RAGE ligand upregulation of VEGF secretion in ARPE-19 cells

PURPOSE: The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGEs]) by its ligands, including AGEs, amyloid-beta peptide (Abeta), and S100B/calgranulins, some of which are known components of drusen and Bruch's membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-kappaB activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-kappaB inhibition was assessed by using parthenolide. RESULTS: ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased VEGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh Abeta, and S100B were less effective in increasing VEGF secretion. Studies with Abeta demonstrated that oligomeric and surface-immobilized forms of Abeta, but not soluble monomeric forms of Abeta, were effective upregulators of VEGF secretion via RAGE. Oxidation of S100B's thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-kappaB, as indicated by studies with parthenolide. CONCLUSIONS: Immobilized or oligomerized ligands for RAGE induce RPE cells to increase VEGF secretion. NF-kappaB plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD, activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.     

The upregulation of angiogenic gene expression in cultured retinal pigment epithelial cells grown on type I collagen

PURPOSE: Increases in matrix proteins, such as type I collagen and fibronectin, are observed with aging in the retinal pigment epithelium (RPE) basement membrane. However, little is known about altered gene expression profiles of RPE associated with increases in matrix proteins. We investigated changes in gene expression profiles of a human RPE cell line (ARPE-19) cultured on type I collagen. METHODS: Visually confluent ARPE-19 cells were grown on either Matrigel (M group) or type I collagen (C group) without serum over 3 days. Total RNA was extracted and reverse transcribed. Gene expression profiles in both groups were compared using microarray analyses. Several angiogenic genes including integrin alpha V, integrin alpha 2, integrin beta 1, integrin beta 3, VEGF-A, VEGF-B, and VEGF-C were subjected to quantitative analyses using real-time PCR. RESULTS: Out of 192 genes examined, angiogenesis-related genes (17.7%) and extracellular matrix-related genes (30.2%) were expressed highly (with more than 1.5-fold difference) in the C group when compared with the M group. In real-time PCR analyses, all VEGF and integrin family genes examined were expressed more in the C group than in the M group. CONCLUSIONS: Type I collagen likely causes an upregulation in a number of angiogenic gene expression patterns as seen in RPE in vitro experiments.     

Differential toxic effect of dissolved triamcinolone and its crystalline deposits on cultured human retinal pigment epithelium (ARPE19) cells

The aim of the study was to evaluate the antiproliferative and cytotoxic properties of triamcinolone acetonide (TA) on human retinal pigment epithelium cells (ARPE19) and the role of epicellular crystalline deposits. Monolayer cultures of ARPE19 cells were used. Purified or unpurified crystalline TA suspension (0.01-1.0 mg/ml) or the vehicle alone (benzyl alcohol, 0.025%-0.00025%), diluted in culture medium, were added to the cells that were either grown on cell culture dishes covered by a protecting membrane filter insert or without a filter. After 1, 3, 5 and 7 days mitochondrial activity was measured using the MTT assay and the morphology assessed microscopically. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays ARPE19 cells were grown to confluence and then cultured in a serum-deficient medium to ensure a static milieu. Annexin V-FITC and propidium iodide co-staining was performed and analyzed by flow cytometry. Exposure to TA without direct cellular contact showed a moderate antiproliferative activity resulting in a dose-dependent suppression of DNA synthesis (maximum 42.7%), but not a cytotoxic effect. In contrast, adherent deposits of crystalline TA particles on top of the cell layer caused a rapid-progressive and dose-dependent cell death preceded by an early phosphatidylserine externalization to the outer leaflet of the plasma membrane. Within a healthy, confluent cell layer the number of viable cells decreased by 14.2, 20.8 and 68.8%, respectively, after one day of direct exposure. Exposure to the vehicle alone caused only a slight growth inhibitory effect in a proliferating cell layer, but early signs of cell death were detected even at the lowest concentration tested. In conclusion, the effect of the vehicle is less pronounced than formerly assumed, but not negligible, thus indicating a beneficial effect of purification. While non-adherent TA, if purified, appears to be safe in clinically used concentrations, direct physical contact with crystalline particles might cause a local, rapid-progressive cytotoxicity that involves the induction of the apoptotic cascade. Therefore, epiretinal deposits after intravitreal TA administration might be critical in terms of long-term biocompatibility.     

