Cataract, types and calcium alternation in cataract formation

The loss of transparency due to cataract development severely impairs the ability of the lens to focus an image on the retina. Cataract is the leading cause of blindness worldwide with the 37 million people affected (48 % of world blindness). Age-related cataract is the most common cataract with the cumulative incidence rates ranging from 4.1% in people aged 43-54 to 56.2% in people 75 years of age or older [1].

Age-related cataract is intensive topic of research. In spite of more than fifty years of basic and clinical research, no non-surgical intervention exist that can prevent or treat age-related cataract. However, there is a better understanding of the complexity of this multifactorial condition in which incidence and progress are modified by factors such as age, sex, radiation, oxidation, physical trauma, diet, diabetes, hypertension, smoking and medications [2]. With aging, non-enzymatic, post-translational modification and the accumulation of fluorescent chromophores occur on lens proteins, increasing susceptibility to oxidation and cross-linking and increased light-scatter [3].

Cataract types: Morphological classification of age-related cataracts distinguishes among following main types of cataracts with approximate percentage: cortical cataract (60%), nuclear cataract (30%), posterior subcapsular cataract (10%). Cataract is a multifactorial disease and different types of cataract have different etiologies. Two major types of cataracts are believed to be formed through very different mechanisms.

Nuclear cataract is located in the centre of the lens and takes place due to slow oxidative changes which may be related to aging [4]. Nuclear cataract is characterized by the accumulation of insoluble proteins which appear to be derived from soluble proteins. It is characterized by steady accumulation of chromophores and formation of complex insoluble crystallin aggregates in the lens nucleus leading to the formation of a brown nuclear cataract. The process is homogeneous and the affected lens fibres retain their gross morphology [3].

Anterior cortical cataract is located in outer layers of the lens and develops due to degeneration and liquefaction of cortical lens fibers [4] due to altered electrolyte and water transport [5]. Cortical opacities are due to changes in membrane permeability and enzyme function with shear-stress damage to lens fibres with continued accommodative effort. Unlike nuclear cataract, progression is intermittent, stepwise and non-uniform [3]. Morphology, molecular composition and prevalence of early cortical opacities were described in detail by Vrensen [6].

Calcium in cataract formation: Calcium, Ca²⁺, plays a specific role in the development of human cataracts. The Ca²⁺ level controls homeostasis of entire lens. The role of Ca²⁺ was brought to the forefront of cataract research in 1975 by the paper of Duncan [7] and is enjoying prominent scientific attention as in detail reviewed by Rhodes [8]. The raised levels of Ca²⁺ in human lenses with cortical cataract have been found to play a major role in the opacification process [7-10]. Nuclear cataracts do not involve major Ca²⁺ alteration in the lens [7,11]. Pure nuclear cataracts have a normal internal ionic content while lenses with cortical cataract (pure or mixed) have increased lenticular Na+ and Ca²⁺ and decreased K+ content [7].

The very large increases in Ca²⁺ recorded in cortical cataract indicate that intracellular Ca²⁺ homeostasis breaks down and influx exceeds the ability of lens cells to remove Ca²⁺ from the cytosol. As stressed by Rhodes [8], it is not clear, whether this is due to an unregulated increase in Ca²⁺ influx, an impaired ability to remove excess Ca²⁺ or a combination of both processes, and these questions remain fundamental to the current hypotheses to explain cortical cataract development.

Lens epithelium

The lens is a cellular structure entirely without blood vessels, lymphatics and nerves. It is enclosed by an avascular thick capsule and is nourished by diffusion from the aqueous humor through the lens capsule and epithelium. The cataract is a result of the functional impairment of the two types of cells that comprise the human lens, epithelial and fibre cells. It is still not known which molecular and metabolic changes of the epithelial cells are the initiating events that lead to biochemical and molecular events changes that compromise the structural integrity of the fiber cells and the lens proteins. Because of its important role in functioning of the lens and its accessibility, the lens epithelium is a subject of numerous studies.

