Molecular Vision 2005; 11:901-908 <http://www.molvis.org/molvis/v11/a107/> Received 7 April 2005 | Accepted 27 October 2005 | Published 1 November 2005

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Lack of fiber cell induction stops normal growth of rat lenses in organ culture

Madhumita P. Ghosh, J. Samuel Zigler, Jr
 
 

Lens and Cataract Biology Section, National Eye Institute, Bethesda, MD

Correspondence to: J. Samuel Zigler, Jr., National Eye Institute, 7 Memorial Drive, MSC 0703, Bethesda, MD, 20892-0703; Phone: (301) 496-6669; FAX: (301) 496-1759; email: ziglers@nei.nih.gov

Abstract

Purpose: Lens organ culture has been widely used as a model system for studying cataract induction and prevention. While rat lenses remain transparent and viable for a week or longer in culture, they do not increase in weight. This study was undertaken to determine what accounts for the lack of weight increase.

Methods: Lenses from 4-week-old Sprague-Dawley rats were cultured using standard methods. Histological analysis was performed on sections from methacrylate embedded tissue. ³⁵S-labeled amino acids were used to metabolically label lenses in culture for the purpose of analyzing protein synthesis. BrdU labeling was used to assess synthesis of DNA in vivo and in vitro.

Results: Lenses from young, rapidly growing rats do not increase in weight after being put into organ culture. Protein synthesis continues in the cultured lenses although at decreased levels as time in culture increases. Lens epithelial cells continue to synthesize DNA as indicated by BrdU labeling, however, the normal migration of epithelial cells from the proliferative zone to the equator does not occur in culture. In the cultured lens, the shape of the lens bow gradually changes, becoming compressed towards the capsule.

Conclusions: The differentiation of lens epithelial cells into fibers is arrested in the cultured lens; consequently lenses in organ culture do not grow normally.

Introduction

The ocular lens would appear to be an ideal organ for maintaining under culture conditions. Lacking nerves and blood vessels, the lens in vivo obtains its nutrients and eliminates waste products via diffusion with the surrounding fluids. Indeed lenses from various species have been incubated successfully since the middle of the last century [1]. This laboratory has utilized rat lenses in organ culture as a model system for studying the effects of various stresses on the lens, mechanisms of cataract formation, and for screening potential anti-cataract agents [2-4]. In certain instances lens opacification induced in vivo by administration of a particular cataractogenic agent can be mimicked in vitro by addition of the same agent to the culture medium [5]. Further, agents which prevent such cataracts in vivo may also be effective in culture [4]. Thus, there is good evidence to support the idea that lenses in culture can be an effective model for the lens in vivo.

Our studies using cultured rat lenses were, until recent years, generally limited in duration to 24-48 h. In investigating the effects of the cholesterol-lowering drug, lovastatin, on the lens we extended this culture time to 7-10 days for rat lenses [6]. We were struck by the fact that, while the lenses were transparent and viable throughout this period and remained capable of responding to stress, after being placed in culture they immediately ceased to increase in weight. Thus, a lens from a rat about 4 weeks of age which would typically weigh about 20 mg would still weigh 20 mg after a week in culture whereas had the lenses remained in the living animal for that additional week their wet weight would have increased by about 20%. The current study was undertaken as an attempt to determine the cause of this lack of normal weight increase. We also hoped to determine whether the failure of normal growth would adversely affect the utility of lens organ culture as a model system for studying cataractogenesis.

Methods

Animals

Sprague-Dawley rats approximately 4 weeks of age (76-100 g) were obtained from Taconic farms (Germantown, MD). The animals were euthanized using CO₂ in compliance with the Guide for the Care and Use of Laboratory Animals (National Academy Press).

