The retinal pigment epithelium in health and disease.

Retinal pigment epithelial cells (RPE) constitute a simple layer of cuboidal cells that are strategically situated behind the photoreceptor (PR) cells. The inconspicuousness of this monolayer contrasts sharply with its importance [1]. The relationship between the RPE and PR cells is crucial to sight; this is evident from basic and clinical studies demonstrating that primary dysfunctioning of the RPE can result in visual cell death and blindness. RPE cells carry out many functions including the conversion and storage of retinoid, the phagocytosis of shed PR outer segment membrane, the absorption of scattered light, ion and fluid transport and RPE-PR apposition. The magnitude of the demands imposed on this single layer of cells in order to execute these tasks, will become apparent to the reader of this review as will the number of clinical disorders that take origin from these cells.

Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy.

Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4-/- mice were injected subretinally with 1 microl ( approximately 5.0 x 10(5) TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4-/- mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4-/- mice accumulated 8-12 pmol per eye (s.d.=2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3-5 times more A2E (27-39 pmol per eye, s.d.=1.5; P=0.001-0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.

A method of drusen measurement based on reconstruction of fundus background reflectance.

BACKGROUND: The hallmarks of age related macular degeneration (AMD) are the subretinal deposits known as drusen. Current manual methods of drusen segmentation and quantification are laborious and subjective. The authors introduced a digital method and tested it for accuracy and reliability. METHODS: Fourteen eyes with drusen were selected. The authors digitally reconstructed the macular background using normal background areas ("dots") fitted to quadratic polynomials in two zones. The model was used to level the reflectance for the purpose of segmenting drusen by a global threshold. Measurements of drusen areas were compared with those of a semi-automated background levelling technique and manual drawings from stereo pairs. RESULTS: Intraobserver reproducibility had standard deviations from 0.1% to 4.1%. Interobserver reproducibility yielded 95% limits of agreement of -2.7% to 6.3%. The dots method compared with manual drawings and with the semi-automated method had 95% limits of agreement of -8.3% to 2.8% and -7.1% to 4.8%, respectively. CONCLUSIONS: The dots method was reproducible and accurate with respect to validated methods. It provided less total operating time and greater precision than that of standard fundus photo grading. With implementation of commercial software, this technique for macular image analysis has potential for use in clinical research.

How much blue light should an IOL transmit?

Older, and even some modern, intraocular lenses (IOLs) transmit potentially hazardous ultraviolet radiation (UVR) to the retina. In addition, IOLs transmit more blue and green light to the retina for scotopic vision than the crystalline lenses they replace, light that is also potentially hazardous. The severity of UVR-blue type phototoxicity increases with decreasing wavelength, unlike the action spectrum of blue-green type retinal phototoxicity and the luminous efficiency of scotopic vision which both peak in the blue-green part of the optical spectrum around 500 nm. Theoretically, UVR+blue absorbing IOLs provide better retinal protection but worse scotopic sensitivity than UVR-only absorbing IOLs, but further study is needed to test this analysis. UVR is potentially hazardous and not useful for vision, so it is prudent to protect the retina from it with chromophores in IOLs. Determining authoritatively how much blue light an optimal IOL should block requires definitive studies to determine (1) the action spectrum of the retinal phototoxicity potentially involved in human retinal ageing, and (2) the amount of shorter wavelength blue light required for older adults to perform essential activities in dimly lit environments.

Fluorescent pigments of the retinal pigment epithelium and age-related macular degeneration.

The major hydrophobic fluorophore of the retinal pigment epithelium (RPE) is A2E, a pyridinium bis-retinoid derived from all-trans-retinal and phosphatidyl-ethanolamine. The accumulation of fluorophores such as A2E is implicated in the pathogenesis of age-related macular degeneration (AMD), a disease associated with the deterioration of central vision and a leading cause of blindness in the elderly. Recent chemical and biological studies have provided insight into the synthesis and biosynthesis of A2E, the spectroscopic properties of this pigment, and the role of A2E and RPE cell death.

Blue light-induced apoptosis of A2E-containing RPE: involvement of caspase-3 and protection by Bcl-2.

