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DOI: 10.1080/01926230601178199
An Overview on the Toxic Morphological Changes in the Retinal Pigment Epithelium after Systemic Compound Administration
1 ALTANA Pharma AG, Institute of Preclinical Drug Safety, Hamburg 22885, Germany Correspondence: Address correspondence to: Lars Mecklenburg, Veterinary Pathologist ALTANA Pharma AG, Institute of Preclinical Drug Safety, Haidkrugsweg 1, 22885 Barsbuettel, Germany; e-mail:lars.mecklenburg{at}altanapharma.com
Many medications that are administered systemically for nonocular conditions may evoke ocular toxicological complications. Therefore, the eye is routinely investigated histopathologically in preclinical in vivo toxicity studies. The retinal pigment epithelium is a likely target for systemically administered compounds, since the underlying choroid is highly vascularized. The specialized pigment epithelium has numerous functions that all maintain the integrity and function of photoreceptors. Consequently, toxic effects on the pigment epithelium will eventually affect the neural retina. The potential of pigment epithelial cells to respond to toxic injury is limited, but a standardized terminology to describe its morphological changes does not exist in the scientific literature. Detailed morphologic analysis, however, might allow early detection of retinotoxicity and may provide evidence on the underlying pathomechanism. We here review toxic effects on the pigment epithelium focusing in particular on the morphology of toxic cell injury. Morphological changes comprise hypertrophy, intracytoplasmic accumulation of cellular components, loss of cell polarity, degeneration, metaplasia, and formation of subretinal membranes. Some of these changes are reversible whereas others are permanent, leading to impaired function of the pigment epithelium and eventually to photoreceptor loss and retinal atrophy.
Key Words: Retina • RPE • eye • subretinal • ocular toxicity Abbreviations: ARMD, age-related macular degeneration • GES, guanidinoethyl sulfonate • PCNA, proliferating cell nuclear antigen • POS, photoreceptor outer segments • RCS, Royal College of Surgeons • RPE, retinal pigment epithelium
The blood-retinal barrier prevents many systemically administered drugs from entering the eye via the circulatory system. Yet many medications that are administered systemically for nonocular conditions may still gain access to the eye and evoke ocular toxicological complications (Zinn and Greenseid, 1975; Koneru et al., 1986; Scroggs and Klintworth, 1994). The mechanisms by which these toxins exert their harmful effects on the eye are varied and in most cases remain poorly understood (Meier-Ruge, 1973; Scroggs and Klintworth, 1994). In man, ocular toxicity associated with systemic drug therapy can result in retinal degeneration (Moorthy and Valluri, 1999), although it most frequently comprises visual disturbances including decreased visual acuity, impaired color perception, visual field defects, scotomata, night blindness, visual perseveration beyond the physiological afterimage (palinopsia), and an illusory movement of the physical environment (oscillopsia) (Scroggs and Klintworth, 1994). Sildenafil, for example, inhibits phosphodiesterase-5 activity, and is associated with transient visual symptoms, typically blue tinge to vision, photophobia light, and blurry vision (Marmor and Kessler, 1999; Laties and Sharlip, 2006). To detect the potential for retinal toxicity, the eye is routinely investigated clinically and histopathologically in pre-clinical toxicity studies conducted in rodent and nonrodent species (Heywood and Gopinath, 1990; Kuiper et al., 1997; Whiteley and Peiffer, 2002). Non-inflammatory degenerative changes in the retina are generally described as retinal atrophy (Greaves and Faccini, 1984; Whiteley and Peiffer, 2002). Whereas the end stage of retinal degeneration is morphologically uniform, early stages may exhibit distinct morphological abnormalities that may provide evidence for the underlying pathomechanism. This is particularly true for the retinal pigment epithelium (RPE). To our knowledge, there is no harmonized nomenclature in terms of morphological abnormalities in the RPE, making it difficult to compare different studies with each other. Moreover, morphological abnormalities in RPE cells cannot be assessed in terms of their functional relevance, since little is known about their pathogenesis, their involvement in disease process, and ultimately in regeneration. A more precise evaluation of RPE pathology might therefore allow a better understanding in terms of pathogenesis and relevance for human risk assessment. Finally, a precise knowledge of RPE pathology may allow earlier recognition of substance-related effects, since retinal atrophy is a late stage disease process whereby photoreceptor cells are lost. We here provide a detailed description of the various forms of drug-induced RPE pathology, providing the reader with an accurate terminology to describe morphological lesions in the RPE.
