Connexin 50 Influences the Physiological Optics of the In Vivo Mouse Lens

Abstract

Purpose: The purpose of this study was to utilize multi-parametric magnetic resonance imaging (MRI) to investigate in vivo age-related changes in the physiology and optics of mouse lenses where Connexin 50 has been deleted (Cx50KO) or replaced by Connexin 46 (Cx50KI46).

Methods: The lenses of transgenic Cx50KO and Cx50KI46 mice were imaged between 3 weeks and 6 months of age using a 7T MRI. Measurements of lens geometry, the T2 (water-bound protein ratios), the refractive index (n), and T1 (free water content) values were calculated by processing the acquired images. The lens power was calculated from an optical model that combined the geometry and the n. All transgenic mice were compared with control mice at the same age.

Results: Cx50KO and Cx50KI46 mice developed smaller lenses compared with control mice. The lens thickness, volume, and surface radii of curvatures all increased with age but were limited to the size of the lenses. Cx50KO lenses exhibited higher lens power than Cx50KI46 lenses at all ages, and this was correlated with significantly lower water content in these lenses, which was probably modulated by the gap junction coupling. The refractive power tended to a steady state with age, similar to the control mice.

Conclusions: The modification of Cx50 gap junctions significantly impacted lens growth and physiological optics as the mouse aged. The lenses showed delayed development growth, and altered optics governed by different lens physiology. This research provides new insights into how gap junctions regulate the development of the lens's physiological optics.

As an avascular organ, the lens relies on a microcirculation to maintain its physiological homeostasis, and gap junctional communication is integral to generating this circulatory system.¹ Gap junctions form intercellular channels facilitating the cell-to-cell communication essential for various physiological processes, including cell cycle synchronization, proliferation, growth, and cellular metabolism.^2,3 The crystalline lens expresses 3 connexins, Cx43, Cx46, and Cx50. Cx43 expression is restricted to the lens epithelium and is absent from the fiber cells.¹ Cx50 is expressed in epithelial and lens fiber cells, whereas Cx46 is restricted to fiber cells. 

To understand the relative roles of Cx46 and Cx50 in the lens, transgenic mouse models that express different levels of the two connexins have been developed.^4–6 These mice were genetically modified by knocking out (KO) either Cx46 or Cx50, or by knocking in (KI) Cx46 into the gene locus of Cx50. Using these models, it has previously been shown that Cx50 modulates the developmental process in epithelial cells and fibers. Deficiency of Cx50 in mice resulted in reduced cell proliferation in the lens epithelium, leading to an undersized lens and smaller eye in both Cx50KO and Cx50KI46 mice.^5–7 Cx50KI46 developed transparent lenses, whereas the Cx50KO mice developed nuclear cataracts.⁸ These previous investigations were performed on ex vivo lenses that lacked active physiological support. Furthermore, the lens optics of these transgenic mice have not been studied owing to a lack of effective methods. 

Mutil-parametric magnetic resonance imaging (MRI) is a technology capable of establishing this link between lens physiology and optics by measuring the tissue-specific MRI parameters of longitudinal relaxation time (T1) and transverse relaxation time (T2). T1 is roughly proportional to the free water content, an emerging biomarker for lens physiology.^9,10 T2, in the context of the lens, is associated with the interactions between water and proteins (water-bound protein ratios), and this ratio, which spatially varies throughout the lens, forms the basis of the gradient of refractive index (GRIN).^11,12 Finally, an MRI can measure lens geometry noninvasively without concerns about optical distortions.^13,14 We have successfully implemented this technology in a variety of settings using different MRIs, including the in vitro bovine lens,¹⁰ the in vivo mouse lens,^15,16 and clinical studies of the human lens.^14,17,18 These MRI studies have contributed substantially to understanding the role of lens water status in lens aging and accommodation.^19,20 