PURPOSE: The lipofuscin fluorophore A2E has been shown to mediate blue light-induced damage to retinal pigment epithelial (RPE) cells. The purpose of this study was to evaluate caspase-3 and Bcl-2 as executor and modulator, respectively, of the cell death program that is initiated in A2E-containing cells in response to blue light. METHODS: Human RPE cells (ARPE-19) that had accumulated A2E were exposed to blue light. Caspase-3 activity was assayed by observing cleavage of a fluorogenic peptide substrate, and the effect of a peptide inhibitor of caspase-3 (Z-DEVD-fmk) on the quantity of apoptotic nuclei was determined. ARPE-19 cells were transfected with either a neomycin-selectable expression vector containing Bcl-2 cDNA or a control neomycin-selectable expression vector without Bcl-2 cDNA. Expression of Bcl-2 transcripts by independently derived clones was established by in situ hybridization, and Bcl-2 protein expression was confirmed by Western blot analysis. Cell viability was assayed by TdT-dUTP terminal nick-end labeling (TUNEL) in conjunction with 4'6'-diamidino-2-phenylindole (DAPI) staining and by fluorescence staining of the nuclei of membrane-compromised cells. RESULTS: In RPE cells that had previously accumulated A2E, caspase-3 activity was detected within 5 hours of blue light exposure. The incidence of apoptotic nuclei was attenuated when A2E-containing RPE cells were exposed to blue light in the presence of caspase-3 inhibitor and in A2E-loaded RPE cells that had been stably transfected with Bcl-2. CONCLUSIONS: Blue light illumination of RPE in the setting of intracellular A2E initiates a cell death program that is executed by a proteolytic caspase cascade and that is regulated by Bcl-2.

The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane.

The autofluorescent lipofuscin that accumulates in retinal pigment epithelial cells with age may contribute to an age-related decline in cell function. The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid. We previously proposed that the biogenesis of A2E involves the following: (i) formation of the Schiff base, N-retinylidene phosphatidylethanolamine from all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane; (ii) further reaction of N-retinylidene phosphatidylethanolamine with retinal to yield phosphatidylethanolamine-bisretinoid, A2-PE; (iii) hydrolysis of A2-PE to generate A2E. To provide evidence for this biogenic scheme, all-trans-retinal was reacted with dipalmitoyl-l-alpha-phosphatidylethanolamine to yield DP-A2-PE (A2-PE), as confirmed by UV, with mass spectrometry revealing the molecular ion at m/z 1222.9 (C(77)H(124)O(8)PN) accompanied by product ion at m/z 672.8, representing the phosphoryl-A2E fragment of A2-PE. In reaction mixtures of retinal and outer segments and in samples of Royal College of Surgeons rat retina containing outer segment membranous debris, A2-PE was detected as a series of high performance liquid chromatography peaks, each with UV similar to reference A2-PE. By mass spectrometry, A2-PE consisted of multiple peaks, representing fatty acids with different chain lengths, and the phosphoryl-A2E moiety, m/z 673. Incubation of the retinal/outer segment reaction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatography, thus confirming A2-PE as the A2E precursor.

The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.

PURPOSE: To determine whether the lipofuscin fluorophore A2E participates in blue light-induced damage to retinal pigmented epithelial (RPE) cells. METHODS: Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 microM concentrations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as assayed by quantitative high-performance liquid chromatography (HPLC). Restricted zones (0.5-mm diameter spots) of confluent cultures were subsequently exposed to 480 +/- 20-nm (blue) or 545 +/- 1-nm (green) light for 15 to 60 seconds. Phototoxicity was quantified at various periods after exposure by fluorescence staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure. RESULTS: Nonviable cells were located in blue light- exposed zones of A2E-containing RPE cells, whereas cells situated outside the illuminated areas remained viable. As shown by fluorescence labeling of the nuclei of membrane-damaged cells and by the presence of TUNEL-positive cells, the numbers of nonviable cells increased with exposure duration and as a function of the concentration of A2E used to load the cells before illumination. The numbers of blue light-induced TUNEL-positive cells also increased in advance of the increase in labeling of membrane-compromised cells, a finding that, together with Annexin V labeling, indicates an apoptotic form of cell death. Conversely, blue light- exposed RPE cells that did not contain A2E remained viable. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells. CONCLUSIONS: These studies implicate A2E as an initiator of blue light-induced apoptosis of RPE cells.

Human immunodeficiency virus type 1 can infect human retinal pigment epithelial cells in culture and alter the ability of the cells to phagocytose rod outer segment membranes.