Induction of interleukin-8 gene expression and protein secretion by C-reactive protein in ARPE-19 cells

C-reactive protein (CRP) is an acute phase reactant and its level rises rapidly during inflammation. Recent studies have suggested the potential involvement of CRP in the pathogenesis of age-related macular degeneration (AMD). To delineate the functional roles of CRP in inflammatory response by the ocular posterior segments, the effects of CRP on ARPE-19, an immortalized human retinal pigment epithelia (hRPE) cell line, were investigated in the present study. Treatment of ARPE-19 cells with CRP resulted in enhanced NF-kB nuclear translocation and dose-dependent transient induction of IL-8 mRNA synthesis and protein secretion. Stimulated expression of VEGF, but not MCP-1 by CRP was also observed. The induced IL-8 expression was transient and peaked at 12 h post stimulation. In the presence of inhibitors for NF-kB, p38, MEK and JNK, the CRP-induced IL-8 production was abolished by 99.5 ± 2.3, 97.8 ± 2.1, 55.3 ± 2.5 and 37.3 ± 1.3%, respectively. Neutralization of Fc gamma receptors by anti-CD32 and CD64 antibodies produced 39.9 ± 1.6 and 59.5 ± 2.6% reduction, respectively, of CRP-stimulated IL-8 secretion, whereas that by anti-CD16 antibody had no effect. This study suggests that the pro-inflammatory effects of CRP in ARPE-19 cells may contribute to the inflammatory retinal diseases by induction of pro-inflammatory cytokines such as IL-8. This induction is mediated by NF-kB and multiple MAPK pathways through Fc gamma receptors.     

Regulation of connective tissue growth factor gene expression in retinal vascular endothelial cells by angiogenic growth factors

Background: Connective tissue growth factor (CTGF) is a novel, cysteine-rich secreted protein, which is implicated in fibrotic disorders and atherosclerosis. To elucidate the role of CTGF in fibrovascular proliferative retinopathy, we investigated the regulation of CTGF gene expression in a cell line of retinal vascular endothelial cells (RVEC) stimulated with fetal calf serum (FCS) and angiogenic growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF-BB), endothelial growth factor (EGF), transforming growth factor-β1 and -β3 (TGF-β1, TGF-β3), and insulin-like growth factor-I (IGF-I). Methods: RVEC derived from Macaca mulatta (CRL-1780; ATCC) were stimulated with 10% FCS as well as with VEGF, bFGF, PDGF-BB, TGF-β1, TGF-β3, EGF, or IGF-I. Time-dependent CTGF gene expression was assessed by northern blot analysis. Results: FCS, TGF-β1, TGF-β3, bFGF, and EGF induced an upregulation of CTGF gene expression in RVEC in a time-dependent manner. Highest expression was induced with TGF-β1. No response on CTGF gene ex- pression could be detected to VEGF, PDGF-BB, or IGF-I. Conclusion: The present study demonstrates for the first time that CTGF mRNA is expressed at high levels in RVEC, and that the level of the temporal pattern of its expression is differentially regulated by angiogenic growth factors, indicating a significant role of CTGF in the pathological course of uncontrolled retinal angiogenesis. Received: 3 November 1998 Revised: 23 May 2000 Accepted: 26 June 2000     

Induction of angiogenic cytokine expression in cultured RPE by ingestion of oxidized photoreceptor outer segments

