Ординатура / Офтальмология / Английские материалы / Development of the Ocular Lens_Lovicu, Lee Robinson_2004

The apical membrane of lens epithelial cells is planar and interfaces with the apical membranes of elongating fiber cells as they migrate to their sutural locations. This epithelialfiber interface (efi) is characterized by transcytotic events (Brown et al., 1990; Kuszak et al., 1995). Numerous examples of micropinocytosis and clathrin-coated vesicles can be found at and immediately subjacent to the efi. Thus, nutrients, ions, essential metabolites, and presumably other receptor-mediated substances can be exchanged across the efi via transcytosis (Figs. 4.16 and 4.17). Both gap junctions and “square array membrane,” areas of membrane characterized by groups of orthogonally arranged aquaporins, are extremely rare at the efi. The function of square array membrane is not known. One hypothesis is that it regulates fluid movement in and out of the extracellular compartment (Zampighi et al., 1989; Costello et al., 1989).

4.5.1.1. Physiologic Significance of the EFI as a Function of Lens Size and Age

The lens has been described as a “functional 3-dimensional syncytium” (Mathias et al., 1981). Though it is clearly not “a multinucleate mass of protoplasm produced by the merging of all cells” (the literal definition of a syncytium), such a characterization is nevertheless not inappropriate when one considers the following: The elongating fibers, or superficial cortex, of any lens form incomplete shells on the lens periphery. As mentioned, when the lens fibers elongate, their apical ends are in close apposition with the apical surfaces of lens epithelial cells, giving rise to the efi. SEM analysis of complete radial cell column exposure along the polar axis reveals that a fiber that breaks contact with either the cz epithelium at the anterior pole or the posterior capsule at the posterior pole is approximately 200 µm deep at the equator (Kuszak and Rae, 1982). Thus, Kuszak and Rae (1982) proposed that this arrangement provides a transport “short circuit” to the lens interior. Substances that enter the above-described fiber can traverse the 200 µm into the lens interior by diffusing in the cytoplasm of this cell rather than by crossing myriad cell boundaries through gap junctions. Such an intracellular diffusion pathway is likely to be at least an order of magnitude less resistive than the gap junctional pathway. Since elongating fibers are electrically coupled to epithelial cells at the efi (Rae and Kuszak, 1983), it has been presumed that small molecules transported by the epithelial cells can diffuse into these fibers through a single set of gap junctions and are thereafter free to diffuse deeper into the lens through the fiber cytoplasm. Indeed, lens fibers are coupled by an unusually high density of fiber-fiber gap junctions as compared with other epithelia (Benedetti et al., 1976; Goodenough, 1979; Kuszak et al., 1978; Kuszak et al., 1982; Costello et al., 1985; Lo and Harding, 1986). Thus, the efi has been considered to be an important component in the transport of essential components into the lens.

4.5.1.2. Correlative Structure Function Analysis of the EFI

Both structural studies (TEM and freeze-etch; Brown et al., 1990; Bassnett et al., 1994; Kuszak et al., 1995) and physiological studies (electrotonic and dye-coupling; Schuetze and Goodenough, 1982; Rae and Kuszak, 1983; Miller and Goodenough, 1986; Prescott et al., 1991; Prescott et al., 1994; Bassnett et al., 1994), which have quantified intercellular communication at the efi, have consistently found that the extent of the coupling at the efi is markedly less than either fiber–fiber or epithelial cell–epithelial cell coupling.

Brown et al. (1990) were the first to define the fiducial markers necessary for unequivocally identifying the efi in freeze-etch replicas, perhaps the most appropriate methodology

Figure 4.17. Transmission electron micrographs showing additional examples of endocytotic events (black squares) at different locations in a lens. (a) Forming endocytotic vesicles (shown enlarged in upper right inset) at the apicolateral membranes of elongating fibers (EF ) two and three layers beneath the central zone epithelium (EC). (b) A clathrin-coated vesicle (shown enlarged in upper left inset) at the apicolateral membrane of a cortical fiber terminated at an anterior suture plane. From Kuszak and Brown, 1993, courtesy of W. B. Saunders Co., Philadephia, PA.