Function: The lens epithelium is the first physical and biological barrier in the lens and the major role of lens epithelial cells is to act as a regulating barrier between the aqueous humour and the lens fibre cells, protecting the inside of the lens. The single-layered lens epithelium underlying the anterior capsule is metabolically the most active part of the lens, acting as a metabolic engine that sustains the physiological health of the tissue. The cells of the lens epithelium regulate most of the homeostatic functions of the lens [12]. Enzymes that protect the epithelium and key antioxidant systems are situated in the epithelial cells. Lens epithelial cells are also responsible for growth of the lens through mitosis, fibre cell differentiation and protein synthesis, and also for secondary capsule opacification. Central, anterior epithelial region is considered to be quiescent, whilst the peripheral germinative region is mitotically active.

The maintenance and development of the epithelium is promoted by influences from the aqueous humor whilst in the posterior part of the lens, to posterior environment that promotes fiber cell differentiation [13,14].

The ocular lens epithelium maintains the lens internal milieu by providing the driving force for the ionic gradients and the fluid circulation within the lens [12,15-19]. The epithelium is the lens layer where many constitutive elements of the mechanisms responsible for the transport of water, ions and nutrients through the lens are situated [16,20,21]. As it is known to be the primary site of active transport and permeability, it plays a crucial role in maintaining the levels of electrolytes and water in the lens that are necessary for preserving of lens transparency [22]. Any factor disturbing the transport processes, morphology or biochemistry of the lens epithelium would result in water accumulation in the lens and the subsequent imbalance of intracellular and extracellular ion concentration that would lead to cataract formation [23-25].

As the function of the lens epithelium is associated with the structural organization of the lens epithelial cells that could be responsible for the functional changes and potentially for the cataract development, here we give a brief summary of the specific structural organization of lens epithelial cells

Structural organization: The lens epithelium is different from many other epithelia as it does not line a lumen or the external surface. The basal surfaces of the epithelial cells are lying on the basal lamina (lens capsule) while the apical surfaces of the epithelial cells contact the apical surface of subjacent lens fibre cells. The cells in the lens epithelium have typical epithelial morphology. They are cuboidal and are tightly packed in a single layer with very little intercellular space. The attachment to adjacent cells occurs by adhesion complexes located in the lateral membranes that include both desmosomes and tight junctions [20,26,27]. The lens epithelium has apical cell-cell junction complexes, including tight and gap junctions as well as adherens junctions, utilizing both E- and N-cadherin [28]. The cadherins are a family of calcium dependent, trans-membrane glycoproteins that mediate cell-cell adhesion via homophilic interactions with cadherins on adjacent cells [29]. Cell-cell junctions are critical for maintaining epithelial integrity and are increasingly becoming recognized as major centers of signalling that impact on cell structure and also on gene transcription and other cell responses such as proliferation and differentiation [29,30].

The epithelium also expresses high levels of the mesenchymal intermediate filament protein vimentin, as well as other intermediate filament proteins such as glial fibrillary acidic protein (GFAP) [31,32], nestin [33,34], cytokeratin-8, cytokeratin-18, cytokeratin-19 and synemin [35].

Intercellular communication between lens cells occurs via gap junction channels, which are formed by integral membrane proteins (connexins). Connexin 43 (Cx43) is uniquely expressed in epithelial cells [36,37], while Cx50 is expressed in both epithelial and fibre cells [38,39]. Cx43, the main component of gap junctions, was found downregulated at both the mRNA and protein levels after exposure of human lens epithelial cells to UVA radiation [40].

Lens epithelial cells express the typical tight junction proteins, claudin, occludin and zonula occludens protein 1 (ZO-1) [41]. ZO-1 has been shown to bind with the lens gap junction proteins Cx43, Cx46 and Cx50 [42,43].

The cytoskeleton of lens epithelial cells includes actin and myosin [44,45].