In our previous MRI study of lens development using wild-type C57BL/6 mice, the lenses showed substantial changes in the free water content, GRIN, and geometry with age, resulting in combined effects on the optics during the early period of lens growth from 3 weeks to 3 months.¹⁶ Afterward, all parameters stabilized from 3 to 12 months.¹⁶ We concluded that the mouse lens showed two distinct development phases, where the first fast-growing stage was probably essential to contribute to the emmetropization of the eye. The mechanism(s) behind these early changes in the first phase remained unclear. To further our investigation, we have used 2 transgenic mouse models with modified Cx50 activity, Cx50KO and Cx50KI46.^5,6 Herein, we study these animals with multi-parametric MRI and optical modeling as a function of age to explore how modifying lens growth and gap junctional communication via genetically altering Cx50 expression levels will influence the early development of the optical properties of the mouse lens. 

Materials and Methods

Animals

Animal use adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research using procedures approved by the Stony Brook University IACUC. All mice were in the C57BL/6 genetic background. Animals from 3 weeks to 6 months of age were initially anesthetized with 5% isoflurane, followed by a bolus of xylazine (6 mg/k, IP) to reduce eye motion, and then maintained with 1% to 1.5% isoflurane.^21,22 In addition, a topical mydriatic eye drop of 1% tropicamide was applied to limit anterior eye motion,²³ and lubricant eye ointment was used to maintain corneal hydration. Animals were placed in a prone position in a cradle with a circulating warm water pad to maintain body temperature, and their heads were fixed with ear and tooth bars. During the scan, mice were continuously given 1% to 1.5% isoflurane from a nose cone under spontaneous breathing. The respiration rate and rectal temperature (approximately 37°C) were monitored to ensure physiological homeostasis. 

MRI Scan Protocols

MRI was performed using a 7T horizontal pre-clinical scanner (Biospec, Brucker, Billerica, MA, USA) equipped with a 650mT/m BGA12S-HP gradient at the Stony Brook University preclinical MRI facility. The left eye of each mouse was studied using a customized circular surface coil transceiver.^24,25 The imaging plane was set on the central axis of the lens by visually locating a slice that bisected the eye into superior and inferior halves using MRI protocols, consistent with our previous studies.¹⁵ Scans consisted of T1 and T2 parametric mapping protocols acquired with a field of view (FOV) = 6.4 × 6.4 mm and matrix size = 64 × 64, with a slice thickness of 0.5 mm. T1 mapping used a refocused echo (RARE) sequence with variable TRs (TR = 200, 380, 620, 950, 1500, and 4000 ms) and TE = 2.62 ms to ensure rapid acquisition. T2 mapping utilized a multi-echo spin-echo sequence with 12 TEs (minimum TE = 2.78 ms and echo spacing = 2.78 ms), with TR = 1600 ms and 10 repetitions.¹⁵ In addition, balanced steady-state free precession (bSSFP) images with higher resolution (FOV = 6.4 × 6.4mm, matrix size = 128 × 128) were acquired that utilized TE = 2.5 ms, TR = 5 ms, and 4 RF phase cycling angles (0, 90, 180, and 270 degrees) to generate a banding-free image to assess lens and eye geometry. 

Extraction of Lens Geometry

The phase-cycled bSSFP images were combined by nonlinear averaging, and the corrected images were used to extract the lens geometry using custom-written routines in Matlab (Math-Works, Natick, MA, USA).^15,26 The anterior and posterior lens radii of curvatures (R_a and R_p) and conic constants (Q_a and Q_p) were calculated from the elliptic geometry as R_{a, p}= a²/b and Q_{a, p} = (a/b)² – 1, where a and b are major and minor axes of the fitted ellipse.²⁷ The lens thickness (LT) and equatorial diameter (ED) were calculated using the distance along the optical and equatorial axis.¹⁶ The axial length (AL) for the mouse eye was calculated by summing up all the thicknesses of the ocular components using our previously described methods.¹⁶ Corresponding geometric parameters from different mouse types are labeled in Figure 1A. Lens volume (LV) was estimated by computing the solid revolution of the cross-sectional plane around the optical axis using the following discrete integration formula²⁸:  

\begin{eqnarray}V = \pi \mathop \int \limits_{ - {{T}_P}}^{{{T}_A}} {{\left[ {h\left( x \right)} \right]}^2}dx\end{eqnarray}

(1)

where T_A is the lens anterior thickness, T_P is the lens posterior thickness, and h(x) is the elliptical fitting of the lens. 