The RPE derives embryologically from the same neural anlage as the neural retina, but is differentiated into a secretory epithelium (Marmorstein et al., 1998). It is a monolayer of cuboidal cells that is closely associated with the rod and cone photoreceptors, separating them from the capillary bed of the choroid (Bok, 1993). The developing RPE cells slowly increase in number throughout fetal development; after birth however, they only increase in size to cover the increased surface area. Mitoses are not seen after birth, and under normal circumstances, the RPE does not undergo cell division (Rizzolo, 1997). Each RPE cell is differentiated into an apical portion facing the photoreceptors, and a basal portion that is situated on Bruch’s membrane (Bok, 1993). The apical side has numerous long microvilli, whereas the basal side shows large infoldings (Figure 1). Apical and basal membranes of RPE cells are distinct with respect to receptoral and ion channel properties (Mamorstein, 2001). In differentiated RPE cells, the electrogenic sodium-potassium pump (Na/K-ATPase) is primarily expressed on the apical membrane (Rizzolo, 1990; Ruiz et al., 1996), whereas the chloride-bicarbonate exchange transporter is located at the basolateral membrane (Lin and Miller, 1994). RPE cells are usually characterized by melanin-containing organelles, i.e., melanosomes (Boulton, 1998; Schraermeyer and Heimann, 1999). Mature melanosomes are located at the apical cell surface, often within microvilli (Figure 1).
The function of melanin in the RPE is not clear yet (Schraermeyer and Heimann, 1999). Melanin absorbs scattered light, which would otherwise disturb visual acuity, and—in cooperation with various anti-oxidative enzymes (Newsome et al., 1994)—protects against reactive oxygen species (i.e., oxidative stress) produced by phagocytosis of shed photoreceptor outer segments (POS) (Miceli et al., 1994). In rats, melanin in the RPE, choroid and iris prevents development of light-induced retinal atrophy (Perez and Perentes, 1994). Another potentially important function of melanin in the RPE could be the storage of zinc, since melanin is the main source of zinc in the eye, and zinc plays an important role in the metabolism of the retina (Schraermeyer and Heimann, 1999). Melanogenesis in the RPE continues to some degree throughout life. With age, however, these granules begin to fuse with lysosomes and break down (Schraermeyer and Heimann, 1999). With regard to their functions and their capacity, RPE cells are virtually unique among all epithelia of the body. They have no photoreceptive or neural function, but are necessary for the support and viability of the photoreceptors (Bok, 1993; Marmor, 1998). The RPE serves various functions that are based on its physical and biochemical properties (Table 1). Beside absorption of stray light, adhesion between neural retina and choroid, and secretion of various growth factors, the most important functions are (i) synthesis and maintenance of the interphotoreceptor matrix, (ii) photoreceptor membrane turnover, and (iii) retinoid metabolism (Bok, 1993). These functions are briefly discussed next (for review see Strauss, 2005).
Synthesis and Maintenance of Interphotoreceptor Matrix In principle, blood-borne metabolites within the choriocapillaris can diffuse out of the vessel lumen, through Bruch’s membrane and into the extracellular spaces both beneath and between the RPE. Free diffusion into the neural retina, however, is prevented by tight junctions between the RPE cells as well as between the retinal vascular endothelial cells and a paucity of intra-endothelial cell vesicles. These features constitute the blood-retinal barrier (Konari et al., 1995). The basal membrane of the RPE is highly convoluted and contains specific receptors that enable metabolites to accumulate actively within the cell (Miller and Edelman, 1990; Joseph and Miller, 1991). These metabolites (such as glucose, nutrients, and retinol) are required by photoreceptors and are transported to the apical surface by special intracellular carrier proteins. The described differences between the apical and basolateral membranes allow net movement of Na+, K+, HCO–3, and water from the subretinal space into the choroid, and net movement of Cl– from the choroid into the subretinal space (reviewed in Strauss, 2005). Carbonic anhydrase regulates the intracellular HCO–3 concentration and thus determines the intracellular pH (Wolfensberger, 1999). Transport of ions is not only required for the long-term maintenance of the interphotoreceptor matrix, but also facilitates spatial buffering of fast occurring changes in the ion composition of the subretinal space, induced by light-absorption (Steinberg, 1985).