Lens organ culture

Lenses were dissected from freshly enucleated eyes by a posterior approach as previously described [7] and were individually placed into 2.0 ml modified TC-199 culture medium [6] in 24 well cluster dishes. The dishes were placed into a 37 °C tissue culture incubator with humidified 95% air/5% CO₂ atmosphere. The osmolarity of the culture medium was adjusted to 298±2 mosmoles. The condition of all lenses was assessed by measuring the protein content of the culture medium 30-60 min after the lens was placed in culture [8]. Lenses found to be leaking protein into the medium were discarded. Lenses were incubated for varying times up to 9 days and the medium was changed every 48 h.

Lens weight determination

Since it is not feasible to obtain an accurate wet weight of the lens and subsequently culture it successfully, we determined the lack of weight increase for lenses placed into organ culture in two ways. In some studies we carefully blotted and weighed one lens from each animal and placed the second lens of each pair into culture. We assumed that the lenses in each pair had essentially identical weight at the time they were dissected from a freshly euthanized animal. Cultured lenses were removed at various times, blotted dry, and carefully weighed. Weights of lenses after 3-9 days in culture were compared with the weights of their respective contralateral lens as measured at zero time. Alternatively, litters of animals were divided into two groups. One group was euthanized and their lenses placed into culture while the second group of animals was maintained in the animal facility until the final day of the culture experiment. These rats were then euthanized. Their lenses and the previously cultured lenses were weighed as described above. This provided data regarding the average weight increase of the lens in vivo during the time period of the culture experiment.

Histological analysis

Lenses were placed in 2.5% glutaraldehyde in 50 mM cacodylate buffer (pH 7.2) containing 4% sucrose and 2 mM CaCl₂ for 72 h, then transferred to 10% buffered formalin. The fixed lenses were oriented by a histology technician with extensive experience working with ocular tissue and embedded in methyl methacrylate. Midsagittal sections of 1-2 μm were cut, mounted and stained with hematoxylin and eosin or with PAS (Periodic Acid Schiff). Samples were photographed using a Nikon Eclipse E800 microscope equipped with a Hamamatsu Digital Camera C4742-95.

Protein synthesis

Lenses were metabolically labeled with ³⁵S-labeled amino acids (Easy Tag Express Protein Labeling Mix; Perkin Elmer Life Sciences, Boston, MA) immediately after being placed into culture or after varying times of culture. Into a 1.5 ml volume of culture medium with a single lens was added 300 μCi ³⁵S. The lenses were removed after 5 h and rinsed to remove radiolabel from the surface, blotted dry, and weighed. For quantification of total incorporation of label, lenses were homogenized in 10% TCA. The protein precipitate was washed twice with 1.0 ml 10% TCA and was then solubilized in 10% SDS containing NaOH to neutralize the TCA. In some instances the capsule/epithelium was separated from the lens fiber mass prior to TCA precipitation. Tissue samples were placed into 1.5 ml tubes of known weight, weighed to obtain wet weight of tissues, and homogenized in the same tubes using fitted plastic pestles. Samples were counted by liquid scintillation and expressed as DPM/mg lens wet weight. For autoradiography, lens samples were homogenized in Nu-PAGE sample preparation buffer (Invitrogen, Carlsbad, CA) and run on 4-12% Nu-PAGE Bis-Tris gels. Gels were stained with Coomassie Brilliant Blue, destained and dried. Bio Max MR film (Kodak, Rochester, NY) was used with exposure times up to 7 days.

BrdU labeling

For the in vivo study, one month old rats were injected with 100 μg/gm body weight 5-Bromo-2-deoxyuridine (BrdU) and 6.7 μg/gm body weight 5 fluoro-deoxyuridine (5 FdU). Animals were euthanized at various times up to 14 days post-injection. For the organ culture study, animals were euthanized 4 h after injection and the lenses placed into modified TC-199. Lenses were removed from culture at various times up to 7 days. Some lenses in organ culture were labeled in vitro with 10 μM BrdU for one h and rinsed in PBS. All lenses were fixed in 10% buffered formalin for 72 h, transferred to PBS, and embedded in paraffin.