Figure 1.

 

Representative geometry measurements and optical models of wild-type, Cx50KO, and Cx50KI46 mouse eyes. (A) Ocular geometric measurements including lens thickness (LT), lens equatorial diameters (ED), and the axial length (AL) of the eye were labeled on the bSSFP image obtained from each mouse type. (B) The lens optical models were constructed with anterior and posterior lenses in ZEMAX using the previously described method. The optical power of each model was calculated from ray tracing in ZEMAX.

T1 and T2 Calculation

Pixel-wise T1 values were fit from the signal intensity, S, at an array of TRs as:  

\begin{eqnarray}{\rm{S}}\left( {{\rm{TR}}} \right) = {{{\rm{S}}}_0}(1 - {{{\rm{e}}}^{ - \frac{{{\rm{TR}}}}{{{\rm{T}}1}}}})\end{eqnarray}

(2)

where S₀ is the signal at infinite TR. 

T2 values were calculated using an optimized phase-correction method to counter the problem of extremely short T2 values in the lens nucleus, as previously described.^29,30 This method has been shown to improve the quality of T2 fittings for the lens nucleus of the older mouse lens.¹⁶ The phase-corrected signals, S_pc are then fitted to the signal equation to calculate T2 values.  

\begin{eqnarray}{{{\rm{S}}}_{{\rm{pc}}}}\left( {{\rm{TE}}} \right) = {{{\rm{S}}}_0}{{e}^{ - \frac{{TE}}{{T2}}}}\end{eqnarray}

(3)

where S₀ is the signal at TE = 0 ms. 

Extraction of GRIN

To convert the T2 values measured by MRI to refractive index (n), a calibration curve was developed using n values obtained in a separate study that used X-ray Talbot interferometry to map changes in the GRIN in wild-type C57BL/6 over a range of ages.³¹ Using this data, we established an age-related T2-n calibration for the mouse lens,¹⁶ where a set of first-order equations were used to convert T2 maps at a given age to n maps using Equation 4:  

\begin{eqnarray}n = {{\beta }_0} + {{\beta }_1} \times \left( {\frac{1}{{{{T}_2}}}} \right)\end{eqnarray}

(4)

β₀ and β₁ used are listed in Table 1 based on the mouse age. 

Table 1.

 

β₀ and β₁ Used are Listed Based on the Mouse Age

 

It should be noted that the age-related T2-n calibration applied to the Cx50KO and Cx50KI46 mice was not directly measured by X-ray Talbot interferometry, but instead used the calibration obtained for wild-type mice. Because the mice used in this study all were from the same C57BL/6 genetic background, we believe that in the absence of actual n values of Cx50KO and Cx50KI46 lenses, the use of the T2-n calibration from wild-type mice is a valid first approximation. 

Optical Modeling

ZEMAX software (ANSYS, Inc., Canonsburg, PA, USA) was used to combine lens geometry and GRIN to create an optical model of transgenic mouse lenses used in this study. The mouse lens was modeled as a doublet design with an anterior and posterior GRIN surface.¹⁶ The boundary between these two surfaces was defined as the plane through the “optical center” of the lens,^16,32 which is used to split the lens thickness and GRIN into anterior and posterior portions, as shown in Figure 1B. The “Gradient 3” model (GRIN 3) was used to characterize the lens in ZEMAX, which formulated rotational symmetry of GRIN distribution based on the Liou and Bernans’ model³³:  

\begin{eqnarray}n\left( {w,z} \right) = {{n}_0} + {{n}_{01}}{{w}^2} + {{n}_{02}}{{z}^1} + {{n}_{03}}{{z}^2}\end{eqnarray}

(5)

In addition,  

\begin{eqnarray}{{w}^2} = {{x}^2} + {{y}^2}\end{eqnarray}

(6)

where x is the equatorial direction (y = 0), and z is the optical axis. The respective anterior and posterior lens geometry and GRIN profiles were input into these two surfaces to create an optical model of the biological lens. Other parameters used to formulate the model included a pupil diameter of 2 mm,³⁴ a polychromatic light source (wavelengths = 486, 587, and 656 nm), and field weighting of 0 degrees = 100%, 2.5 degrees = 40%, and 5 degrees = 20%. After reconstructing the initial model, we optimized the model using contrast as the criterion for further improvement. The optical power of the lens in diopters (D) was extracted from the model using the power field map function in ZEMAX. Representative schematic drawings of three different transgenic mice are shown in Figure 1B. 

