A method for determining cell number in the undisturbed epithelium of the mouse lens

Abstract

The anterior face of the mouse lens is covered by a layer of epithelial cells. The epithelial cells serve a barrier function at the lens surface and as a progenitor population from which lens fiber cells, the predominant cell type of the lens, are derived. Decreased epithelial cell density is commonly observed during aging and cataract formation in humans and animal models and may contribute directly to tissue opacification. However, the loss of cells from the epithelium is often not easy to quantify, in part because the cells are arrayed across a near-spherical surface and, as a consequence, are difficult to image and count. Here, we describe a technique for determining epithelial cell number in the undisturbed lens of the mouse, a popular cataract model. The method utilizes orthographic projections of confocal images collected from the anterior and equatorial regions of the lens. The overlapping projections are brought into register using the unique distribution of proliferating cells as fiduciary points. Cell counts are performed using a computer-assisted method. This approach offers several advantages over flat-mount methods employed previously.

Figure 1

Glass-bottomed chamber for positioning and viewing mouse lenses. Oblique view (A) and a plan view (B). The base of the chamber contains a layer of solidified agar from which a wedge-shaped piece has been removed. To image the anterior pole (P), the lens, resting on its epithelium, is positioned in the base of the wedge. To visualize the equatorial region (E), the lens is turned on its side and positioned such that its anterior and posterior faces are supported by the gently tapering walls of the wedge and the equatorial cells are exposed.

Figure 2

Orthographic projections. A: Orthographic azimuthal projection of the globe, polar aspect. B: Orthographic azimuthal projection of the globe, equatorial aspect. C: Orthographic projection of a lens, showing an oblique view of the anterior surface. The epithelium is arbitrarily divided into two regions: a spherical cap (yellow) and an equatorial band (blue). The number of cells in the spherical cap is calculated from cell counts made on a 60°-wide anterior sector (AS). The number of cells in the equatorial band is determined by cell counts made on a 10°-wide equatorial sector (ES). The border between AS and ES is marked by the position of a fiduciary, EdU-positive nucleus (red dot). See text for details.

Figure 3

Orthographic projections of anterior or equatorial regions of the mouse lens. A: Three-dimensional rendering of an anterior lens quadrant, showing EdU-positive nuclei (yellow) and total draq5-stained nuclei (red). An animated version of this panel can be viewed at animation1. EdU-positive cells are relatively numerous near the lens equator but rare or absent in the anterior polar (AP) region. B: Two-dimensional orthographic projection of the data shown in A. The EdU-positive cells form recognizable “constellations.” One such constellation is formed by four nuclei: a, b, c, d. C: Two-dimensional orthographic projection of the lens equator. The constellation a, b, c, d, is also discernible in this orientation. D: High magnification view of the boxed region shown in C. A change in the size and orientation of the fiber cell nuclei (arrow) indicates that fiber cell differentiation has commenced and that cells have entered the meridional rows (MR). Scale bars in B and C are 250 µm, and in D is 50 µm.

Figure 4

Defining the anterior sector (AS). An orthographic projection of an anterior lens quadrant is generated. A circle (1) of diameter D is fitted by eye to the arc of the quadrant. A square (2) with sides equal to D is fitted around the circle. The intercept of diagonals (3 and 4) defines the center of the circle. The fiduciary nucleus, d, in the constellation a, b, c, d is identified in the projection. The distance between d and the center of the circle is measured and used as the radius of a second, smaller circle (5), concentric with the first. An anterior sector (yellow) is drawn in the smaller circle, with a central angle (θ) of 60°.

Figure 5

Computer-assisted identification and quantification of nuclei in a 60° sector (AS) of the anterior lens epithelium. A: Nuclei are identified using the count nuclei application. Colors indicate nuclear size (large nuclei are shown in warmer colors). B: Following image segmentation, nuclei lying within sector AS are counted (green). The arc of the sector is located at d, the fiduciary nucleus (see Figure 3 and Figure 4). C: A higher magnification view of the boxed area in B. The position of the centroid (center of mass) of each nucleus is calculated (indicated by a white dot). Only nuclei with centroids located within AS are included in the analysis.

Figure 6

Quantification of epithelial nuclei in orthographic projections of the equatorial lens region. A: Projection of double-labeled lens tissue showing Edu-positive nuclei (yellow) and Draq5-stained nuclei (red). B: Three-dimensional editing of the original image stack removes the underlying fiber cell nuclei and clarifies epithelial nuclear fluorescence, facilitating automated counting. An arrow indicates the border between the epithelial cell nuclei and those of young fiber cells in the meridional rows (MR). C: Epithelial nuclei are identified automatically and color-coded according to size. Note the densely packed nuclei (blue green color) near the border of the epithelium. D: An isosceles trapezoid is defined in the center of the projection. The width of the trapezoid corresponds to 10° of longitude (See Figure 2). Sides a and b are oriented parallel to the lens equator. Side a is positioned level with the fiduciary cell, d (see Figure 4) and side b is located at the border between the epithelium and the meridional row cells. Nuclei (green) with centroids lying within the trapezoid are included in the count.