Human immunodeficiency virus type 1 (HIV-1) has been found in the vitreous of persons with AIDS. Here we investigated the susceptibility of human retinal pigment epithelial (RPE) cells to HIV-1 infection in culture and the effects of HIV-1 on the phagocytic function of the RPE. We found that 10 of 11 populations of RPE cells isolated from different fetal or adult eyes were susceptible to low-level replication of HIV-1/NL4-3 as determined by the detection of viral DNA and spliced viral RNA encoding envelope. HIV-1 infection was not inhibited by recombinant soluble CD4, suggesting that CD4 is not required for virus entry into RPE cells. RPE cells fused with target cells constitutively expressing HIV-1 envelope glycoproteins, indicating that HIV-1 enters cells by receptor-mediated fusion. Exposure to HIV-1 or recombinant gp120 caused a two- to four-fold increase in the binding and uptake of isolated rod outer segments by RPE cells. These findings introduce a new cell target of HIV-1 replication in the eye and indicate that RPE cells function aberrantly when exposed to HIV-1 or its envelope glycoprotein.

Trk C signaling is required for retinal progenitor cell proliferation.

Although neurotrophin actions in the survival of specific retinal cell types have been identified, the biological functions for neurotrophin-3 (NT-3) in early retinal development remain unclear. Having localized NT-3 and trk C expression at early developmental stages when retinal neuroepithelial progenitor cells predominate, we sought to modulate NT-3 signaling in these cells by overexpressing a truncated isoform of the NT-3 receptor, trk C. We have demonstrated that this non-catalytic receptor can inhibit NT-3 signaling when coexpressed with the full-length kinase-active trk C receptor. Using a replication-deficient retrovirus to ectopically express the truncated trk C receptor to limited numbers of progenitor cells in ovo, we examined the effects of disrupted trk C signaling on the proliferation or differentiation of retinal cells. Clones expressing truncated trk C exhibited a 70% reduction in clone size, compared with clones infected with a control virus, indicating that inhibition of trk C signaling decreased the clonal expansion of cells derived from a single retinal progenitor cell. Additionally, impaired NT-3 signaling resulted in a reduction of all retinal cell types, suggesting that NT-3 targets retinal precursor cells rather than differentiated cell types. BrdU labeling studies performed at E6 indicate that this reduction in cell number occurs through a decrease in cell proliferation. These studies suggest that NT-3 is an important mitogen early in retinal development and serves to establish the size of the progenitor pool from which all future differentiated cells arise.

A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture.

PURPOSE: To study A2E, a component of retinal pigmented epithelial (RPE) cell lipofuscin, after its internalization by cultured human RPE cells. METHODS: A2E was synthesized and incubated with an adult RPE cell line devoid of native lipofuscin. To investigate the cellular compartmentalization of A2E, cells were incubated simultaneously with A2E and a fluorescent acidotropic probe, (Lysotracker Red DND-99; Molecular Probes, Eugene, OR). Plasma membrane integrity was evaluated by assaying for leakage of the cytoplasmic enzyme lactate dehydrogenase (LDH), by fluorescence nuclear staining with a membrane-impermeant dye and by morphologic criteria. The emission spectrum of internalized A2E was also determined. The levels of A2E accumulated by the cultured cells were quantified by high-performance liquid chromatography and compared with amounts present in RPE isolated from human eyes. RESULTS: Internalization of A2E by the RPE cells was evidenced by the acquisition of intracellular granules detectable by fluorescence confocal imaging. Internalized A2E had an emission maxima of 565 to 570 nm. The levels of A2E accumulating in cells incubated with 10 to 25 microM A2E were comparable to the amounts of A2E present in equal numbers of RPE cells harvested from human eyes. Colocalization of A2E and the Lysotracker probe revealed a preferential accumulation in acidic organelles. The elevated LDH levels that were measured after exposure to 50 and 100 microM A2E were attributable to membrane damage in a subpopulation of the A2E-accumulating cells, determined by fluorescence nuclear labeling. CONCLUSIONS: Internalized A2E has an affinity for acidic organelles. The membrane damage exhibited by A2E-accumulating RPE is dependent on the concentration of A2E and reflects the ability of this amphiphilic compound to exert detergent-like effects.

Immunohistochemical analysis of the neurotrophins BDNF and NT-3 and their receptors trk B, trk C, and p75 in the developing chick retina.