Statistical Analysis

To facilitate comparison among age groups, T1, T2, and n values were extracted from the equatorial axis using an averaging band of three pixels in width and then plotted against relative distance, r/a¹⁵ (0 refers to the lens center and ±1 refers to the lens boundary).¹⁶ Error bars indicate the standard error of the mean at each location in the lens. Statistical comparisons were performed by 2-way analysis of variance (ANOVA) with age and mouse group as factors. Tukey’s post hoc testing was used to assess the differences between groups, with P values < 0.05 considered significant. 

Results

Age-Related Changes in the Lens Geometry of Cx50KO and Cx50KI46 Mice

Geometry measurements, including the LT, ED, and AL of the eye, were extracted from the bFFSP images. The radii of curvatures of the anterior (R_a) and posterior (R_p) lens were calculated by fitting the lens edge surfaces into an ellipsoid, and LV was calculated from Equation 1. All geometric parameters changed with age for all studied mouse groups (Fig. 2). The Cx50KO and Cx50KI46 mice displayed smaller LT (see Fig. 2A) and ED (see Fig. 2B) at all ages. In contrast, the absolute values of R_a and R_p (see Fig. 2C) increased with age for all studied groups and exhibited similar patterns to each other. The AL was reduced in Cx50KO and Cx50KI46 animals. Subsequent calculation of LV (Fig. 2E) showed that lenses from wild-type mice had a larger volume than transgenic animals at all ages. As seen previously,¹⁶ the lenses of wild-type mice exhibited a rapid growth phase from 3 weeks to 3 months, as revealed by dramatic increases in the LT, ED, and LV that slowed over the next 3 months. Rapid growth trends were also observed in Cx50KO and Cx50KI46 lenses, but with distinct growth rates during the early period. For example, from 3 to 6 weeks of age, Cx50KO mice had more significant increases in the lens LT (21%) and ED (23%) when compared with the increases in LT (8%) and ED (11%) obtained from Cx50KI46 mice at this age. The aspect ratios (ARs; see Fig. 2F), given by the ratio of ED/LT, were all greater than one for all studied mice, suggesting that the lens retained the same aspherical shape, regardless of their ages or genetic modifications. 

Figure 2.

 

Age-related changes in the lens size, shape, and surface curvatures of the different transgenic models. (A) The lens thickness (LT), (B) the equatorial diameter (ED), (C) the anterior and (R_a) posterior radii ofsurface curvature (R_p), (D) the axial length (AL), (E) the lens volume (LV), and (F) the aspect ratio (AR) were processed from bSSFP images. Data are plotted against age for each group. AR is calculated from ED/LT, where an AR of 1 represents a perfectly round lens. Data are mean ± SD.

The observed differences in the geometric parameters at any specific age could be because the loss or replacement of Cx50 significantly reduced the physical size of the lens. To test this, we re-plotted all the two-dimensional geometric parameters (R_a, R_p, LT, AL, and ED) against lens volume instead of lens age (Fig. 3). Data from these plots showed the same overlapping trends, suggesting that the observed differences in LT (see Fig. 3A), ED (see Fig. 3B), R_a/R_p (see Fig. 3C), and AL (see Fig. 3D) resulted from the previously characterized lens growth defect observed during the first postnatal week that permanently reduced the volume of Cx50KO and Cx50KI46 lenses throughout the rest of their lifespans.^35,36 

Figure 3.