Photoreceptor Membrane Turnover
Retinoid Metabolism The RPE is also crucially involved in regeneration of 11-cis-retinaldehyde, which is known as the "visual cycle" (Bok, 1993). The 11-cis-retinaldehyde is the chromophore for the visual pigments in rod and cone outer segments. Upon photostimulation, it is isomerized to all-trans retinal, which leaves the POS and enters the RPE. Within the RPE, all-trans retinal is reisomerized to 11-cis retinaldehyde, a process that requires the RPE-specific protein RPE65 (Redmond et al., 1998). Thereafter, the recycled 11-cis retinaldehyde is transported back to the photoreceptors. These transport processes between RPE and POS involve cellular (CRALBP) and intercellular retinoid binding proteins (IRBP) (Bernstein et al., 1987).
Photoreceptors, particularly the rods, are lost during aging. However, the ratio of photoreceptors to RPE cells remains largely the same, suggesting a parallel loss of these closely apposed cells (Gao and Hollyfield, 1992). Due to this close relationship, morphological or clinical distinction between RPE and photoreceptor dysfunction is often impossible. A number of spontaneous diseases in mammals are associated with RPE pathology that comprises the following morphological features. Hypertrophy (i.e., increased cell size, due to an increase in intracellular structural components) of the RPE can occur for no apparent reason and without any clinical impact. Congenital hypertrophy of the RPE is a common finding in man with familial adenomatous polyposis of the intestine (Lloyd et al., 1990; McKay, 1993) or neuroepithelial tumors in the central nervous system (Munden et al., 1991). In all species, hypertrophy of the RPE is commonly associated with detachment of the neural retina, or with trauma leading to photoreceptor loss. This RPE pathology is clinically known as "pseudoretinitis pigmentosa" (Lee, 2002). Injured RPE cells exhibit retraction of pigment granules from the cell apices and become rounded (Fite et al., 1985). Although it is believed that RPE cells are postmitotic and cannot be replaced, trauma and detachment are well known to result in hyperplasia (i.e., an increase in cell number) (Kalnins et al., 1995; Lee, 2002; Men et al., 2003). RPE cells can proliferate, but they may also migrate into the subretinal space or undergo metaplasia to fibroblasts or osteoblasts (Lee, 2002; Toyran et al., 2005). As a consequence of fibroblastic metaplasia, the pigment epithelium may form a multilayered accumulation of RPE cells, known as epiretinal or subretinal membranes (Grierson et al., 1994). Dedifferentiation of RPE cells and metaplasia, also known as epithelial-mesenchymal transition, involves TGF-β/Smad-signaling (Saika et al., 2004), as well as expression of bone morphogenetic proteins and growth differentiation factor (Toyran et al., 2005). RPE metaplasia and proliferation may complicate rhegmatogenous retinal detachment and may lead to proliferative vitreoretinopathy, a serious complication of retinal detachment in man (Kirchhof and Sorgente, 1989). Morphologic changes in the RPE (such as those described above) are frequently involved in hereditary retinopathies in man and animals. In man, this diverse group of clinical entities with progressive degeneration of rods and cones and a secondary involvement of the RPE is referred to as "retinitis pigmentosa" (Lee, 2002). Similar diseases known as "progressive retinal atrophy" or "photoreceptor dysplasia" exist in animals such as dogs, cats, monkeys and rodents (Aguirre et al., 1998). As in man, the RPE only shows changes in advanced stages of the disease process. A classic example of an inherited retinal dysplasia is the "Royal College of Surgeons" (RCS) rat. These animals carry a spontaneous mutation in the Mertk gene that encodes for the receptor tyrosine kinase c-mer, which is crucial for phagocytosis of shed outer segments (Vollrath et al., 2001). Consequently, RPE cells of RCS rats are unable to phagocytize outer segment membranes and lack phagosomes, leading to photoreceptor loss and retinal dystrophy (Herron et al., 1969; Strauss et al., 1998). Another characteristic morphologic finding in RPE cells is the accumulation of normal or abnormal cellular components. Those components may be mucopolysaccharides or glycoproteins, such as in inherited storage diseases, or lipofuscin. The latter occurs in hereditary and acquired diseases of various species, such as retinal pigment epithelial dystrophy in dogs (Aguirre et al., 1998), and is a normal feature of aging, particularly in primates (Dorey et al., 1989). Age-related macular degeneration (ARMD) is an important RPE-related cause of visual deterioration in aging man (Garner et al., 1994). Although oxidative stress is thought to play an important role in the pathogenesis of AMRD, its development is still poorly understood. RPE cells either degenerate, or they are hypertrophic and hyperplastic, often