The neurotrophins are trophic and mitogenic factors critical for the development of specific classes of neurons in the central and peripheral nervous systems. In the retina, BDNF and NT-3 have been shown to promote the survival of differentiated ganglion cells (Rodriguez-Tebar et al., 1989; De La Rosa et al., 1994). NT-3 has also been demonstrated to support the survival of amacrine cells and facilitates the differentiation of retinal neurons in culture (De La Rosa et al., 1994). Here, we examine immunohistochemically the expression of BDNF and NT-3 proteins, their cognate receptors, trk B and trk C, respectively, and the p75 neurotrophin receptor in the developing chick retina. At E8, the earliest stage of retinal development examined, all of these proteins exhibit diffuse expression throughout the width of the retina, with the strongest reactivity in the innermost layers. A gradual restriction in expression to ganglion cells and amacrine cells, the staining of which is most prominent at E15, is followed by a downregulation of expression with the strongest immunoreactivity persisting in the ganglion cell layer. Overlapping patterns of expression throughout embryonic development indicate a colocalization of the neurotrophins and their receptors, although NT-3 and p75 alone are present in the inner plexiform layer and only p75 is observed in the outer plexiform layer. Although some of the immunoreactivity for BDNF, NT-3, and their receptors in retina may reflect trophic mechanisms operating in association with the optic tectum and isthmo-optic nucleus, the colocalization of ligands and receptors in retina strengthens the assertion that these neurotrophins function locally during development.

CD36 expression is altered in retinal pigment epithelial cells of the RCS rat.

The retinal pigment epithelial cell has several important functions, one of which is the phagocytosis of photoreceptor outer segments which are discarded diurnally. We previously provided evidence in human retinal pigment epithelium that CD36, an 88 kDa integral membrane glycoprotein, participates in the phagocytosis of photoreceptor outer segments. Since in the Royal College of Surgeons dystrophic rat, retinal pigment epithelial cells fail to perform this function and as a result the photoreceptor cells degenerate, the expression of CD36 has now been examined by retinal pigment epithelial cells of the dystrophic rat. Consistent with earlier work using human retinal pigment epithelial cells, expression of CD36 by freshly isolated retinal pigment epithelial cells of Long Evans rats was confirmed by immunoblotting and immunocytochemistry with antibody to rat CD36. The protein was also present in lysates of cultured retinal pigment epithelium. Furthermore, with an in vitro phagocytosis assay using 125I-labeled outer segments, it was demonstrated that the binding and ingestion of outer segments by rat retinal pigment epithelial cells was reduced by 64% in the presence of antibodies to rat CD36. In contrast to observations in the Long Evans rat, immunoblotting of retinal pigment epithelial cells isolated from the adult Royal College of Surgeons rat revealed that CD36 protein was not present. This appeared to be a tissue-specific absence since CD36 protein was present in peritoneal macrophages harvested from the adult Royal College of Surgeons rat. A developmental study of CD36 expression also demonstrated an absence of the protein on the day of birth and at 1 and 2 weeks postnatally. By reverse transcriptase-polymerase chain reaction, CD36 mRNA was detected in freshly harvested retinal pigment epithelial cells of the Royal College of Surgeons rat at only PN1, 1 week and 10 days. Significantly, at 2 weeks of age and in the adult Royal College of Surgeons rat. CD36 transcripts were no longer present. Nevertheless, by Northern blot analysis CD36 mRNA was detected in various other tissues shown previously to express CD36. We conclude that in RPE cells of the Royal College of Surgeons rat, CD36 protein is not expressed while CD36 mRNA is present only transiently during postnatal development.

Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36.

The specific recognition of negatively charged phospholipids in cell membranes has been suggested to play an important role in a variety of physiological and pathophysiological processes. Recent work (Rigotti, A., Acton, S. L., and Krieger, M. (1995) J. Biol. Chem. 270, 16221-16224) has described specific and tight binding of anionic phospholipids, such as phosphatidylserine (PS) and phosphatidylinositol (PI), to the class B scavenger receptors, CD36 and SR-B1. We have previously reported that CD36 is present on retinal pigment epithelium (RPE) and plays a role in the phagocytosis of photoreceptor outer segments (ROS), a function critical to the normal visual process (Ryeom, S. W., Sparrow, J. R., and Silverstein, R. L. (1996) J. Cell Sci. 109, 387-395). We now report that phospholipid liposomes PS and PI, but not phosphatidylethanolamine, bind specifically to RPE. Cross-competition experiments suggest that PS and PI recognize the same receptor on RPE, while immunoinhibition studies indicate that the receptor is CD36. RPE cells isolated from a mutant rat strain, the RPE of which does not express CD36 ( Sparrow, J. R., Ryeom, S. W. , Abumrad, N., Ibrahimi, A., and Silverstein, R. L. (1996) Exp. Eye Res., in press), did not bind PS or PI, further confirming the role of CD36. We also showed that purified ROS blocked binding and uptake of anionic phospholipid liposomes by RPE and that PS and PI liposomes blocked ROS uptake by RPE, suggesting that PS and PI on the ROS membrane may be the ligands on ROS recognized by CD36. This is the first demonstration that CD36-phospholipid interactions may play a role in normal physiology.