 

Changes in the lens shape plotted against lens volume. To investigate the size effects on the lens shape, the two-dimensional geometric measurements: LT (A), ED (B), R_a and R_p (C), and AL (D) were replotted against the lens volume for each transgenic mouse group.

Effect of Removal of Cx50 on Free Water Content During Lens Growth

In our previous study that utilized wild-type lenses, we showed that a reduction in the free water content of the central lens nucleus was correlated with the observed age-related changes to lens GRIN and power. Because the volume of Cx50KO and Cx50KI46 lenses is smaller than that of wild-type lenses, we first assessed the effect of removing Cx50 expression on the free water distribution in these transgenic lenses. Representative T1 maps of all mouse types at each age illustrated the smaller lens size of the two transgenic mice relative to the wild-type, and how the internal water distributions differed (Fig. 4). This is more evident in the T1 line profiles extracted from the equatorial axis of wild-type, Cx50KO, and Cx50KI46 lenses at each age and then plotted against the normalized radial distance (r/a; Figs. 5A–D). The profiles for Cx50KO and Cx50KI46 mice demonstrated similar age-dependent changes in magnitude and shape, a central decrease in T1 values, and an extension of the central plateau. T1 values were extracted from the lens nucleus and plotted as a function of age (Fig. 5E) and volume (Fig. 5F). A significant increase in water content was observed from 3 to 6 weeks in Cx50KO and Cx50KI46, followed by a consistent decrease with age (see Fig. 5E). In contrast, a continual reduction in free water content was observed across all age groups in wild-type mice. Finally, although the free water content ultimately decreased with aging in all lenses, water content was always significantly higher in Cx50KI46 lenses than in wild-type, or Cx50KO lenses. Lens size was not a determining factor for lens water content, because Cx50KO and Cx50KI46 exhibited distinct changes, which differed from both the wild-type pattern and each other (see Fig. 5F). Taken together, this analysis showed that, despite having a smaller volume than wild-type, Cx50KI46 lenses had substantially higher free water than lenses from wild-type or Cx50KO animals. Next, we wanted to check how the different baseline water content and age-dependent changes in the water distribution induced by the removal of Cx50 expression affected the ability of the lens to alter its power as the eye grew. To achieve this, we first determined the effect of Cx50 removal on the development of the GRIN by measuring age-related changes to the T2 values. 

Figure 4.

 

Representative T1 maps were obtained from mice at 0.75 (A–C), 1.5 (D–F), 3 (G–I), and 6 (J–L) months of age from wild-type (A, D, G, J), Cx50KO (B, E, H, K), and Cx50KI46 (C, F, I, L) mice. The T1 maps reveal the free water distribution across the lens.

Figure 5.

 

Age and size-related changes in the lens T1 (free water content). Trend analysis was performed for the lenses of each mouse genotype at (A) 0.75, (B) 1.5, (C) 3, and (D) 6 months of age, averaged and then plotted against the normalized radial distance (r/a). The minimum T1 (ms) extracted from r/a = 0 was plotted against age (E), or lens volume (F). The change in T1 for Cx50KO and Cx50KI46 mice was different from the wild-type control and was independent of lens size. Data in panels A to D are mean ± SEM. Data in panels E and F are mean ± SD.

