Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

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III

MECHANISMS UNDERLYING RETINAL

DEGENERATIONS

are also affected by diabetes. The present chapter expands on this and reviews recent literature about involvement of various retinal cell types in diabetic retinopathy with an emphasis on work published in the last 3–5 yr.

GLIAL CELL INVOLVEMENT IN DIABETIC RETINOPATHY

Müller cells are the chief retinal glial cell (3) and play numerous roles in maintaining normal retinal function, including modulating levels of excitatory neurotransmitters in the retina, transporting nutrients and ions, mediating glycogen metabolism, and facilitating aerobic and anaerobic glycolysis. These cells play a crucial role in neuronal survival by providing trophic substances and precursors of neurotransmitters to neurons (4). Müller cells form the primary scaffolding of the retina, spanning the entire thickness of the retina, contacting and ensheathing every type of neuronal cell body and process in the retina. Virtually every disease of the retina is associated with a reactive Müller cell gliosis. Müller cells are the only glial cells in the outer half of the retina, but in the inner portions accessory glial cells are present. These include astrocytes, microglia, and perivascular glia. Müller cells, astrocytes, and perivascular glia are of glioblast origin deriving from the primitive neural tube, whereas microglia are derived from mesoderm. Frequently, studies that examine Müller cells will analyze astrocytes and microglia as well.

Evidence that Müller cells may be involved very early in the pathogenesis of diabetic retinopathy comes from studies by Li and co-workers (5), in which diabetic rats were monitored for changes in retinal function using electroretinograms (ERGs). The b-wave activity that originates from Müller cells is altered as early as 2 wk postonset of diabetes. These observations preceded changes in expression of glial fibrillary acid protein (GFAP), which is associated with severe Müller cell stress. Others have reported structural gliosis as early as 4 wk postonset in the diabetic rat model (6). The expression of GFAP in the diabetic rat model has been reported by several laboratories to be increased by about 3 mo of diabetes. Interesting metabolic studies from Lieth and colleagues reported the increased glial reactivity as well as impaired glutamate metabolism in diabetic rats (7). They observed a marked reduction in the capacity of Müller cells to convert glutamate to glutamine in diabetes and hypothesized that altered glutamate metabolism could lead to elevated glutamate during diabetes. Elevated glutamate levels have been reported in retinas of diabetic rat models (8) and in the vitreous of patients with diabeties (9). Significantly, changes in the b-wave have been reported in studies of human patients with diabetic retinopathy as well as elevation of GFAP (10).

The altered levels of glutamate observed in retinas of diabetic humans and rats caused investigators from the Puro laboratory to analyze the function of the sodiumdependent glutamate transporter in Müller cells of diabetic rats (11). Müller cells were freshly isolated from normal and diabetic rat retinas and Müller cell sodium-dependent glutamate transporter activity was monitored using a perforated-patch-clamp technique. As early as 4 wk postonset of diabetes, significant dysfunction of the Müller cell glutamate transporter was observed and by 3 mo its activity was reduced by nearly 70%. Our laboratory has used an in vitro system to study the effects of hyperglycemia on the uptake of radiolabeled glutamate in primary Müller cells isolated from mice. Interestingly, short periods of hyperglycemia alone do not seem sufficient to alter glutamate uptake (12); however, experiments are underway to dissect the myriad factors associated with diabetes

that can compromise the function of the EAAT1 (GLAST) transporter in these cells as well as the function of system xc, the sodium-independent glutamate/cystine exchanger. Altered function of glutamate transporters has implications for neuronal toxicity in the retina because of the possible accumulation of glutamate in the extracellular milieu. The intracellular glutamate concentration in neurons, which lack glutamine synthetase (the enzyme that converts glutamate to glutamine), is as high as 10 mM, whereas the extracellular glutamate concentration is in the micromolar range (13). Clearance of extracellular glutamate is a key function of Müller cells and alterations of this function could have significant implications on the function of neuronal cells in the retina.