The paraffin sections were dehydrated in xylene and ethanol. The sections were treated with 2 N HCl at 37 °C for one h to denature the DNA. The acid treatment was neutralized by 0.1 M sodium borate for 10 min. The sections were blocked in 5% normal goat serum for 20 min and washed in ICC buffer (0.5% BSA, 0.05% NaN₃, 0.2% Tween 20 in PBS; pH 7.4). Primary antibody, mouse monoclonal anti-BrdU (Ab-2; Oncogene Research Products, Cambridge, MA) at a concentration of 25 μg/ml prepared in ICC buffer was applied to the sections and incubated overnight. Negative control sections were incubated with ICC buffer during primary incubations. Sections were rinsed with ICC buffer 5 times. The secondary antibody, Cy3 conjugated goat anti mouse IgG (1:200, Alexa Fluor 568; Molecular Probes, Eugene, OR) prepared in ICC buffer was applied to the sections and incubated for one h. Sections were washed 5 times with ICC buffer, mounted with an antifading gel, observed under epifluorescent Nikon Eclipse E800 and photographed using Hamamatsu Digital Camera C4742-95 microscope.

Sections were also stained with DAPI (4¹, 6-diamidens-2-phenylindole dihyrochloride, Molecular Probes) to visualize all nuclei.

Results

Weight of cultured lenses

As previously reported [6] rat lenses cultured under the conditions used in this study could be kept transparent and viable for up to 10 days. As we extended the duration of culture studies from the 1-2 days typically used in the past to 7-10 days more recently, it became apparent that lenses stopped increasing in weight when placed in culture. Table 1 gives representative data demonstrating this phenomenon. For the data in this Table a group of littermates were used, some were euthanized at day 0 and their lenses placed in culture while others were euthanized on days 3, 6, and 9. The weights of the lenses from the freshly euthanized animals were compared with the weights of lenses removed from culture on the same day. Lenses removed from culture after 3, 6, or 9 days all weighed essentially the same, approximately 19-20 mg which was the typical weight of lenses from the 4-week-old rats that we used. By day 9 of culture the lens weight in the live animals typically increased to 24-25 mg. Since it is not feasible to get an accurate weight for the lens and subsequently maintain it in culture, each entry in Table 1 represents a different lens. To confirm the results since there is a certain degree of variability in lens weight even among age and gender matched animals, we also cultured a single lens from some animals and weighed the contralateral lens at T=0. Lenses weighed after 7-10 days of culture were never found to have increased in weight relative to their contralaterals (data not shown).

Morphology

To determine whether the cultured lenses differed histologically from freshly isolated lenses, we compared methacrylate sections from lenses cultured for 7 days with sections from freshly extracted lenses. Figure 1 shows PAS stained sections of the bow region of fresh and 7 day cultured lenses. The most consistent finding observed in the cultured lenses is narrowing of the bow which is compressed toward the capsule. The orientation and appearance of nuclei in the lens equatorial epithelium is also altered relative to the fresh lens and often vacuoles are evident in the epithelial cells. It should be noted however that vacuoles, presumably artifactual, are also often seen in epithelial cells in sections from fresh lenses (see top region of left panel, Figure 1). A second feature commonly observed in cultured lenses was multilayering of the lens epithelium. This typically was not a generalized phenomenon, but was limited to the area near the lens equator where most cell division takes place in the normal lens. The DAPI-stained section of a 1 week cultured lens in Figure 2 demonstrates the multilayering with a portion of the germinative zone shown magnified. The appearance of the central epithelium and the region of the lens containing mature fiber cells was not abnormal in the cultured lenses. In Figure 1 it appears that the lens capsule in the cultured lens is thicker than in the control lens, however in reviewing sections from other cultured lenses this was not generally found to be the case. Therefore we do not believe that a thicker lens capsule is characteristic of lenses maintained in organ culture.