PURPOSE: Normal aging is associated with accumulation of lipofuscin pigment in the retinal pigment epithelium (RPE). This may occur as a result of phagocytosis and incomplete degradation of oxidized photoreceptor outer segments (POS). This study was undertaken to determine whether phagocytosis of UV-irradiated POS (artificial lipofuscin) would increase expression in the RPE of various chemotactic and angiogenic cytokines. METHODS: ARPE-19 cells were exposed to latex beads (0.76 micro m), na?ve bovine POS, and UV-irradiated POS (Ox-POS; 2 x 10(7)/mL), and supernatants were collected at 18 and 36 hours. The supernatants were assayed for IL-8, monocyte chemotactic protein-(MCP)-1, and TNF-alpha by ELISA. Protein synthesis and NFkappaB activity were inhibited by actinomycin D and SN50, respectively. Phagocytosis and generation of intracellular reactive oxygen species were assessed by flow cytometry. Confocal and electron microscopy studies were also performed to verify phagocytosis and cellular integrity. RESULTS: IL-8 and MCP-1 levels were decreased in the na?ve POS group (IL-8: 473.76 +/- 66.9 pg/mL, P = 0.0005; MCP-1: 550.1 +/- 21.8 pg/mL, P = 0.0001), but were increased in the Ox-POS group (IL-8: 1348.8 +/- 164.9 pg/mL; MCP-1: 1772.28 +/- 65.19 pg/mL) compared with the control (IL-8: 741.09 +/- 39.8 pg/mL; MCP-1: 1413.47 +/- 38.4 pg/mL) and latex bead groups (data not shown). TNF-alpha levels were not affected. At 12 hours (but not at 6 hours), ROS were increased in the Ox-POS group. The cytokine increases observed were dependent on de novo protein synthesis and were NF-kappaB dependent. CONCLUSIONS: Ingestion by RPE of oxidized bovine POS stimulates expression of the chemotactic and angiogenic factors IL-8 and MCP-1 that have the capability to promote angiogenesis directly, or indirectly through the accumulation of immune cells such as macrophages, which themselves may release angiogenic promoters and degrade Bruch's membrane. This may be of significance in the development of exudative AMD.     

The toxic and stress responses of cultured human retinal pigment epithelium (ARPE19) and human glial cells (SVG) in the presence of triamcinolone

PURPOSE: To compare the cytotoxic effect of TA on human retinal pigment epithelium (ARPE19) and human glial (SVG) cells over a range of concentrations and durations of exposure. METHODS: TA (0.01-1 mg/mL) or vehicle (benzyl alcohol, 0.025%) was added to the ARPE19 and SVG cultures on day 0 and then subsequently for 1, 3, or 5 days. The amount of cell proliferations with or without TA treatment was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All samples were read in triplicate (n = 4 in all cases). c-Fos, c-jun, caspase-3, c-myc, and p53 expression was determined after TA treatments after 0, 10, 20, 30, 40, 50, 60, and 90 minutes. All results were analyzed with ANOVA. RESULTS: TA (0.01-1 mg/mL) caused a significant reduction in ARPE19 cells that had been exposed to it for more than 1 day. Significant reductions in the number of SVG cells were observed as early as day 1 at 0.1 and 1 mg/mL TA. In general, the level of remaining SVG cells was less than that of the APRE19 cells over the 5 days. SVG cells appeared more susceptible to TA. Caspase-3 was elevated in both ARPE19 and SVG cells after TA treatment. c-Fos and c-jun expression was also increased in ARPE19 cells but not in SVG. The vehicle of TA had no effect, and there was no change in p53 or c-myc expression. CONCLUSIONS: TA was cytotoxic to both SVG and ARPE19 cells, with higher efficacy on SVG. TA caused the activation of the caspase-3 pathway more readily than the cell-protective c-fos and c-jun pathways in SVG cells, making those cells more vulnerable than the ARPE19 cells. The results suggest that TA toxicity in one cell type may not reliably indicate its toxicity in other cells. Different cells within the retina may react to TA differently, or TA may cause changes in the gene expressions differentially with different concentrations of the same stimulus.     

Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1beta in cultured lacrimal gland acinar cells

PURPOSE: Inflammation of the lacrimal gland is one of the major causative factors in aqueous tear-deficient dry eye syndrome. Pro-inflammatory cytokine production is upregulated in lacrimal gland autoimmune disease (i.e. Sj?gren's syndrome) and is associated with cell death. The expression of inducible nitric oxide synthase (iNOS/NOS-2) is known to be induced in the presence of pro-inflammatory cytokines in several secretory epithelial cell types. We hypothesize that pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta), cause a marked increase in nitric oxide (NO) production via induction of iNOS in lacrimal gland epithelial cells and that this may be a significant pathophysiological pathway of dry eye syndrome. METHODS: Cultured immortalized rabbit lacrimal gland acinar cells were incubated with IL-1beta, iNOS inhibitor, or IL-1 receptor antagonist (IL-1ra). Colorimetric detection of NO(2)(-) and NO(3)(-) in the media, measured by the Griess reaction, was used as an index of NO production. Expression of iNOS was determined by SDS-PAGE and Western blot. RESULTS: IL-1beta stimulated a concentration-dependent and time-dependent increase in NO production. IL-1beta-induced NO production was significantly antagonized by co-incubation with IL-1ra or the iNOS-specific inhibitor, 1400W. Expression of iNOS protein was greatest at 4hr after addition of IL-1beta, and was nearly undetectable at 12hr. IL-1ra greatly reduced IL-1beta-induced iNOS expression. CONCLUSIONS: Lacrimal gland acinar cells are able to produce iNOS in response to the pro-inflammatory cytokine IL-1beta. The amount of iNOS expressed and the subsequent levels of NO that are produced by lacrimal cells are far lower than those seen in macrophages, but are consistent with those reported for other cell types in the literature. This pathway of iNOS induction and overproduction of NO may be a factor in lacrimal gland cell death in dry eye syndrome. Inhibitors of iNOS or IL-1 receptor may be beneficial for controlling lacrimal gland inflammation.     

Expression of angiogenic and fibrogenic factors in proliferative vitreoretinal disorders

Purpose To investigate the expression of connective tissue growth factor (CTGF) in the retina of human subjects with diabetes mellitus, and CTGF, CD105, and gelatinase B in proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR) epiretinal membranes. Methods Twelve donor eyes from six subjects with diabetes mellitus, 10 eyes from five nondiabetic subjects, 14 PDR membranes, and five PVR membranes were studied by immunohistochemical techniques. In situ zymography was used to examine gelatinolytic activity in four PDR membranes. Results In nondiabetic retinas, there was no immunoreactivity for CTGF. Diabetic retinas showed immunoreactivity for CTGF in ganglion cells and microglia. Vascular endothelial cells in PDR membranes expressed CTGF, CD105, and gelatinase B in 10 (71.4%), 11 (78.6%), and 5 (35.7%) membranes, respectively. Myofibroblasts in PDR membranes expressed CTGF, and gelatinase B in 14 (100%), and 6 (42.9%) membranes, respectively. There was a significant correlation between the number of blood vessels expressing the panendothelial marker CD34 and the number of blood vessels expressing CTGF (r = 0.7884; P = 0.0008), and CD105 (r = 0.6901; P = 0.0063), and the number of myofibroblasts expressing CTGF (r = 0.5922; P = 0.0257). There was a significant correlation between the number of myofibroblasts expressing α-smooth muscle actin and the number of myofibroblaasts expressing CTGF (r = 0.8393; P = 0.0002). In situ zymography showed the presence of gelatinolytic activity in vascular endothelial cells in PDR membranes. Myofibroblasts in PVR membranes expressed CTGF, and gelatinase B. Conclusions These results suggest a possible role of CTGF, CD105, and gelatinase B in the pathogenesis of proliferative vitreoretinal disorders.     