Effect of calcium and its regulation on lens epithelial cells and the opacification process

Lately the focus of a significant portion of lens epithelial cells research has shifted to the role of the altered Ca²⁺ signalling in lens epithelial cells in and subsequent effects this may have in cataract formation [46-48]. It was also shown that in cortical cataracts, the epithelial cell breakdown causes dysfunction of active transport of electrolytes, causing passive inward movement of water [49]. Intracellular overload with Ca²⁺ in the lens epithelial cells triggers a series of events such as activation of Ca²⁺-dependent enzymes, irreversible breakdown of important structural proteins and cell death [4]. Although there is still a controversy on a mechanism of lens epithelial cell death, it is widely accepted that agents that induce cataract in animal models also induce lens epithelial cell damage [50]. We have studied ultrastructure of the lens epithelial cells in the patients with white intumescent cataracts and have shown structural changes that might explain loss of integrity of the barrier function associated with possible hydropic degeneration of the lens epithelial cells [51].

Calcium is universal intracellular messenger involved in essential cellular functions and is a key mediator of signalling within lens cells. As the proper maintenance of Ca²⁺ levels by regulating activity of Ca²⁺- pumps and Ca²⁺-channels and inhibition of Ca²⁺-dependent enzymes is necessary for its homeostasis, we will give the brief overview concerning [Ca²⁺]_i regulation in lens epithelial cells and its correlation to cataract.

The Ca²⁺ gradients between compartments are essential to the cellular processes regulated by Ca²⁺. The free Ca²⁺ concentration in human aqueous humour is approximately 1 mM while free cytosolic Ca²⁺ concentration in lens epithelial cells is approximately 100 nM, so there is a large, 10,000 fold inward-directed gradient across the plasma membrane. Certain intracellular organelles, such as the endoplasmic reticulum (ER) have a free luminal Ca²⁺ concentration ranging from 0.2-1 mM. The total Ca²⁺ in the lens epithelial cells is approximately 0.1 mM. Such a wide difference in the concentrations of free and bound Ca²⁺ is maintained either by sequestration of Ca²⁺ in the ER including the nuclear envelope, the Golgi complex and mitochondria or by preferential binding of Ca²⁺ to complex protein molecules [4,52]. The role of lens epithelial cells in controlling the lenticular Ca²⁺ is interesting since other components of lens do not possess intracellular Ca²⁺-store such as endoplasmic reticulum and mitochondria. In order for Ca²⁺ to act as a signalling molecule and to prevent the toxic effects of Ca²⁺ overload, intracellular Ca²⁺ is tightly regulated and the concentration of Ca²⁺ in the cytoplasm is kept low.

Ca²⁺-pumps and Ca²⁺-channels: Some integral membrane proteins provide structural channels through which ions, such as Ca²⁺, can diffuse following an electrochemical gradient [53]. In the lens, cellular calcium homeostasis is attained by a delicate balance [54] between passive inward movement from the extracellular milieu through such membrane channels [55], extrusion by plasma membrane calcium ATPase (PMCA) [56], sodium calcium exchange [57] and internal sequestration by sarcoplasmic/endoplasmic reticular calcium ATPase (SERCA) [58]. PMCA and SERCA are pumps, powered by the hydrolysis of adenosine-5'-triphosphate (ATP), which drive Ca²⁺ against its concentration gradient. In the human lens, the Ca²⁺-ATPase pumps are found only in the epithelium [59-62]. Both PMCA and SERCA pumps play an important role in Ca²⁺ homeostasis in lens epithelial cells [58] and each has been shown to contribute approximately 50% of the total activity [63]. It is important to define the role of this pump in the human lens, especially in the light of the study showing that Ca²⁺ ATPase activity is reduced by 50% in human cataractous lenses [59]. On the other hand, upregulation of PMCA2 was reported in the epithelia of cataractous lenses, presumably in compensation for increased [Ca²⁺]_i levels [64].

Oxidation is a major factor in cataract development [65-70]. The lens Ca²⁺-ATPase pumps are very sensitive to oxidation [71-73] and oxidative inhibition of the lens Ca²⁺- ATPase can be reversed [73]. Human lens epithelial cells exhibit a profound response to the oxidative stress, which manifests itself, among other, by a significant increase in the expression of calcium regulatory proteins: PMCA1, PMCA2, SERCA2 and SERCA3 [54].