Effect of Removal of Cx50 on the Development of the GRIN

T2 values are a surrogate for the water-bound protein ratios in the lens and are inversely proportional to the refractive index, n.¹¹ Previously, we had shown in wild-type mice that the T2 of the lens decreases with age.¹⁶ In this study, we performed similar experiments on Cx50KO and Cx50KI46 mice to extract T2 maps from animals ranging from 3 weeks to 6 months of age (Fig. 6). These images show the effect of the removal or replacement of Cx50 on the extracted T2 maps. To facilitate the visualization of these age-dependent changes in T2 values in the different mice, line profiles extracted from the equatorial axis were plotted on a logarithmic scale against the normalized radial distance (r/a; Figs. 7A–D). This analysis confirmed that the significant changes in the T2 profile for Cx50KO and Cx50KI46 with age were localized to the central region of the lens (± 0.6 r/a) and the largest changes occurred between the ages of 0.75 to 1.5 months before becoming less noticeable from 3 to 6 months of age. To enable comparison between the groups of lenses, the mean T2 from a region of interest in the lens nucleus (r/a = 0) was extracted and plotted against age (Fig. 7E). As shown for wild-type lenses in our previous study,¹⁶ T2 in the central lens nucleus decreased at a faster rate at earlier ages before stabilizing at approximately 3 months of age. Relative to wild-type and Cx50KO mice, the T2 of the nucleus of Cx50KI46 lenses was 58.7% and 68.1% higher, respectively, in lenses at 3 weeks of age. However, with advancing age, the T2 of the nucleus for the Cx50KI46 lens moved closer to both wild-type and Cx50KO T2 values, and by 6 months, Cx50KI46 lenses were not significantly different from the wild-type lenses. In contrast, from 3 weeks to 6 months, the central T2 of Cx50KO lenses was consistently lower than that of wild-type lenses. 

Figure 6.

 

Representative T2 maps were obtained from mice at 0.75 (A–C), 1.5 (D–F), 3 (G–I), and 6 (J–L) months of age from wild-type (A, D, G, J), Cx50KO (B, E, H, K), and Cx50KI46 (C, F, I, L) animals. The T2 maps represent water-bound protein ratios across the lens.

Figure 7.

 

Age and size-related changes in the lens T2 (water-bound protein ratios). Trend analysis was performed for the lenses of each mouse genotype at (A) 0.75, (B) 1.5, (C) 3, and (D) 6 months of age, averaged and then plotted on a log₁₀ scale against the normalized radial distance (r/a). The minimum T2 (ms) extracted from r/a = 0 was plotted against age (E), or lens volume (F). Data in panels A to D are mean ± SEM. Data in panels E and F are mean ± SD.

To investigate the effects of lens size on these differences in the age-dependent changes in T2 values from the lens nucleus, we re-plotted the values against lens volume (Fig. 7F). This analysis showed that despite dramatically different initial volumes, all lenses exhibited the same age-dependent decrease in the T2 values in the lens nucleus. The initial rate of decrease of T2 in Cx50KI46 lenses was initially steeper than the rates shown by both wild-type and Cx50KO lenses. This suggested that regardless of the lenses’ actual size, modulating gap junction coupling altered the rate at which the water-bound-protein ratios, represented by the T2 values, were reduced during these 6 months of lens growth. 

Becuase T2 is inversely correlated with n, T2 maps were converted into GRIN maps (Fig. 8) using Equations 3 and 4, from which GRIN profiles were extracted and plotted against the normalized radial distance (Figs. 9A–D) for wild-type, Cx50KO, and Cx50KI46 mice. The conversion of T2 values to n produced GRIN profiles that exhibited more pronounced age-related changes in magnitude and shape with the emergence of a plateau in the GRIN similar to that observed previously in wild-type lenses.¹⁶ By extracting the maximal refractive index (Max n) from the lens nucleus and plotting it against age, it became apparent that there was a large increase in Max n observed in all 3 mouse groups between 0.75 and 1.5 months of age (Fig. 9E). The Max n values extracted from Cx50KO mice were consistently higher than those from wild-type or Cx50KI46 lenses at all ages. Plotting Max n as a function of lens volume revealed similar shapes of the rate of change in Max n, but with differences in the value of n, and accentuated the differences between Cx50KO and Cx50KI46 mice (Fig. 9F). 

Figure 8.

 

Representative GRIN maps were obtained from mice at 0.75 (A–C), 1.5 (D–F), 3 (G–I), and 6 (J–L) months of age from wild-type (A, D, G, J), Cx50KO (B, E, H, K), and Cx50KI46 (C, F, I, L) mice. The GRIN maps reveal the refractive index distribution across the lens. The refractive index values were converted from the T2 values using age-dependent calibration equations.

Figure 9.