with loss of melanin and intracellular accumulation of lipofuscin (Garner et al., 1994). Necrotic RPE cells, with massive lipofuscin content, are discharged into the subretinal space, and gaps within the RPE layer may result. Cells that are shed into the subretinal space may retain RPE characteristics, such as tight junctions with the overlaying cells, stubby microvilli, and abundant smooth endoplasmic reticulum, which distinguish them from macrophages (Ishikawa et al., 1983). One characteristic of ARMD is the formation of deposits between the RPE cell basal membrane and the inner collagenous layer of Bruch’s membrane. These deposits are known either as basal linear deposits, "soft drusen" or "hard drusen" (colloid bodies), depending on their size (Garner et al., 1994; Hageman and Mullins, 1999; Russell et al., 2000). They are composed of numerous extracellular matrix components, such as vitronectin, apolipoprotein E, complement proteins and amyloid proteins (Mullins et al., 1997; Hageman et al., 1999; Mullins et al., 2000; Anderson et al., 2004) and likely result from cellular remnants and debris derived from degenerated RPE cells. This may induce local inflammation and finally lead to choroidal neovascularization (Anderson et al., 2002). In addition to RPE cell pathology, inner and outer segments of photoreceptors are gradually lost, and the outer nuclear layer is eventually replaced by glial cells (Garner et al., 1994). Drusen-like bodies and a macular pigmentary anomaly may occur spontaneously in the eye of Rhesus and Cynomolgus macaques (Fine and Kwapien, 1978; El-Mofty et al., 1980, Feeney-Burns et al., 1981; Stafford et al., 1984; Ulshafer et al., 1987; Engel et al., 1988). The incidence is reported to be between 6% and 84%, clearly increasing with age (Fine and Kwapien, 1978). Beside the typical drusen, vacuolated and lipid-laden RPE cells ("lipoidal degeneration") are found, which can be detected by indirect ophthalmoscopy as yellowish-white irregular dots of variable size.
Toxic compounds affecting the retina can be subdivided into those primarily affecting the photoreceptors or ganglion cells and those affecting the pigment epithelium (Heywood, 1982; Heywood and Gopinath, 1990). However, since the RPE is closely associated with the photoreceptors, damage to the rods and cones will also affect RPE cells (and vice versa), and determination of the primary target cell may be impossible. In addition, not all functional impairments of the RPE will result in significant morphological alterations, suggesting that sophisticated electrophysiological methods need to be applied in order to detect very early stages of toxicity. Although ocular structure and function are similar between mammalian species, differences in ocular anatomy should be taken into account when choosing an animal species for pre-clinical toxicity testing. For example, vascularization—and hence blood flow—differs significantly between mammalian species (Buttery et al., 1990). Moreover, the retinal organization into macula and fovea is restricted to primates (Provis, 2001). In fact, there are large topographical differences in RPE pathology throughout the ocular fundus, both in spontaneous and in toxic diseases (Aguirre et al., 1998). Another complication of preclinical toxicity testing in animals is the fact that dogs, but not primates, rodents or swine, have a tapetum lucidum, a focally extensive specialized structure within the choroid that improves night vision. Some xenobiotics induce toxicity of tapetal cells that secondarily affects the RPE (Heywood and Gopinath, 1990; Dillberger et al., 1996; Aguirre et al., 1998), and further challenges risk assessment for humans. Finally, the anatomy of Bruch’s membrane varies between species, being poorly defined in the dog (Aguirre et al., 1998). Pigmentation of the eye may also affect the outcome of toxicity testing, since many compounds show significant affinity to melanin and therefore accumulate in the pigmented compartments of the eye, particularly the RPE (Leblanc et al., 1998). Binding to melanin, however, is not predictive of ocular toxicity. In fact, some drugs, such as vigabatrin (Butler et al., 1987) are detoxified by melanin binding, whereas others may accumulate in the eye and exert intrinsic toxicity (Eves et al., 1999; Dayhaw-Barker, 2002). Drugs that bind to melanosomes can alter their ion composition (particularly the concentration of calcium) and their morphology, resulting in the expulsion from RPE cells (Schraermeyer, unpublished data). There is a large variety of different coumpounds that affect the retina after systemic administration, associated with particular morphological alterations of the pigment epithelium (Grant and Schuman, 1993). Shown below is a list of compounds compiled from data that is publically available, that specifically have shown morphological RPE toxicity in in vivo animal studies.