Ca²⁺ entry mechanism and possible receptors involved: Entry of Ca²⁺ into the lens epithelial cells and Ca²⁺ signalling are regulated by stimulation of cell surface receptors. As highlighted by Duncan [46], the lens possesses an impressive array of G-protein receptors that are coupled to the release of intracellular calcium. They include members of the muscarinic, adrenergic and purinergic families and activation of the former has been implicated in cataract for some time. There are several possible mechanisms whereby activation of such receptors could give rise to cataract. A prolonged increase in intracellular calcium would be expected to activate proteases such as calpain and so could induce unscheduled and irreversible breakdown of important structural proteins. In vivo and in vitro animal models have implicated the Ca²⁺- activated protease calpain in the mechanism of cataractogenesis [74].

As was found previously [75-77], the lens epithelial cells respond to the bath application of acetylcholine (ACh) with a rise in intracellular calcium concentration, [Ca²⁺]_i. In human anterior lens epithelial cells ACh binds to M1 muscarinic receptors and induces a rise in [Ca²⁺]_i [8,77,78]. Exposure to ACh opens up the store-operated channel pathway which is in the lens insensitive to the voltage-activated channel antagonist nifedipine, but is blocked by lanthanum [79].

Many cellular agonists evoke increases in cytoplasmic Ca²⁺, which consist of an initial and transient release of Ca²⁺ from intracellular stores followed by a more sustained Ca²⁺ entry from the extracellular medium through Ca²⁺-permeable channels [80]. This Ca²⁺-release activated Ca²⁺ entry, also termed capacitative or store-operated Ca²⁺ entry (SOCE), is a major mechanism for Ca²⁺ influx [81]. Store-operated Ca²⁺ entry has been a focus of lens cell signalling research since the initial experiments that identified such a pathway in lens epithelial cells exposed to thapsigargin [58]. In human lens epithelial cells, Williams et al. [82] found that, SOCE was blocked by zinc (Zn2+) but was unaffected by nifedipine. SOCE has the dual function of enabling refilling of the store and prolonging the intracellular Ca²⁺ increase initiated by store release [81]. For many cell functions what is important is not the transient increase caused by the initial store release but the long lasting increase in Ca²⁺ during this phase of the response [83].

The receptors that are activated by thrombin which increase [Ca²⁺]_i via protease activated receptor type 1 (PAR1) were discovered in both central anterior epithelial and equatorial cells of the intact human lens [84]. Thrombin receptors are unusual in that they are activated when thrombin, a protease most commonly associated with blood coagulation, cleaves a peptide bond which releases an integral, tethered ligand allowing it to interact with the receptor binding site. Their expression exemplifies the surprising diversity of Ca²⁺-coupled receptors in the lens.

The lens preparations and the physiological studies of the lens epithelium

The methodology to study physiology of human lens epithelial cells and particularly calcium homeostasis in these cells is getting renewed attention in research of mechanisms of cataractogenesis. The structure and the function of the lens epithelium and the role of the lens epithelium in the formation of cataract as well as correlation with calcium, were studied on different types of preparations: human and animal lens epithelium, cultured lens epithelial cells, intact lens (human or animal), and in vivo in animals. Each of these preparations have some advantages (for example: for tissue manipulation, application of different techniques, pointing on specific questions) for the studies and increases the knowledge about the different aspects of the lens epithelial cells.