 

Age and size-related changes in the lens refractive index (n). Refractive indices were converted from the T2 values using age-dependent calibration equations. Trend analysis was performed for the lenses of each mouse genotype at (A) 0.75, (B) 1.5, (C) 3, and (D) 6 months of age, averaged, and then plotted against the normalized radial distance (r/a). The maximum n, corresponding to the reciprocal of the minimum of T2 values was extracted from r/a = 0 and was plotted against age (E), or lens volume (F). Data in panels A to D are mean ± SEM. Data in panels E and F are mean ± SD.

Effect of Removal of Cx50 on Lens Power

Because the geometry (see Fig. 1) and GRIN (see Fig. 8) determine lens power, we next utilized the optical modeling software ZEMAX to calculate lens power for each mouse strain (Fig. 10). Using this approach, it was evident that the power of Cx50KO lenses was significantly higher than that of wild-type or Cx50KI46 lenses at all ages because of the smaller size and greater Max n exhibited by Cx50KO lenses (see Figs. 8, 9). When comparing lens power as a function of age (see Fig. 10A), two distinct phases of development are observed. In the initial phase of 3 to 6 weeks, all lenses exhibited an increase in power, followed by a tendency to stabilize at older ages. The lens power was also plotted against the AL (see Fig. 10B) and the lens volume (see Fig. 10C) of the eyes to determine if the size of the lens and/or the eye were limiting factors in the development of lens optics. The plots obtained from Cx50KO and Cx50KI46 mice were parallel to each other and different from the pattern seen in the wild-type mice. The lens power had large differences between groups regardless of either the lens or the eye size. Compared with the normal development of the lens optics in wild-type mice, genetic manipulation of Cx50 leads to different lens optics in the growing eyes, and this is not only due to the geometric factors, but potentially relates to the major physiological factors of water content, and changes in gap junctional coupling. 

Figure 10.

 

Changes in lens optics for Cx50KO and Cx46KI50 transgenic mice. Lens optical power in diopters was calculated from the lens model built in ZEMAX and plotted against mouse age (A), eye axial length (B), or lens volume (C), obtained from different transgenic mouse groups. Data are mean ± SD.

Discussion

In this study, we have used our in vivo MRI-optical modeling approach¹⁶ to study transgenic mouse models where changing the expression of the gap junction protein Cx50, either by removing (Cx50KO) or replacing it with Cx46 (Cx50KI46), altered both lens growth and gap junctional coupling. Our previous study showed that the wild-type mouse lens displayed two distinct phases of changes in water distribution. The first phase showed dramatic changes in shape, volume, water content, GRIN, and lens power from 3 weeks to 3 months, which was then followed by a gradual transition over 3 to 12 months to a second phase where all conditions achieved a steady state.¹⁶ These findings suggested that the changes in the lens’ physiological optics that occurred in the early phase could contribute to the emmetropization of the mouse eye. To better elucidate the role of lens circulation that mediates water transport in these processes, we utilized our optimized MRI protocols to extract the shape, water content, and GRIN from lenses of Cx50KO and Cx50KI46 mice across a range of different ages. 

MRI offers the advantage of imaging the lens without optical distortions so that the geometry of the mouse lens and eyes can be accurately studied in vivo. Our bSSFP protocol and post-processing method were consistent with the previous results obtained from the ex vivo lens using light microscopy. Cx50 is known to be essential for normal lens growth,^5,6 and our lens geometry data confirmed that the lenses of Cx50KI46 and Cx50KO mice had significantly smaller volumes than wild-type mice at all the time points examined. For other geometric parameters, we revised our analysis to include plots of geometric parameters against both lens volume and age. This revealed that all the other lens geometric parameters (thickness, equatorial diameter, and radii of curvature) were more tightly linked to lens size than age. Thus, although the lenses were significantly smaller in Cx50KO and Cx50KI46 mice at every age, they still developed the same aspherical geometry as wild-type animals when corrected for total lens volume. 