Aluminium
Aminophenoxyalkanes Intact RPE cells accumulate disrupted lamellar discs and myelin bodies. By four days, both necrotic and regenerated immature RPE cells are found. Immature RPE cells are characterized by a large vesicular nucleus, prominent nucleoli, scant cytoplasmic organelles with few melanosomes, and a lack of long apical microvilli. By seven days, the RPE shows marked hyperplasia, forming up to seven cell layers between Bruch’s membrane and a partially detached and folded neural retina with excessive outer segment disruption. After another seven days, RPE hyperplasia has regressed to a single cell layer. Few pigmented cells are found in the subretinal space, and outer segments start to regenerate. 57 days after treatment, the retina has regained normal morphology. If a higher dose is administered, RPE and POS changes are more severe and photoreceptors are permanently lost, resulting in irreversible retinal atrophy (Lee and Valentine, 1991).
Cationic Amphophilic Drugs (Tricyclic Antidepressants)
Desferrioxamine
DL-(p-trifluoromethylphenyl) Isopropylamine Hydrochloride
Fluoride
Iodate
Iodoacetate
Lead
Methanol/Formic Acid
4,4'-Methylenedianiline
N-Methyl-N-nitrosurea
Naphthalene
Napthol
Nitroaniline
Organophosphates
Oxalate
Phenothiazines
Quinolines Chloroquine-induced retinal toxicity has been reproduced in several animal species, including rat, cat, dog, rabbit, pig, and monkey (Meier-Ruge, 1965; Gleiser et al., 1969; Abraham and Hendy, 1970; Berson, 1970; Gregory et al., 1970; Rosenthal et al., 1978). Morphological hallmark is the intracellular accumulation of membranous phospholipid inclusions (myeloid bodies) that occur in ganglion cells and (apparently to a lesser degree) in photoreceptors and RPE (Abraham and Hendy, 1970; Gregory et al., 1970; Perasalo et al., 1973; Ivanina et al., 1983; Duncker et al., 1995). RPE cells largely remain intact, but eventually the POS deteriorate (Ivanina et al., 1983). Although it has been speculated that oxidative stress is a primary mechanism of chloroquine-induced retinal toxicity (Ivanina et al., 1987; Toler, 2004), there is extensive evidence suggesting that chloroquine primarily inhibits lysosomal degradation (Ivanina et al., 1989; Schraermeyer, 1992; Mahon et al., 2004; Peters et al., 2005). The high affinity of chloroquine for melanin and its potential to accumulate within the eye are thought to be an important pathogenic mechanism, but its significance is still obscure.
Streptozotocin
Taurine Deficiency Adult female albino rats treated with GES orally for 8 weeks exhibit vacuolization and swelling of the RPE. This is associated with disorganization of POS and finally with photoreceptor cell degeneration, formation of few retinal rosettes, and migration of RPE cells into the subretinal space (Lake and Malik, 1987). The mechanism of toxicity is still unknown, but impaired protection against oxidative stress is one proposed mechanism (Lake and Malik, 1987).
Urethane
Zinc Deficiency Caused by Metal Chelators
As summarized here, the RPE can exhibit distinct morphological features, which may depend not only on the duration and severity of the toxic injury, but also on the pathomechanism of injury. The following characteristics can be used to describe morphological abnormalities in the pigment epithelium of the retina as assessed by light and transmission electron microscopy.
