, the IRBP KO animals also showed a trend for longer axial length from P8 onward using the LED non-contact micrometer that became statistically significant at P10 (Appendix 1, panels 3B and 3C). We found that the IRBP KO and WT mouse eyelids opened at the same time (mean = 13.0±0.93 days for the KO mice, n = 8 litters; for the WT mice, 13.8±0.67 days, n = 9 litters; p = 0.0717, unpaired two-tailed Student t test with the Welch correction). Thus, in the IRBP KO animals, the eyes were heavier and generally longer on the optical axis 6 days before the eyelids opened than in the WT animals.

Morphology of eyes in IRBP KO and WT mice

We did not detect any major histological changes in the structure of the eye, including the retina, lens, cornea, iris, ciliary body, and optic nerve head, with the exception of a small constant thinning of the retinal layers, which began at P7. This thinning was in proportion to the expansion of the eye surface area over which the retina was stretched in a comparison of the IRBP KO and WT mice. These thicknesses are illustrated in Figure 4A (the retina) and Figure 4B-E in Appendix 2.

Developmental maturation in the IRBP KO and WT mice appeared to be similar with the exception of the appearance of the OPL at P5 in the IRBP KO mice. The OPL appeared more distinct, better formed, and more mature in the IRBP KO mice than in the WT mice at P5 (Figure 5; Table 3). However, a day later, the OPL appeared equally mature in the IRBP KO and WT mice, and the same morphologically as assessed with toluidine blue staining of the plastic sections and H&E staining of the paraffin sections (data not shown). Retinal sections from P5 WT (n = 16) and IRBP KO (n = 8) mice were ranked by three blinded observers. Grade 1 was for a distinct, regular, uniform, and continuous OPL. Grade 2 was for a less distinctive, slightly undulating, and interrupted OPL, and grade 3 was for little to no evidence of an OPL. Table 3 shows the scoring of the differences in grading between the WT and IRBP KO mice, and a significant difference between the two strains according to the Mann–Whitney U-test (p = 0.041). Subtle changes in specific cell types that required immunostaining are discussed in the following sections.

DA and DOPAC measurements

We measured the DA and DOPAC levels with HPLC. Contrary to expectations, neither DA nor DOPAC decreased in the IRBP KO mice compared to the WT mice (Appendix 3, Figure 6A,B). The ratio of DOPAC to DA remained unaltered (Appendix 3, Figure 6C).

More TH-positive amacrine cells in IRBP KO mouse retinas compared to the WT controls

We examined TH-positive cells in retinal flatmounts to obtain a much larger sampling of TH-positive cells than in standard cross sections. In these flatmounts, we detected about 28% more TH-positive cells in the P30 IRBP KO mice than in the WT mice (Figure 7). These results correlated temporally with our previous studies in which IRBP KO mice did not exhibit any ONL degeneration until P23 and older [22].

Lack of form deprivation myopia in IRBP KO mice

Form deprivation in WT mice results in a myopic shift in refraction [60-62]. In contrast, form deprivation in the IRBP KO mice for 4 weeks (from P28 to P56) produced no myopic shift, and the refractive error in the goggled eyes was not significantly different from that in the ungoggled fellow eyes or naïve controls (Figure 8). During the 4 weeks of goggling in the IRBP KO mice, the refractive errors shifted toward more myopia in the goggled eyes, the unmanipulated contralateral eyes, and the eyes of the naïve IRBP KO animals. This age-dependent myopic shift is the same value (about 4 D) as previously observed with ungoggled IRBP KO mice at P30 to P60 [22].

Visual acuity thresholds

The visual acuity of the IRBP KO and WT mice was measured with OKT and compared at several ages (P26–P265). There were no differences in visual acuity between the strains at any of the indicated ages. The responses of the WT mice at all ages were close to 0.383±0.024 c/d. The IRBP KO mice had similar visual acuity (0.386±0.001 c/d) at P26, with a gradual taper to about 0.347±0.002 c/d at P265, representing a slight loss. The overall visual behavior at P265 was unaffected by the reduced thickness of the ONL by about 50%, and by a significant myopic refractive error of about 15 D at the same age. This indicated great resilience of reflexive visual behavior despite these two substantial abnormalities.

Future experiments beyond the intended scope of this article

We aim to formally test for genetic epistasis. Does the Rbp3 gene act upstream from putative downstream genes known to be part of visually-driven refractive development, including potential candidates such as Egr-1 [70]?

Experiments with agonists and antagonists of morphogens may help to test whether vision-dependent mechanisms and the earlier non-image forming development of eye size rely on the same or different pathways to adjust eye size as neonatal mice mature to adulthood. This could include the interference or delivery of excess potential IRBP substrates to determine their role in morphogenesis.

Form vision–dependent emmetropization is subordinate to previous eye growth mediated by IRBP according to our goggling experiment, which employed diffusers. It could be that hyperopic lenses instead of diffusers could be more informative.

Conclusions

The lack of IRBP before eye opening prevents subsequent adjustments to achieve a normal eye size and response to form deprivation in the mouse eye. The absence of IRBP affects downstream mechanisms that might be expected to correct for the abnormally elongated eye and myopia of the IRBP KO mouse during emmetropization. Another explanation is that there are separate mechanisms altogether, and the mutation in IRBP has such a massive effect that the visually-driven pathways cannot compensate. There are more total TH-positive amacrine cells in the IRBP KO mouse retina than in the WT mouse retina. This may suggest that the lack of IRBP manifests problems that precede the use of the dopaminergic amacrine cells in emmetropization. Although there are subtle changes in retina morphology when the IRBP KO mouse retina is compared to the WT mouse retina, there were no major changes in the functional behavioral measure of visual acuity with OKT, which implies that optokinetic responses are robust and unaffected by severe myopic shifts. Ultimately, the experiments suggested that the abnormal increased eye elongation that starts at P7 in the IRBP KO mouse is unaffected by and precedes the processes of emmetropization.

The most interesting implication is that in myopia, ocular development may be generally accelerated. For whatever reason, by the time focus-dependent processes become possible, too much growth has already occurred. That is, there is a balance between the rate of growth and the rate of development of focus-dependent growth suppression. Possibly, myopic eyes simply develop too quickly before the onset of focus capabilities. This may be an imbalance, or the timing is off, with growth coming too early and focus coming too late. Thus, it may be that the loss of IRBP simply allows too rapid growth due to too much free morphogen (which normally would be bound to IRBP) or too late an onset of focus capability, again, possibly due to too much free morphogen being accessible to cells when it ought to be sequestered by IRBP.