The stability of perfluoro-N-octane during vitreoretinal procedures.

OBJECTIVES: To examine the propensity for intraoperative procedures, such as endolaser, to generate polar impurities in perfluorocarbon liquids, either by degradation of the compound or by dissolution of materials contacting the liquid, given the value of these liquids as adjuncts to vitreoretinal procedures and the importance of using pure and inert liquid. METHODS: Perfluoro-N-octane liquid recovered from patients after vitreoretinal procedures was analyzed by gas chromatography, nuclear magnetic resonance, ultraviolet spectroscopy, and a cell proliferation assay. Similar analyses were performed on pure and impure perfluoro-N-octane exposed in vitro to superclinical energy levels of argon and YAG laser, endodiathermy, and endoillumination. RESULTS: No change in chemical structure and only minor (parts per million) increases in dissolved contaminants were observed. The perfluoro-N-octane liquid retained its inertness as indicated by the inability of fibroblasts to attach and proliferate on its surface. CONCLUSION: The structure and biologic inactivity of perfluoro-N-octane are unaffected by vitreoretinal surgical manipulations.

CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium.

Mechanisms of phagocytosis are complex and incompletely understood. The retinal pigment epithelium provides an ideal system to study the specific aspects of phagocytosis since an important function of this cell is the ingestion of packets of membranous discs that are normally discarded at the apical ends of rod and cone cells during outer segment renewal. Here we provide evidence that rod outer segment phagocytosis by retinal pigment epithelium is mediated by CD36, a transmembrane glycoprotein which has been previously characterized on hematopoietic cells as a receptor for apoptotic neutrophils and oxidized low density lipoprotein. Immunocytochemical staining with monoclonal and polyclonal antibodies demonstrated CD36 expression by both human and rat retinal pigment epithelium in transverse cryostat sections of normal retina and in primary cultured cells. By western blot analysis of retinal pigment epithelial cell lysates, polyclonal and monoclonal antibodies to CD36 recognized an 88 kDa protein which comigrated with platelet CD36. Furthermore, the synthesis of CD36 mRNA by retinal pigment epithelium was confirmed by reverse transcriptase-PCR using specific CD36 oligonucleotides. The addition of CD36 antibodies to cultured retinal pigment epithelial cells reduced the binding and internalization of 125I-labeled rod outer segments by 60%. Immunofluorescence confocal microscopy confirmed that outer segment uptake was significantly diminished by an antibody to CD36. Moreover, we found that transfection of a human melanoma cell line with CD36 cDNA enabled these cells to bind and internalize isolated photoreceptor outer segments as seen by double immunofluorescent staining for surface bound and total cell-associated rod outer segments, and by measurement of cell-associated 125I-labeled rod outer segments. We conclude that the multifunctional scavenger receptor CD36 participates in the clearance of photoreceptor outer segments by retinal pigment epithelium and thus, participates in the visual process.

Effect of intravitreal tissue plasminogen activator on experimental subretinal hemorrhage.

PURPOSE: To study the effect of intravitreally injected tissue plasminogen activator (tPA) in an experimental model of subretinal hemorrhage. METHODS: Autologous blood was transsclerally injected into the subretinal space in 34 albino rabbits. One day later tPA was injected into the posterior vitreous in 24 eyes and saline was injected into 10 control eyes. Lysis of the subretinal blood was assessed ophthalmoscopically and retinal function was evaluated electroretinographically. RESULTS: In all eyes in which tPA was injected intravitreally 1 day after subretinal injection of blood, the formed subretinal clots was not visible within 24 hours of treatment. Liquefied subretinal blood that formed from clot lysis disappeared within 6 days. Conversely, in all saline-injected control animals, the subretinal clots were unchanged at 24 hours and were observed for at least 3 days after injection. As a result of the presence of subretinal blood, scotopic electroretinogram amplitudes were markedly reduced in the tPA and saline-injected groups. In many eyes, blood migrated from the subretinal space into the vitreous, but it was detected later, was less severe, and cleared more rapidly after tPA injection. CONCLUSION: Intravitreal injection of tPA 1 day after subretinal injection of blood in rabbits facilitated more rapid lysis of the clotted blood, however, retinal damage was not prevented.