Hypertrophy and Intracellular Accumulations
Similar lysosomes containing abundant concentric lamellae typically accumulate in aminoglycoside intoxication or intoxication with other cationic amphophilic compounds and are frequently named "residual bodies" (D’Amico et al., 1984). Other deposits found in RPE cells are lipofuscin (Figure 5), crystals or other yet undetermined inclusions. Lipofuscin accumulation is usually seen with age and can be enhanced by intoxication (Hass et al., 1964; Brown, 1974; Hughes and Coogan, 1974; Fox and Chu, 1988). Calcium oxalate crystals accumulate in the RPE following subcutaneous administration of dibutyl oxalate to rabbits (Caine et al., 1975). The crystals have a needle-like shape and are best demonstrated under polarized light (Caine et al., 1975). Zinc deficiency has been associated with the intracellular accumulation of irregularly shaped osmiophilic non-membrane bound inclusion bodies in the basal portion of the RPE (Leure-duPree and McClain, 1982). The nature of these inclusions is unknown, but they are morphologically distinct from phagosomes, lysosomes, or lipofuscin granules (Leure-duPree and McClain, 1982).
Loss of Polarity/Dedifferentiation
Depigmentation RPE cells may loose their melanosomes and become depigmented. Depigmentation is usually associated with degradation of melanosomes that fuse within lysosomes.
Degeneration/Necrosis/Atrophy
Hyperplasia Although RPE cells have a very limited potential to replicate, it has repeatedly been shown that they can proliferate and replace gaps within the RPE cell layer. The RPE is prone to exhibit hyperplasia in response to a variety of insults, including injury, chronic inflammation in adjacent structures and long-standing retinal detachment with traction (Machemer and Laqua, 1975). Proliferation of RPE cells has been demonstrated by PCNA expression (Kiuchi et al., 2002) and is characterized by immature RPE cells with a large vesicular nucleus, scant cytoplasmic organelles, few melanosomes, and a lack of apical microvilli (Lee and Valentine, 1991). They may form multiple layers between the choroid and the POS (Figure 8).
Cellular Infiltration into the Subretinal Space Many cases of retinal toxicity show a cellular infiltrate into the subretinal space, i.e., the space between the POS and the RPE (Figure 9). Those cells are usually heavily pigmented. In most cases, it is not easy to differentiate between detached RPE cells and macrophages. Macrophages are characterized by a lack of cell junctions, whereas RPE cells may retain their junctions, showing stubby microvilli, and abundant smooth endoplasmic reticulum (Ishikawa et al., 1983). Immunohistochemical phenotyping using macrophage-specific antibodies or typical markers of RPE cells (such as ZO-1, RPE65, CRALBP, and Mertk) may help to further differentiate between these two cell types, if appropriately fixed tissue is available. Photoreceptor nuclei may also be displaced into the subretinal space, but their characteristic round shape and small size allows easy distinction from the cell populations mentioned here (Aguirre et al., 1998).
Subretinal Membranes As mentioned previously, RPE cells do have a potential to transform into fibroblasts and as such they can form a collagenous extracellular matrix. This may result in the formation of membranes that are located between the RPE and Bruch’s membrane. In rare cases, these membranes consist of multiple cell layers, interspersed with collagen-rich extracellular matrix (Figure 10). If neovascularization is initiated, those membranes can be highly vascularized, but avascular deposits occur as well.