The human anterior lens capsule preparation: The anterior lens capsule preparation consists of the monolayer of anterior lens epithelial cells lying on the basal lamina. Human anterior lens capsules are excised during routine cataract surgeries by the capsulorhexis technique, and are a potential source of the human lens epithelium that offers the possibility of using this material to study the physiology and pathophysiology of the lens epithelial cells. The method is explained in detail in Andjelic et al. [85]. Working on the human preparation obtained during the surgery allows direct studies of the tissue of interest in different forms of cataract and the obtained results are directly applicable to enhance our understanding of the cataract pathophysiology. The most widely applied technique to investigate cellular Ca²⁺ dynamics is the use of Ca²⁺ indicator dyes that allow the tracking of changes in [Ca²⁺]_i in real time. Fura-2 AM, a cell permeable dye ester derivative, is often used to import the [Ca²⁺]_i indicator dye Fura-2 into the cell and we have used it on the human anterior lens capsule preparation showing that the human anterior lens capsule preparation is an adequate source for investigating cellular Ca²⁺ dynamics of lens epithelial cells. We have recently described fast contractions of lens epithelial cells [86] and found that these occur as a nonspecific response to ACh, water jet or mechanical contact. We have also found that [Ca²⁺]_i may not be directly involved in the changes of the shape of epithelial cells described. During contraction, the cells stay connected to each other at several points, presumably representing regions containing desmosomes and/ or gap junctions. The gaps forming between the epithelial cells would very likely cause influx of water and seriously impair the normal function of the lens epithelium in situ. Water influx through these gaps may be linked to cataractogenesis, for example in injury or cataracts associated with phakic intraocular lenses which frequently touch the lens or cause altered fluid movement. As contractions may be a new mechanism associated with with cataractogenesis, there is a need for their further studies to assess the underlying physiological mechanisms and eventual therapeutic or prophylactic possibilities.

A number of other studies used the anterior capsule preparation after cataract surgery. Yeh et al. [44] studied the actin filaments in human lens epithelial cells after extracapsular cataract extraction whilst Li et al. used this preparation to study of the lens epithelial cell apoptosis [87]. Rae [88] used a similar starting preparation of human anterior lens capsule as Yeh but then dissociated lens epithelial cells to measure the whole-cell current without being subjected to culture media or serum.

As decreased epithelial cell density is commonly observed during aging and cataract formation in humans and animal models and may contribute directly to tissue opacification [89], cell density was studied using different types of preparations including the flat human lens capsule preparation. With progressive involvement of the cortex in the formation of a cataract, there is a tendency towards a decrease in the number of epithelial cells [90]. Vasavada et al. [91], used the same preparation to study the epithelial cell density and morphology in different types of cataracts and found that mature cataracts had significantly lower cell counts than other cataracts. The major cataractous changes in all types involved vacuolization of the cytoplasm. The distribution of lens epithelial cells in the majority of nuclear cataracts was similar to the normal human lens epithelium. Tseng et al. [92] found a marked reduction in lens epithelial cell density in patients with advanced cataracts. Lens capsule preparations obtained after cataract operations of patients with different types of cataracts were also used by Kalariya et al. [93] in combination with lens capsule preparations obtained from eye bank donors. Changes in the lens epithelium with respect to cataractogenesis were also studied on the lens capsule preparation [94].

We have used this preparation to study ultrastructure of anterior lens capsule in white intumescent cataracts where major changes were found in the integrity of lens epithelial cells [51]. In most of these studies, integrity of epithelium was better in nuclear than in cortical cataracts.

Other preparations used for studying the human lens epithelial cells: Another way for obtaining the human lens epithelial cells on the basal lamina is from the eye bank, by dissection from the lens obtained after enucleation and after the cornea is removed for transplantation surgery. The eye bank source of the human anterior lens capsule preparation gives the possibility of having healthy epithelial cell layer because the one obtained during the cataract surgery are from patients suffering from some form of cataract. The epithelium preparations from human donor lenses were used for immunohistochemical investigations of both normal epithelial cell cytoskeletal structure, and of structural changes induced by increasing cell calcium which led to a loss of cytoskeletal organization and included depolymerisation of cytoplasmic actin filaments, disaggregation of microtubules and initial thickening [95]. This source of human epithelia was also used by Collison et al. [78] in combination with lens cell culture for a comparative study concerning muscarinic receptor expression and function and the calcium response.