Arsenic trioxide initiates ER stress responses,perturbs calcium signalling and promotes apoptosis in human lens epithelial cells

Identification of novel agents to eradicate the residual lens cell population following cataract surgery provides one mode of preventing PCO formation. The present study investigated the biological mechanism of As₂O₃ cytotoxicity in a human lens cell line and capsular bag system. FHL 124 cell survival was assessed by quantification of total protein content, a cell population measure. Gene changes were detected by Real-time PCR; apoptosis by TUNEL assays. Intracellular calcium was measured by real-time fluorimetric single-cell digital imaging techniques after Fura-2 incorporation. In vitro human capsular bags were generated from donor eyes, which involved sham cataract surgery then use of the Perfect Capsule device to form a closed system to deliver As₂O₃ for 2 min. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence immunocytochemistry. FHL 124 cells demonstrated a dose-dependent sensitivity to As₂O₃ exposure. A 2 min exposure of As₂O₃ to cells within the capsular bag, using the perfect capsule system, resulted in total cell death when used at 100 mM. As₂O₃ provoked an ER stress response identified through an upregulation of known genes. As₂O₃ depleted the calcium store and consequently lead to reduced calcium signalling. As₂O₃ increased rates of apoptosis. Arsenic trioxide provokes ER stress that leads to down-regulation of calcium signalling resulting in apoptosis. The application of As₂O₃ to cells within the capsular bag for a 2 min window using the Perfect Capsule system predicts putative therapeutic benefit in vivo.     

Arsenic trioxide initiates ER stress responses, perturbs calcium signalling and promotes apoptosis in human lens epithelial cells

Identification of novel agents to eradicate the residual lens cell population following cataract surgery provides one mode of preventing PCO formation. The present study investigated the biological mechanism of As(2)O(3) cytotoxicity in a human lens cell line and capsular bag system. FHL 124 cell survival was assessed by quantification of total protein content, a cell population measure. Gene changes were detected by Real-time PCR; apoptosis by TUNEL assays. Intracellular calcium was measured by real-time fluorimetric single-cell digital imaging techniques after Fura-2 incorporation. In vitro human capsular bags were generated from donor eyes, which involved sham cataract surgery then use of the Perfect Capsule device to form a closed system to deliver As(2)O(3) for 2min. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence immunocytochemistry. FHL 124 cells demonstrated a dose-dependent sensitivity to As(2)O(3) exposure. A 2min exposure of As(2)O(3) to cells within the capsular bag, using the perfect capsule system, resulted in total cell death when used at 100mM. As(2)O(3) provoked an ER stress response identified through an upregulation of known genes. As(2)O(3) depleted the calcium store and consequently lead to reduced calcium signalling. As(2)O(3) increased rates of apoptosis. Arsenic trioxide provokes ER stress that leads to down-regulation of calcium signalling resulting in apoptosis. The application of As(2)O(3) to cells within the capsular bag for a 2min window using the Perfect Capsule system predicts putative therapeutic benefit in vivo.     

Inhibition of calcium of beta adrenoceptor mediated cAMP responses in isolated bovine corneal epithelial cells

The increases in adenosine 3',5' monophosphate (cAMP) content were measured in isolated bovine corneal epithelial cells in response to either adrenergic agonists or adenylate cyclase stimulation. The beta selective adrenergic agonist, isoproterenol, and the adrenergic agonists, norepinephrine as well as epinephrine elicited large increases in cAMP accumulation. At their maximum effective concentrations, the respective increases were 16-fold, 6.6-fold and 4.7-fold. These stimulatory effects were completely inhibited by the beta selective adrenergic antagonist, propranolol. Similarly forskolin increased cAMP content more than 3-fold. These increases and the previous identification of beta adrenoceptors in fresh intact bovine corneas as well as cells in culture indicate that this enzymatic dissociation procedure does not affect the cAMP responses to either adrenergic agonists or forskolin. The relationship was considered between increases in Ca2+ concentration and the effects of either isoproterenol or forskolin, on cAMP accumulation. There were no changes in any of the cAMP responses at bathing solution Ca2+ concentrations between 0.01 microM and 1 microM. However, in cells permeabilized to Ca2+ with 10 microM ionomycin, increases within this concentration range depressed the baseline levels of cAMP content. Furthermore, the stimulatory effects of both forskolin and isoproterenol on cAMP accumulation were significantly blunted in this concentration range. These blunting effects by Ca2+ were not the result of any measurable decrease in ATP content. This negative relationship between increases in Ca2+ concentration and increases in cAMP content indicates that changes in intracellular Ca2+ concentration could modulate the second messenger function of cAMP linked to these agents.     