There have been a number of studies that have evaluated oxidative stress, thought to be a key player in the pathogenesis of diabetic retinopathy (14) on Müller cell function (15–17). The development of a rat Müller cell line (rMC-1) by Sarthy and colleagues (18) has benefited the field of retinal research considerably, particularly studies of diabetic retinopathy. Culturing the rMC-1 under hyperglycemic conditions led to increased production of nitric oxide (NO), prostaglandin E(2) (PGE[2]), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (15). Human subjects with diabetes have also demonstrated increased expression of iNOS (16). Interestingly, the immunoreactivity was associated with Müller cells in retinas of these subjects, suggesting that high levels of NO produced by neural NOS could contribute to neurotoxicity and angiogenesis that occur in diabetic retinopathy. Studies from the Kern laboratory have shown that culturing Müller cells in hyperglycemia leads to increased production of superoxide as well (17).

In addition to elevated levels of glutamate and NO in retinas of patients with diabetes and subsequent effects on Müller cells, Inokuchi and colleagues reported that insulin-like growth factor (IGF)-1 levels were elevated significantly in vitreous obtained from patients with diabetes and suggested that at least one source of IGF-1 was the Müller cell (19). Similar observations have been reported independently from other laboratories (20). The observation is intriguing in light of a 2004 report from Ruberte and co-workers showing that increasing the ocular levels of IGF-1 in a transgenic mouse model leads to diabetes-like alteration in the eye including thickening of the basement membrane of capillaries, microvascular abnormalities, neovascularization, increased GFAP expression, and cataract (21). IGF has been shown to generate tractional forces by Müller cells. Recent studies from the Guidry laboratory (22,23) have focused on the role of Müller cells in proliferative diabetic retinopathy (PDR), which is an end-stage complication of diabetes. In PDR, fibrovascular tissues grow into the vitreous and tractional forces originating within these tissues threaten retinal anatomy. Guidry and co-workers (24) have data that suggest that Müller cells play a key role in fibrocontractive retinal disorders and that they function as an effector cell type in traction retinal detachment associated with PDR. The group has exploited the ability to isolate pure cultures of Müller cells permitting characterization of altered cell function and changes in proteins. They show that Müller cells have remarkable capacity to alter their shape becoming polygonal and fibroblast-like over a period of weeks and that the myofibroblastic Müller cell phenotype observed in culture is in fact present in human fibrocontractive disorders. The relevance of their findings to human patients with diabetes was demonstrated recently when vitreous samples of

patients with diabetes had considerably greater activity in a Müller cell tractional force generation bioassay than patients without diabetes (24).

Given the abundance of data implicating Müller cells in the pathogenesis of diabetic retinopathy, Gerhardinger and co-workers have initiated a systematic assessment of alterations in gene expression in Müller cells as a consequence of diabetes (25). Their work has used the streptozotocin-induced diabetic rat model and focused on changes that occurred 6 mo postonset of diabetes. Their gene expression profile studies identified 78 genes that were differentially expressed in Müller cells isolated from diabetic rats. Notable among these were acute-phase response proteins including α2-macroglobulin, ceruloplasmin, complement components, lipocalin-2, metallothionein, serine protease inhibitor-2, transferrin, tissue inhibitor of metalloproteases-1, transthyretin, and the transcription factor C/EBPδ. The acute-phase response of Müller cells in diabetes was associated with upregulation of interleukin (IL)-1β in the retina suggesting this cytokine as a mediator of the acute-phase response.

Regarding the alterations of microglia in diabetic retinopathy, there have been a few reports in the literature. Rungger-Brandle et al. (6) studied diabetic rats from 2 wk postonset of diabetes through 20 wk. They reported not only a significant increase in Müller cells with 4 wk of diabetes, but also an increase in microglia. Interestingly, the number of astrocytes was significantly reduced. Thus, microglial activation with astrocytes regression was an early event in diabetes, which the authors believe may contribute to the onset and development of neuropathy in the diabetic retina. A study by Kuiper and colleagues (26) described differential expression of connective tissue grown factor (CTGF) in microglia and pericytes in humans with diabetic retinopathy. CTGF stimulates extracellular matrix formation, fibrosis, and angiogenesis. Immunohistochemical analysis of CTGF expression patterns in human control and diabetic retinas revealed distinct and specific staining of CTGF in microglia of control retinas and a shift to microvascular pericytes in retinas from human subjects with diabetes.

In summary, Müller cells show changes in function early in experimental diabetes as evidenced by alterations in the ERG, altered capacity to transport glutamate, increased oxidative stressors including NO and superoxide, production of IGF-1, and involvement in the tractional forces involved in PDR. A few additional reports have noted changes in microglial and astrocytic cells during diabetes as well.