Intact human lenses obtained from donors to the eye bank were used to study functionality of ACh receptors throughout life [96], regional distribution of functional receptors and calcium mobilization in the lens epithelium [77], the calcium activation of small conductance calcium-activated potassium channels (SK Channels) [97] and the lens epithelium store-operated Ca²⁺ entry [98].

Human culture methods used to study the lens were reviewed by Wormstone et al. [99]. Tissue-cultured human and bovine lens epithelial cells were first used to study calcium regulation by Duncan et al. [58]. Agonist-induced rise in intracellular calcium of lens epithelial cells and the effects on the actin cytoskeleton were studied on the primary cultures of rabbit and skate lens epithelia [76]. Human lens epithelial cell cultures from the donor eyes from the eye bank were used to study the calcium mobilization in human lens epithelial cells after activation of histamine H1 and purinergic P2u receptors [100]. The cultures of human lens epithelial cells were used to study the role of endoplasmic reticulum in shaping calcium dynamics [82]. Human lens epithelial cells primary cultures prepared from the donors of the eye bank were used to study lens epithelial cell apoptosis and [Ca²⁺]_i increase in the presence of xanthurenic acid [101].

Preparations used for studying animal lens epithelial cells: Animal lens preparations were often used in research of lens epithelial cells. Cell count was done on the flat-mount preparation of rodent lens epithelium [102-104]. Mouse lenses were used by Bassnett and Shi [89] for determining cell number in the undisturbed epithelium. Intact rat lenses were used to study the influence of external calcium and glucose on internal total and ionized calcium [105]. The role of thapsigargin in potassium conductance and calcium influx was studied in intact rat lens [106]. Lens epithelial cell proliferation in whole mice lenses was visualized by Wiley et al. [107].

Animal lenses were often used for making the lens cultures. Piper et al. [108] showed the changes in the energy metabolism of cultured bovine lens epithelial cells in comparison with the fresh bovine lens. Knorr et al. [109] recorded [Ca²⁺]_i increases in cultured bovine lens epithelial cells in suspension, after the application of the receptor tyrosine kinase (RTK) ligand, platelet derived growth factor (PDGF). Primary cultures of rabbit and skate lens epithelia were used to investigate the effect of calcium release from intracellular stores upon actin cytoskeleton [76]. Primary cultures of cells isolated from the equatorial region of fresh ovine lens and Fura-2 imaging were used to study cell-to-cell calcium signaling and the influence of mechanical stimulation to cell-to-cell calcium signaling [110] as well as the Ca²⁺ regulation of gap junctional coupling in lens epithelial cells [47] while those isolated from the equatorial region of fresh sheep lenses were used to study intracellular calcium stores by Fura-2 imaging [111].

Animal lens capsules were obtained from bovine eyeballs and the differences in gene expression between the central and the peripheral epithelia was assessed [112].

Mutant mouse was also used to study lens epithelial cells [113,114]. Chicken embryos lenses were used to study the communication between epithelial and fiber cells [115]. In isolated chick embryo lenses contractions of lenses and lens epithelial cells as well as Ca²⁺ mobilization were described [116].

The comparison of advantages and disadvantages of the human anterior capsule preparation and other preparations

The advantages of the human anterior capsule preparation: There are a number of advantages for using this particular preparation. First, the capsules are regularly excised and normally discarded during cataract surgery, so there is a steady supply of human lens capsule material. Secondly, the lens capsule preparation has the advantage of preserving the epithelium in a fairly "intact" configuration, i.e. all, or at least most, of the connections between neighboring cells are preserved and if the preparation was not mishandled they should behave as they do in their normal environment. This is not the case with cells separated from each other during primary culturing procedure or with cloned epithelial cells from cell culture collections. The cells remain connected to neighboring cells and to the underlying basement membrane. This is of importance for studying of the structural characteristics of the lens epithelial cells.

The advantage of our preparation is also that the source of the tissue is human cataract tissue so that the different types of cataract and how the epithelial tissue is involved in that type of cataract can be studied. The preparation allows the study of human anterior lens epithelial cells and their contribution to pathological conditions such as cataract formation. The results obtained from human lens tissue can be directly related to the human lens pathophysiology [86,94].