Cytokine regulation of nitric oxide synthase in mouse retinal pigment epithelial cells in culture.

Nitric oxide is an intercellular signaling molecule whose numerous functions include regulation of vascular tone, mediation of the cytotoxic effects of macrophages and potentiation of synaptic transmission. For some cellular functions, nitric oxide synthesis is mediated by the inducible form of nitric oxide synthase. We now show that cultured mouse retinal pigment epithelial cells exposed to interferon-gamma and lipopolysaccharide, express inducible nitric oxide synthase. The latter was detected immuno-cytochemically in interferon-gamma-lipopolysaccharide-treated retinal pigment epithelium using rabbit antiserum to a synthetic peptide of mouse nitric oxide synthase. Untreated cultures of retinal pigment epithelium or cultures treated with either interferon-gamma or or lipopolysaccharide alone were not immunoreactive. Induction of iNOS in gamma-interferon-lipopolysaccharide-stimulated retinal pigment epithelial cells was also evidenced by the presence of nitric oxide synthase enzyme activity in lysates of stimulated but not unstimulated retinal pigment epithelial cells. On immunoblots of lysates of stimulated murine retinal pigment epithelial cells, rabbit antiserum to iNOS recognized a 130-kDa protein which comigrated with the inducible nitric oxide synthase of macrophages and which was not detectable in lysates of unstimulated retinal pigment epithelial cells nor in lysates of cells treated with only interferon-gamma or lipopolysaccharide alone. Nitrite, a stable endproduct of NO formation by cells, was detectable in the culture supernatants after 18-24 hr of exposure to interferon-gamma and lipopolysaccharide, and continued to accumulate in a linear fashion for at least 96 hr. Treatment of cultured retinal pigment epithelium with interferon-gamma, lipopolysaccharide and either basic fibroblast growth factor or epidermal growth factor as third signals augmented inducible nitric oxide synthase expression as evidenced by intensified signals on immunoblots, enhanced accumulation of nitrite and increased iNOS enzyme activity. Conversely, when transforming growth factor-beta was present in the culture medium, gamma-interferon-LPS-induced expression of nitric oxide synthase and NO release were reduced. We conclude that interferon-gamma synergizes with lipopolysaccharide to induce synthesis of inducible nitric oxide synthase and production of nitric oxide by murine retinal pigment epithelium and that this induction can be modulated by basic fibroblast growth factor, epidermal growth factor or transforming growth factor-beta.

Inducible nitric oxide synthase in the central nervous system.

There is an accumulation of evidence indicating that induction of the calcium-independent isoform of nitric oxide synthase (iNOS) in glial cells can contribute to nitric oxide-mediated neural-cell damage. Elucidation of iNOS inducing signals and mechanisms regulating its augmentation and suppression may have implications for our understanding of basic processes underlying some forms of central nervous system disease.

Retinal tolerance to intravitreal perfluoroethylcyclohexane liquid in the rabbit.

To evaluate the use of perfluoroethylcyclohexane (PFE) liquid during vitreoretinal surgery, retinal tolerance was tested by electroretinographic (ERG) and histologic study of rabbit eyes undergoing intravitreal placement of PFE for 48 hours. When PFE occupied the vitreous cavity, ERG amplitudes were decreased, probably because of electrical insulation by the liquid. Immediately after removal of the liquid, elevations of a and b waves occurred. Further improvements in the waveforms were recorded when tested between 5 days and 2 months after PFE removal, such that the eyes injected with PFE exhibited ERG amplitudes comparable to contralateral control eyes and preoperative eyes. Histologic examination of the eyes 2 months after PFE removal also revealed normal morphologic features. Small residual amounts of PFE produced no adverse histologic changes after 6 months. When PFE remained intravitreally for longer than 1 week, dispersion of the liquid and preretinal accumulation of macrophages occurred, and in inferior retina, distortions of photoreceptor outer segments and narrowing of outer plexiform layer were observed in the rabbit model.