NEURONAL CELL LOSS IN DIABETIC RETINOPATHY

The involvement of retinal neurons in diabetic retinopathy has gained considerable attention over the past several years, although the first studies documenting the histological loss of neurons in retinas of patients with diabetic retinopathy were actually published in the early 1960s (27,28). The histological observations of neuronal loss have been corroborated by more recent electrophysiological studies of human patients with diabetes in which loss of color and contrast sensitivity was observed within 2 yr of diabetes onset (29,30). Studies using focal ERGs, which detect electrical responses from ganglion and amacrine cells, have revealed dysfunction of these cells early in diabetes (31–33). In the late 1990s Barber and colleagues published important observations of apoptotic neurons in retinas of patients with diabetes compared to controls (43). Similar results were observed by Bek et al. (35) and by Kerrigan et al. (36).

GANGLION CELLS

Ganglion cells are among the retinal neurons most studied in diabetic retinopathy. Recently, a comprehensive immunohistochemical analysis of markers of apoptosis was undertaken in retinas of human subjects with diabetes mellitus and compared to retinas of nondiabetic subjects (37). Antibodies against GFAP, caspase-3, Fas, Fas ligand (FasL), Bax, Bcl-2, survivin, p53, extracellular signal-regulated kinases (ERK1/2), and p38 were used in the study. All diabetic retinas showed cytoplasmic immunoreactivity for caspase-3, Fas, and Bax in ganglion cells. The authors concluded that ganglion cells in diabetic retinas express several proapoptosis molecules, suggesting that these cells are the most vulnerable population of cell types in the diabetic retina for apoptosis. Interestingly, they found that glial cells in diabetic retinas are activated and express several antiapoptosis molecules, but also the cytotoxic effector molecule FasL, suggesting a possible role of glial cells in induction of apoptosis in ganglion cells. Given that ganglion cell axons form the nerve fiber layer of the retina, it is perhaps not surprising that a reduction in the retinal nerve fiber layer has been reported in human patients with poorly controlled diabetes (38,39).

The studies of Barber and colleagues (34) reported not only ganglion cell apoptosis in retinas of humans with diabetes, but also a 10% reduction in cells of the ganglion cell layer in streptozotocin-induced diabetic rats. They also reported marked reductions in the thickness of the inner plexiform layers (IPLs) and inner nuclear layers (INLs) and a 10-fold increase in the numbers of apoptotic nonvascular cells. The data lend strong support to the notion that diabetic retinopathy has a significant neurodegenerative component. Interestingly, treating the rats with insulin largely prevented this neuronal cell death (34). Others using the streptozotocin rat model of diabetes have confirmed the loss of ganglion cells (40–47). Some of these studies have demonstrated a loss of the ganglion cell axonal fibers in diabetic rat retinas (41,43,44). The studies by Zhang were particularly interesting because they showed impairment of retrograde axonal transport especially in type 1 diabetes, with less impairment in type 2 diabetes (41). The authors speculated that impaired retrograde transport may precede or be a consequence of metabolic dysfunction of the large and medium-sized ganglion cells eventually leading to optic nerve atrophy. Additional electrophysiological experiments in diabetic rats detected reduced ERG responses as early as 2 wk postonset of diabetes (48).

The observations that ganglion cells die in diabetic retinopathy are not restricted to humans and to rats. Mice have been used also in studies of diabetic retinopathy and it is clear that they develop features of diabetic retinopathy. Kern and co-workers have described vascular changes in galactose-induced and streptozotocin-induced mouse models of diabetes (49). Hammes and colleagues have confirmed vascular changes in diabetic mice (50). Mohr et al. reported increased levels of caspase activation, a marker of apoptosis in retinas of diabetic mice (51). Recently, we performed a comprehensive analysis of apoptosis in the retina of streptozotocin-induced diabetic mice (52). The mice were made diabetic at 3 wk and studied over the subsequent 14 wk of diabetes. They were not maintained on insulin. The eyes were subjected to morphometric analysis and detection of apoptotic cells by TUNEL analysis, activated caspase-3 and electron microscopic analysis of the ultrastructural features of apoptosis. The morphometric analysis of retinal cross sections of mice that had been diabetic 14 wk showed approx