The RPE represents a layer of highly specialized complex cells that are not part of the photoreceptive retina, but are essential for its function (Marmor, 1998; Strauss, 2005). The RPE controls a multitude of physical and biochemical processes and as such can easily be influenced by a diversity of pharmaceutical compounds that gain access to the eye via the circulation (Scroggs and Klintworth, 1994). Most of these compounds will only have access to the RPE but not the neural retina, since the RPE provides the inner levels of the blood-retinal barrier. This makes the RPE relatively vulnerable to toxic effects. RPE cells have a limited potential to respond to toxic or any other kind of insult. Hyperplasia of the endoplasmic reticulum in RPE cells is indicative of increased metabolic capacity in the cell and is likely not to be of any functional significance for vision. Accumulation of intracytoplasmic inclusions such as phospholipids or crystals may also be of no functional consequence if its extent is limited. Drug-induced phospholipid inclusions are partially reversible (Lee and Valentine, 1991). First morphological features of an impaired RPE cell function are loss of apical microvilli and basal infoldings (Suyama, 1967; Nilsson et al., 1977a; Kiuchi et al., 2002). Since the apical and basolateral membrane contain specific transport proteins for ions and other substances (such as taurine, glucose, etc.), these morphological features suggest an impaired transport function. The apical microvilli of the RPE embrace the POS and are essential for their maintenance (Bok, 1993). It must be assumed that POS function becomes impaired if RPE microvilli regress. Indeed, most cases in which apical microvilli of the RPE are lost also show ultrastructural disorganization of POS (Nilsson et al., 1977b; Korte, 1984b). Further features of RPE toxicity are swelling and vacuolization of mitochondria and the endoplasmic reticulum. As in other cell types, these are early features of cell degeneration that are considered to be reversible (Lee and Valentine, 1991; Kumar et al., 2005). If RPE function is impaired beyond a "point of no return," the cell is irreversibly damaged and undergoes degeneration, finally resulting in atrophy of the RPE cell layer, loss of photoreceptors, and retinal atrophy (Lee and Valentine, 1991). Since RPE cells are highly specialized cells, their capacity for regeneration appears to be very limited. However, very little is actually known about RPE regeneration (Korte et al., 1994). RPE cells can undergo hyperplasia, which may lead to RPE regeneration comprising reorganization of the polarized plasma membranes and reestablishment of the blood-retinal barrier (Korte et al., 1994). In contrast, RPE hyperplasia may result in the formation of a multilayered epithelium between Bruch’s membrane and the photoreceptors (Machemer and Laqua, 1975). The factors that govern differentiation of newly generated RPE cells are still obscure, but it is known that extracellular matrix components influence RPE membrane polarity (reviewed in Korte et al., 1994). RPE cells that do not differentiate can undergo metaplasia into fibroblasts and as such are capable of producing a collagen rich extracellular matrix. This leads to the formation of collagenous membranes between the RPE and Bruch’s membrane (Lee, 2002). It must be assumed that this membrane significantly impaires the retinal function and therefore has impact on vision. A relatively common finding in retinal toxicity is the occurrence of nucleated, often pigmented cells within the sub-retinal space (Delahunt et al., 1963; Lake and Malik, 1987; Nakajima et al., 1996; Kiuchi et al., 2002). This space between the photoreceptor nuclei and the RPE is usually filled with photoreceptor inner and outer segments and does not contain cell nuclei. Cells that have migrated into the subretinal space are frequently pigmented and could either represent detached RPE cells or macrophages that have infiltrated the retina and have phagocytozed melanin from degenerating RPE cells. In most cases, it is difficult to distinguish between these two cell types, but TEM and immunohistology may help in the differentiation. Clinically, the RPE can be assessed by indirect ophthalmoscopy, fluorescein angiography, and electrophysiology (Grant and Schuman, 1993). Impairment of fluid transport across the RPE will likely result in retinal edema, which can be assessed by indirect ophthalmoscopy, similar to a pigmentary retinopathy. The latter describes abnormal pigmentation of the ocular fundus and is most likely a correlate of RPE cell hyperpigmentation, necrosis and hyperplasia (Laqua and Machemer, 1975). In addition to the above-mentioned techniques, morphology, particularly using transmission electron microscopy, is a very sensitive method to detect RPE cell injury (Heywood and Gopinath, 1990; Whiteley and Peiffer, 2002). Detailed knowledge on the pathophysiology of the retinal pigment epithelium will enable early detection of the potential of a compound to adversely affect the eye, which is of major importance for preclinical toxicity studies, given the prominence of vision as a cognitive sense in humans (Whitley and Peiffer, 2002).
We are grateful to Prof. Dr. Dr. h. c. mult. W. Drommer for providing some electron micrographs of dog retina, and to Dr. Maria Wendt for editorial assistance. We also thank the technical staff from the Institute of Preclinical Drug Safety, ALTANA Pharma AG, and from the Section of Experimental Vitreoretinal Surgery, University of Tübingen, for their excellent technical assistance.
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Abstract
Introduction
V β5-integrin (