for studying membrane ultrastructure (Kreutziger, 1968; Chalcroft and Bullivant, 1970; McNutt and Weinstein, 1970). In this study, freeze-etch replicas containing >20,000 µm2 of adult chick lens efi revealed only two gap junctions. This analysis included the ultrastructural characterization of numerous complete apical surfaces of elongating fibers that had interfaced with cz epithelial cells in situ. Thus, the possibility that gap junctions went undetected due to limited membrane exposure, as can be the case with thin-section analysis (Revel et al., 1971; Gabella, 1979; Ryerse and Nagel, 1991), was minimized. Additional correlative structural and physiological studies of embryonic chick (Bassnett et al., 1994) and adult frog lens efi (Prescott et al., 1991; Prescott et al., 1994) also confirmed that intercellular communication via gap junctions at the efi is very limited. In a study of primate lenses (Kuszak et al., 1995), quantitative analysis of >10,000 µm2 of cz efi revealed no gap junctions. Correlative TEM thin-sections of >1,500 linear microns of cz efi from this region confirmed the presence of epithelial-epithelial gap junctions and elongating fiber– elongating fiber gap junctions but an extreme paucity of epithelial–elongating fiber gap junctions. In contrast, TEM thin-sections of >1,000 linear microns of pgz, gz, and tz efi revealed a number of epithelial–elongating fiber gap junctions. This finding is confirmed by freeze-etch analysis of mouse lens tz efi (Figs. 4.18 to 4.20).

The results of studies using electrotonic and dye-coupling techniques to assess intercellular coupling at the efi also reveal very limited communication at the efi. Bassnett et al. (1994) demonstrated that fluorescent dye (carboxyfluorescein diacetate) is retained by lens epithelial cells over an extended period of time without any significant evidence of dye

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Figure 4.18. Low-magnification scanning electron micrographs of (a) the apical (AMs), lateral (LMs), and basal membranes (BMs) of nascent elongating fibers at the transitional zone, or bow region efi, in an adult rat lens and (b) the apical membranes of elongating fibers in an adult monkey lens at the central zone efi. Note that while the apical membranes of nascent elongating fibers are of relatively uniform size and shape (hexagonal), the apical membranes of elongating fibers at the cz efi are of neither uniform shape nor size. (a) From Kuszak, Brown and Peterson, 1996, courtesy of John Wiley & Sons, Inc. New York, NY.

(b) From Kuszak, Novak and Brown, 1995, courtesy of Academic Press, London, UK.

uptake by the underlying elongating fibers. However, there was a small amount of dye transfer recorded from elongating fibers to epithelium. These results are consistent with comparable physiologic analyses that have shown either a lack of or restricted dye coupling at the efi (Goodenough et al., 1980; Rae and Kuszak, 1983; Miller and Goodenough, 1986). Finally, a study that specifically targeted dye coupling at the lens efi (Rae et al., 1996) presented evidence suggesting that less than 1 in 10 epithelial cells were coupled to underlying elongating fibers. This study also confirmed previous findings of Baldo and Mathias (1992) that showed gz epithelial cells are better coupled to underlying nascent elongating fibers than cz epithelial cells are to elongating fibers in the latter stages of terminal differentiation or to almost fully elongated immature fibers just prior to their detaching from the epithelium anteriorly and the capsule posteriorly.

In spite of the results of numerous studies that have shown only limited communication across the efi, it can still be reasonably argued that cell-cell coupling across this interface is important to lens physiology (Rae et al., 1996). Epithelial-epithelial and fiber-fiber coupling has been shown by correlative structural and functional studies to be essentially 1:1. Therefore, every epithelial cell does not need to be conjoined directly to an underlying elongating fiber.