Protein synthesis

Previous studies had demonstrated significant protein synthesis in lenses in organ culture for short periods of time [9]. The lack of growth of cultured lenses led us to compare the protein synthesis in lenses cultured for longer periods of time with that of freshly excised lenses. Figure 3A gives the total DPM incorporated into protein in lenses pulsed for 5 h with ³⁵S-amino acids immediately after being placed in culture or after 7 days in culture. After being cultured for 7 days the lenses continue to synthesize protein although at a significantly reduced rate. Separation of the capsule/epithelium from the fiber mass revealed that protein synthesis in the lens fibers is decreased to a greater extent than in the epithelium. This is depicted on a DPM/mg tissue basis in Figure 3B showing nearly 50% reduction in the fibers, but about 30% reduction in the epithelium.

To determine whether the changes in ³⁵S-incorporation reflected a generalized decrease in the synthesis of proteins or could be ascribed to specific species, samples were run on SDS-PAGE and the stained gels were dried and autoradiographed. The autoradiographs showing the pattern of newly synthesized protein in 4 fresh lenses and 4 lenses that had been cultured for 7 days are seen in Figure 4. Each lane was loaded with an equal amount of lens protein. The heavy bands at and below 30 kDa are the crystallins which are expressed primarily in the fibers. In general it is apparent that the complement of proteins expressed in the fresh lens is still being synthesized by lenses after a week in culture. Since the gel was run to optimize visualization of the noncrystallin proteins (>30 kDa) it is difficult to appreciate that the crystallins exhibit greater decrease in labeling than the other proteins. The βB1-crystallin band (band 7) demonstrated this best with a 33% decrease in intensity as determined densitometrically. The noncrystallin proteins in the cultured lenses tend to show much lower decreases in incorporation of label and in some cases there is increased intensity. One such band (arrowhead) identified by MALDI-TOF mass spectroscopy as vimentin was increased in intensity by 15% after culture.

BrdU labeling

To determine whether culture conditions affect the ability of epithelial cells in the intact lens to synthesize DNA, lenses were labeled with BrdU after different times in culture. As can be seen in Table 2, cells continue to synthesize DNA in the cultured lens throughout 7 days in culture, although by the fourth day there is a definite decrease in the number of labeled cells when compared to the freshly excised lens. In all the lenses analyzed, both cultured and fresh, there were occasional labeled cells in the central epithelium, but the great majority of labeled cells were found near the equator in the germinative zone. This distribution did not change as a function of time in culture.

Migration and differentiation

To compare the migration and differentiation of epithelial cells in the cultured lens with the lens in vivo, we injected BrdU intraperitoneally (100 μg/gm body weight) as described above into 1 month old Sprague-Dawley rats. After 4 h some animals were euthanized and their lenses placed into organ culture or processed as T=0 samples. The remainder of the injected animals were maintained in the vivarium and euthanized at different times up to 14 days after injection. The location of BrdU labeled cells is demonstrated as a function of time after exposure to BrdU for both groups of lenses in Figure 5. The T=0 lenses for the two groups are equivalent. Sections from two representative lenses are shown with BrdU labeled cells clustered in the germinative zone (arrows). For lenses maintained in the living rat after BrdU exposure, labeled cells are present in the transition zone near the bottom of the bow by 7 days and by 14 days elongating fibers are labeled. In contrast, there appears to be little or no change in the position of labeled cells in those lenses maintained in organ culture. By 7 days the characteristic compression of the bow is apparent and the only labeled cells in the lens epithelium are still located in the germinative region. In none of the lenses analyzed after culture were there any labeled cells located in the transition zone where labeled cells are seen after 7 days in the in vivo lenses.

Discussion

The ocular lens is an organ that is relatively easy to maintain in organ culture. Unlike lens epithelial cell cultures [10] or lens epithelium explants [11] which have been widely used as model systems to study aspects of lens biology such as cell proliferation and differentiation, culture of the intact lens allows one to approach the fundamental issues of cataractogenesis (i.e., transparency) in an in vitro system. This laboratory has utilized lens organ culture over many years. Opacification has been induced by a variety of cataractogenic agents providing opportunities to probe molecular processes underlying cataract formation [5]. These cataract model systems have also allowed us to test potential anti-cataract agents for their ability to inhibit or prevent lens opacification in vitro [4]. Further, the effects of specific insults, such as oxidative stress [12] or hyperglycemia [13], have been studied extensively in the organ culture system. Data from many investigators suggests that in general the lens in organ culture responds to such stresses in ways similar to the lens in vivo.