Prior functional studies of these mouse models have established that Cx50KO lenses had decreased fiber cell gap junctional coupling and increased intracellular hydrostatic pressure gradients.³⁷ In contrast, Cx50KI46 lenses displayed increased fiber cell gap junctional coupling³⁸ that was associated with a decreased intracellular hydrostatic pressure gradient.³⁹ These opposing trends in physiological parameters regulating water transport suggested that the water content of Cx50KO and Cx50KI46 lenses should be substantially different (summarized in Table 2). This idea was partially supported by our analysis of T1 values, which showed significantly higher water content in Cx50KI46 lenses than Cx50KO or wild-type animals (see Fig. 5E). In contrast, Cx50KO lenses only had lower T1 values at 3 weeks of age, the youngest time point studied. 

Table 2.

 

Comparative Summary of Mouse Models

 

It is also worth noting that Cx50KO lenses develop a central cataract before eye opening at 2 weeks of age,^5,8 whereas Cx50KI46 lenses remain cataract-free⁶ throughout life. Attempts to analyze lenses from animals younger than 3 weeks of age by MRI were precluded by the difficulty of administering and monitoring the anesthesia necessary to limit movement in young pups. Thus, interpretation of our results with respect to cataract development following the loss of Cx50 was hindered by technical limitations. 

The higher water content observed in Cx50KI46 lenses would be expected to affect the T2 values, and therefore the GRIN. Consistent with this view, the minimum T2 (see Fig. 7E) and Max n values (see Fig. 9E) observed in the lens nucleus of young Cx50KI46 mice were noticeably higher and lower, respectively, than the values observed in either wild-type or Cx50KO lenses of the same age. However, as the animals got older, and the lens and the eyes grew, T2 values and Max n values both decreased and increased, respectively, in all 3 groups in similar patterns, although the final magnitudes differed from each other. These results tend to suggest that despite changes in the initial distribution of free water content (T1) caused by the loss or replacement of Cx50, the lens was still able to modulate its water content as it grew to reach a similar steady GRIN distribution in adult animals that contributes to lens power. 

Overall, Cx50KO and Cx50KI46 mice exhibited an apparent delay in lens water regulation as it related to lens optics. One possibility is that feedback from the developing optics could adjust the lens water content to modulate the lens GRIN to improve optical properties. In the absence of Cx50, the T1, T2, and n values eventually reach plateaus, but at values different from those of wild-type mice. This could be due to differences in water transport caused by the genetic modulation of gap junctional coupling (see Table 2). It is possible that additional Cx46 channels provided by the knock-in allele expressed on the Cx50 locus influenced water transport in Cx50KI46 lenses in addition to these animals simply lacking Cx50 channels. It is known that central gap junctional coupling is increased more than 2-fold⁷ in Cx50KI46 lenses, and this could facilitate much more efficient water movement out of the lens center. In addition, Cx46 does not undergo normal c-terminal cleavage in Cx50KI46 lenses.³⁸ C-terminal cleavage of lens connexins reduces their functional activity,¹ so this would be expected to further increase water flux through gap junctions. Future MRI studies of Cx46 KO mice⁴ using our established protocols could help further explore these possibilities. 

Accumulating evidence supports the hypothesis that water content is vitally important to regulate lens optics, and this is strongly coupled to the lens microcirculation system.^19,20 Cx50 is a key component of the lens microcirculation,¹ and our present study shows that its loss, or replacement by Cx46, influences the physiological optics of the in vivo mouse lens. We observed that Cx50KO lenses exhibited higher lens power than Cx50KI46 lenses at all examined ages, and this was correlated with significantly lower water content in these lenses. Because Cx50KO and Cx50KI46 lenses are considerably smaller than wild-type, these data suggest that the magnitude of gap junctional coupling, and not only lens size, could influence lens power. 

In conclusion, our study demonstrated that the Cx50 channels played crucial roles in lens development by regulating physiological water transport processes, consequently influencing lens optics. These results provide new mechanistic insight into the regulatory process behind the development of lens physiological optics and potentially open new avenues of research into the processes underlying emmetropization of the eye. 

Acknowledgments

Supported by the National Institutes of Health grant EYO26911 (T.W.W.). 

Disclosure: X. Pan, None; E.R. Muir, None; C. Sellitto, None; Z. Jiang, None; P.J. Donaldson, None; T.W. White, None