4.5.1.3. Percentage of Fibers in Direct Contact with the EFI

Although the equatorial diameters of adult chicken (Gallus domesticus), frog (Rana pipiens) and rat (Wistar and Sprague-Dawley) lenses are, respectively, 7, 4.5, and 3 mm, SEM

analysis of all of these lenses shows that a fiber that breaks contact with either the cz epithelium at the anterior pole or the posterior capsule at the posterior pole is approximately 200 µm deep at the equator (Kuszak and Rae, 1982; Kuszak et al., 1986). The consistent depth of the elongating fiber zone, or superficial cortex, is a function of the fact that elongating fibers are thinnest in their midportions, widest and most flared at their anterior ends, and intermediate in these parameters in their posterior segments (Kuszak et al., 1986). It has also been demonstrated in the primate lens that elongating fibers extend to a depth of approximately 200 µm at the equator (Bassnett and Beebe, 1992; Bassnett, 1992). The equatorial diameter of the adult primate lens is approximately 9 mm. Thus, the percentage of fibers in direct contact with the epithelium at the efi will vary as a function of size and/or age. Estimates of the percentage of fibers in direct contact with the epithelium at the efi can be derived from standard mensuration formulas for spheroidal geometry (Eves, 1978). When calculating such estimates, it is important to take into account that lenses are asymmetrical, oblate spheroids and not spheres. Failure to accurately factor in lens shape results in a gross overestimation of lens volume.

The shapes and relevant axial dimensions of mouse and human lenses used to estimate the percentage of fibers in direct contact with the epithelium at the efi are shown in Figure 4.21.

Since all vertebrate lenses are asymmetrical, oblate spheroids, the total volume estimate for any lens can be calculated as the sum of the anterior lens volume, defined as (4/3 πa12b1)/2, and the posterior lens volume, defined as (4/3 πa1b22)/2, where a1 is the major equatorial radius, b1 is the minor anterior radius, and b2 is the minor posterior radius. The same formulae can be used to estimate the percentage of fibers in direct contact with the epithelium at the efi. However, in this case, a1 is replaced by a2, the major equatorial radius minus the 200 µm contribution of the elongating fibers. Of course, in the case of a lens like the mouse lens at birth, subtracting out the 200 µm contribution of the elongating fibers effectively changes the major and minor axes of the lens. Therefore, in this instance, the volume estimate for the percentage of fibers not in direct contact with the epithelium at the efi must be calculated as the sum of the anterior and posterior lens volume. Calculations of the volumes and percentages for human, and mouse lenses are summarized in Table 4.3.

If the size of the elongating fiber zone is constant throughout life and between species, as suggested by morphological studies to date (Kuszak and Rae, 1982; Kuszak et al., 1986; Bassnett and Beebe, 1992; Bassnett, 1992), then more fibers are in direct contact with epithelial cells at the efi in smaller and rounder lenses (e.g., frog, mouse, and rat) than in larger and flatter lenses (e.g., cat, dog, monkey, baboon, and human). It is important to note that presuming a lens such as a mouse lens to be essentially spherical results in a gross overestimation of the percentage of fibers that are in direct contact with epithelial cells at the efi (the spheroidal estimates are 45% at birth and 29% in an adult lens; the spherical estimates are 69% at birth and 59% in an adult lens).

4.5.2. Fibers

Because every fiber is maintained for a lifetime, the lens presents an ideal model for studies of cellular senescence. In this section, the morphology of the oldest fibers, the primary fibers of the embryonic nucleus, is compared to the morphology of the younger secondary fibers.

In normal transparent lenses, the cytoplasm of fibers in all the developmental regions is smooth and homogeneous. This fact can be confirmed by Fourier transform analysis. A selected region of an adult human embryonic nuclear fiber in Fig. 4.22a is displayed as a transform and a radially averaged plot in Figure 4.22b. The smoothly varying curve and minimal

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Figure 4.21. Scaled schematic diagrams showing the relevant axial dimensions required to estimate the percentage of the lens superficial cortex in direct contact with the lens epithelium in juvenile and adult mouse and human lenses.

intensity near the center suggest that the cytoplasmic proteins and water are packed into an amorphous medium with minimal fluctuations in refractive index (Freel et al., 2002; Taylor and Costello, 1999). The principal components of fiber cytoplasm are the specialized lens crystallin proteins and the lens cytoskeleton. The crystallins provide a medium of high refractive index. With increased age, a variable concentration of the different types of crystallins is related to the higher water content in the lens cortex compared with the