While the lens can be placed into organ culture without the need of severing any vascular or neural connections or changing the structure of the organ itself, it must be recognized that the environment of the lens, in particular its proximity to, and interaction with, other ocular structures is destroyed in the process. In assessing the validity of lens organ culture as a model system for studying cataractogenesis, it is desirable to understand what aspects of normal lens biology are altered under culture conditions.

Our interest in such questions arose as we began to extend the duration of rat lens organ culture experiments from 1-2 days to 7-10 days. We demonstrated [6] that under our standard protocol young rat lenses could be maintained transparent and viable for 8-10 days. In analyzing lenses from these longer experiments it became apparent to us that (1) the lenses ceased to increase in weight when placed into culture and (2) the cultured lenses differed somewhat from fresh lenses histologically, most notably in the shape of the equatorial bow. Since the average growth in terms of weight increase in the rat lens in vivo between the ages of 4 weeks and 5 weeks is approximately 4 mg (or 20% of the starting weight), the lack of increase in weight in organ culture indicates a marked alteration in lens cell biology. Our goal in this study was to determine which major processes required for lens growth (e.g., protein synthesis, cell proliferation, and fiber cell differentiation) were limiting in the cultured lenses.

The bulk content of the young rat lens is 60-65% water with the remainder being more than 95% protein. To determine lens water content, lenses were dried to constant weight in an oven at 105 °C. After culture for 7 days we found no significant difference between cultured and fresh lenses in water content, indicating that loss of water could not account for the lack of weight increase in the cultured lenses (data not shown). Using ³⁵S-labeled amino acids we found that on a DPM/mg tissue basis the cultured lens incorporated about 60% as much label as did lenses freshly removed. In the fresh lenses approximately 75% of incorporation was in the fibers and about 25% in the epithelium. After 7 days of culture, the decrease in incorporation of label was primarily in the lens fibers, which on a DPM/mg tissue basis was reduced by nearly 50%, whereas the epithelium had about a 30% decrease in incorporation. Clearly, while protein synthesis is decreased after a week in culture, there is still a considerable level of protein being synthesized.

BrdU labeling studies demonstrated that epithelial cells do continue to replicate their DNA in cultured lenses although the number of BrdU positive cells in most cultured lenses analyzed decreases by 4 days in culture as compared with freshly excised lenses which were exposed to BrdU immediately after being placed into culture. There was no apparent difference in the distribution of BrdU positive cells in the cultured lenses relative to fresh lenses, with the large majority being in the germinative zone, occasional cells in the central epithelium were labeled in both cultured and fresh lenses.

BrdU labeling was also utilized to evaluate the migration and differentiation of epithelial cells in the cultured lenses. For comparison we labeled cells in vivo by injecting BrdU into one month old rats and then examined lenses for BrdU labeling at various times up to 2 weeks after injection. By day 7 labeled cells had migrated along the capsule to the base of the bow and by 14 days elongating fibers had labeled nuclei (Figure 5). In contrast, in those lenses labeled in vivo as above, but placed in culture 4 h after the animals were injected, all labeled cells remained in the germinative zone after 7 days with no indication of migration towards the bow.

The primary conclusion from these studies is that when the young rat lens is placed into organ culture the process of differentiation whereby lens epithelial cells become lens fibers ceases to occur. Epithelial cells do continue to synthesize DNA in the germinative zone and based on the epithelial multilayering observed in the germinative zone (Figure 2) it seems apparent that cells are proliferating in the cultured lens; they do not however migrate along the capsule toward the bow, nor do the nuclei reorient as they do in vivo as cells approach the bow. This behavior accounts for the abnormal shape of the bow in the cultured lenses (Figure 1). The failure of the lens to add new fibers causes the bow to become progressively compressed towards the lens capsule. The compression of the bow also implies that immature lens fibers formed before the lens is placed in culture do continue to elongate, if they did not the shape of the bow would be expected to remain static in the absence of lens growth. This conclusion is also consistent with the fact that the majority of protein synthesis in the lens after 7 days in culture is still occurring in the fibers (Figure 3A). Mature fibers do not synthesize protein [14,15], thus that synthesis must be occurring in the thin zone of immature fibers.