Induction of apoptosis by the calcium antagonist mibefradil correlates with depolarization of the membrane potential and decreased integrin expression in human lens epithelial cells

Background Posterior capsule opacification is still the major complication in cataract surgery and is caused by migration and proliferation of residual lens epithelial cells. The challenge of a suitable therapy to inhibit capsule opacification is to specifically interfere with cellular mechanisms. Our approach using the T-calcium channel antagonist mibefradil is based on the hypothesis that this drug inhibits the signaling pathways mediated by cell adhesion.Methods The influence of mibefradil dihydrochloride was investigated on primary human lens epithelial cells (hLEC) from cataract surgery and on the human lens cell line HLE-B3. Apoptosis was quantitatively analyzed by flow cytometry (% increase of the sub-G1 peak), and verified by confocal microscopy (annexin V-biotin, TUNEL reaction). The membrane potential was detected by a membrane potential-sensitive dye. Integrin expression and proliferation were measured by flow cytometry. T-calcium channels in hLEC were verified by the whole-cell configuration of the patch-clamp technique.Results Mibefradil induced apoptosis in hLEC. Early signs of apoptosis were observed after only 4 h of incubation with mibefradil, accompanied by a significantly reduced cell area. Apoptosis correlated with inhibited integrin expression, reduced proliferation and the depolarization of the membrane potential. We could identify calcium channels of the T-type in our primary hLEC.Conclusions We suggest that depolarization of the membrane potential and the inhibition of integrin expression leads to the loss of cell adhesion, which is the reason for the induction of apoptosis. Thus, mibefradil seems to be a suitable drug to prevent cell adhesion, migration and proliferation.     

GABA and choline accumulation by cultures of chick embryo neuroretinal cells

GABA and choline accumulation has been investigated in 8-day cultures of 10-day chick embryo neuroretinal cells. Accumulation of (³H)-labelled GABA or choline (1 μm) was linear for about 5 min under most conditions. Kinetic analysis suggested that high affinity uptake systems exist for both GABA and choline, with apparent K_m values of 0·5–1·0 μm for GABA and 0·2–0·4 μm for choline. Accumulation of GABA was strongly temperature-sensitive and sodium-dependent, and was largely eliminated by 50 μm-nipecotic acid, though not by the GABA analogues β-alanine or l-2,4-diaminobutyric acid at concentrations up to 5 mm. Choline accumulation was also temperature-sensitive, but was only about 40% inhibited by sodium-free medium or by 20 μm-hemicholinium III.Comparison of standard cultures (both neurones and glia present) with both glial-enriched cultures and neurone-enriched cultures revealed the following contrasts. GABA accumulation was much greater in neurone-enriehed than in standard cultures, and was very low in glial-enriched cultures. Choline accumulation, however, was lower in neurone-enriched than in standard cultures, and in glial-enriched cultures reached higher levels than did GABA uptake. These results suggest a predominantly neuronal uptake system for GABA, while choline accumulation seems less specific and may include a large glial component (some choline perhaps being used for membrane biosynthesis). These interpretations were confirmed by autoradiographic studies. (³H)-GABA labelled many (but not all) neuronal cell bodies and processes in standard and especially in neurone-enriched cultures. A few glial cells were also labelled in standard cultures, and all labelling was abolished by Na⁺-free medium. (³H)-Choline, by contrast, labelled only a small proportion of neuronal cells prominently, and most silver grains were widely distributed over the background of glial cells.