The disadvantages of the human anterior capsule preparation: An obvious disadvantage of lens capsule preparation is that it is not possible to study the communication between epithelial and fiber cells of the eye lens as the contacts between the epithelial and fiber cells are destroyed during capsulorhexis. Another disadvantage of the human anterior capsule preparation is that the control, human lens epithelial cells from the non-cataract tissue are rarely obtained. In most cases this can be done after the cornea is removed for transplantation surgery and the eye was of a healthy donor.

The disadvantages of other preparations: The experimental conclusions that are obtained on animal species can not be directly applied to the humans. No single animal species is a complete model of the human lens. Even in the cases when the same agonist induces responses in different species, there are differences in receptor subtype expression. The example is the case of muscarinic receptors where the native human lens cells express the M₁ subtype, while rat and rabbit express the M₃ as the dominant subtype [117].

The species and age differences exist also in the response of lenses to increases in intracellular Ca²⁺ [118]. In young rodent lenses, both in vitro and in vivo, nuclear opacities developed in response to treatments that increase lens [Ca²⁺]_i, whereas rabbit and bovine lenses, as well as older rodent lenses, developed cortical opacification [74,118,119]. The cultured primate lens cells were shown by Zigler et al. [120] to be less sensitive to oxidative insult than the cultured rodent lens cells. The human lenses were reported to contain only 3% of the calpain activity found in the rat lens, and no activity could be measured in human lens homogenates unless the endogenous inhibitor calpastatin was removed [121]. Hightower and Farnum [122] demonstrated calcium induced opacities in cultured human lenses. To extrapolate the data from animal models to the process of cataract formation in man, information is needed from human experimental systems [43].

As already mentioned, the connections between neighboring cells are not preserved in culture as it is in natural conditions. There is also the difference in receptor subtype expression found between native and cultured cells. In the human lens cell line, human lens epithelial (HLE) B-3 cell line, (HLE-B3), it is the M₃ subtype that predominates and not M₁ as in the native epithelium [78]. The experimental conclusions obtained on cell cultures can not be directly applied to the humans. Other difficulties with lens epithelial cells in culture are described by Bhat [20].

The advantages of other preparations: Other preparations also have their advantages. Sometimes, it is their easier availability; they may be more suitable for tissue manipulation, application of specific techniques, pointing on specific questions. The reduced dependency on external stimuli in comparison to other tissues, which typically require a blood supply, permits successful lens organ/tissue culture [99].

Some questions can not be answered by using the human preparations. Transgenic as well as gene-targeted animals are useful experimental tools to study different aspects of lens physiology [123]. Intact animal lenses are easier available than human and permit functional and structural studies of intact lens. Together with human preparations, these models allow the biological events of the lens to be further understood.

Lens epithelial cell cultures provide important information concerning the role of epithelium in normal lens and cataract formation. Experimentally, cell lines have advantages in providing a homogenous population of cells. The consistent nature of a cell line, therefore, allows a continuous series of experiments to be carried out. Cell lines also allow reliable comparisons to be made and facilitate the use of robust statistical analysis of the data. Human lens cell lines also provide an additional, more readily available, experimental tool to complement the use of native tissue and can progress the rate of investigations [99].

As we can conclude from this review, significant insights in lens physiology were recently revealed using human lens capsule preparation obtained by capsulorexis, in particular concerning calcium studies. Important function of the lens epithelium and the role of calcium homeostasis in the epithelial function were presented, considering both advantages and disadvantages of this preparation. We conclude that the human lens capsule preparation is an valuable tool with potential for the study of cataractogenesis. The anterior capsules of the human lens isolated during the cataract surgery are an adequate source for the studies of lens epithelial cell function and can provide important insights into the pathophysiology of the cataract.

The work was supported by The Slovenian Research Agency (program P3- 0333 and postdoctoral grant Z3-9689).

No author has a financial or proprietary interest in any material or method mentioned.