As noted above, lens organ culture has been heavily utilized for many years, however little attention has been given to the growth, or lack thereof, of cultured lenses. In many instances culture studies have been of short duration or have used lenses from older animals where lens growth is slow such that significant growth during the time in culture would not be expected. Investigators have used various indicators of lens condition including transparency, membrane transport parameters [12], protein leakage [8], and lactate production [16]. There have been a number of studies demonstrating that mitosis continues to occur in lenses placed in culture [17]. One series of elegant studies [18,19] succeeded in producing growth in cultured neonatal rat lenses by pulsate delivery of medium containing PDGF or EGF. The lenses used were from rat pups 3 days of age. Continuous delivery of the same medium failed to produce growth and the lenses developed opacities. Brewitt, et al. [20] concluded that these findings suggested that lens growth depends on factors external to the lens and that growth occurs in an oscillatory fashion with times corresponding to intervals in the lens epithelial cell cycle. To our knowledge there has been no follow-up of these studies and it is not clear whether the results would apply to lenses of the age used in our study. One month old rat lenses, while still in a stage of rapid growth are fully developed and fully functional, in contrast to the 3 day lenses which are still developing in eyes that are not yet open. In addition, at 3 days of age regression of the hyaloid vasculature has not yet occurred and the lens in vivo is still surrounded and nourished by the tunica vasculosa lentis.

Under the conditions used in this study, rat lenses clearly do not increase in weight during 7-10 days in organ culture. This is in spite of the fact that by other measures of growth, including macromolecular synthesis, elongation of immature lens fibers and proliferation of epithelial cells, the cultured lenses remain active. These processes, while continuing, are limited in the cultured lens and diminish with increasing time in culture. This decrease has been documented for both protein and DNA synthesis and the cessation of new fiber induction limits the elongation of fibers to those immature fibers already present when the lens is placed into culture. The increase in lens mass in vivo results from the formation and maturation of lens fiber cells. The failure of fiber cell induction in culture is certainly the root cause of the failure of the cultured lens to increase in weight.

While the lack of growth, in terms of weight increase, by lenses in vitro may have a negative impact on the use of lens organ culture for certain types of studies, the available evidence suggests that this model system can still be valid and useful for studying cataractogenesis. The extensive literature on induction of cataract and prevention of cataract in vitro and the similarities with in vivo cataract in such models as sugar cataract [2] and naphthalene-induced cataract [5] support this conclusion. It should also be noted that in vivo it is well-known that cataract induction is easier in younger animals than in older ones and that this is also true in vitro. Lenses from younger rats are more susceptible to sugar cataracts, for example, than are lenses from older animals. It has been suggested that the more rapid growth of the lens of younger animals might account for this difference, but since neither young nor older lenses increase in weight in culture, it must not be actual growth that determines the susceptibility to opacification. Thus we believe that lens organ culture is a valid model for the study of lens opacification and other aspects of lens biology, albeit a model in which the induction of new lens fibers has ceased to occur.

If fiber cell induction and differentiation could be made to occur in the cultured lens, the system would provide major advantages over the cell and explant systems currently used to study this crucial process. It is clear from the work of numerous laboratories that multiple growth factors and signaling cascades are involved in regulating fiber cell differentiation. In vivo these factors likely originate from a variety of ocular tissues and are present in the eye in gradients which probably change significantly during development, maturation, and aging. It will be a major challenge to produce a comparable environment for the lens in culture.

Acknowledgements

This work was supported by the Intramural Research Program of the National Eye Institute, National Institutes of Health (Bethesda, MD). The authors thank Kimisha Lowery-Scott for her expert assistance in preparing the manuscript.

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