Mechanisms that regulate the function of the selectins and their ligands.

Selectins are a family of three cell adhesion molecules (L-, E-, and P-selectin) specialized in capturing leukocytes from the bloodstream to the blood vessel wall. This initial cell contact is followed by the selectin-mediated rolling of leukocytes on the endothelial cell surface. This represents the first step in a cascade of molecular interactions that lead to leukocyte extravasation, enabling the processes of lymphocyte recirculation and leukocyte migration into inflamed tissue. The central importance of the selectins in these processes has been well documented in vivo by the use of adhesion-blocking antibodies as well as by studies on selectin gene-deficient mice. This review focuses on the molecular mechanisms that regulate expression and function(s) of the selectins and their ligands. Cell-surface expression of the selectins is regulated by a variety of different mechanisms. The selectins bind to carbohydrate structures on glycoproteins, glycolipids, and proteoglycans. Glycoproteins are the most likely candidates for physiologically relevant ligands. Only a few glycoproteins are appropriately glycosylated to allow strong binding to the selectins. Recently, more knowledge about the structure and the regulated expression of some of the carbohydrates on these ligands necessary for selectin binding has been accumulated. For at least one of these ligands, the physiological function is now well established. A novel and exciting aspect is the signaling function of the selectins and their ligands. Especially in the last two years, convincing data have been published supporting the idea that selectins and glycoprotein ligands of the selectins participate in the activation of leukocyte integrins.

The Onchocerciasis Elimination Program for the Americas: a history of partnership.

The decision in 1987 by the pharmaceutical firm Merck & Co. to provide Mectizan (ivermectin) free of charge to river blindness control programs has challenged the international public health community to find effective ways to distribute the drug to rural populations most affected by onchocerciasis. In the Americas, PAHO responded to that challenge by calling for the elimination of all morbidity from onchocerciasis from the Region by the year 2007 through mass distribution of ivermectin. Since 1991, a multinational, multiagency partnership (consisting of PAHO, the endemic countries, nongovernmental development organizations, the Centers for Disease Control and Prevention in Atlanta, Georgia, as well as academic institutions and funding agencies) has developed the political, financial, and technical support needed to move toward the realization of that goal. This partnership is embodied in the Onchocerciasis Elimination Program for the Americas (OEPA), which is supported by the River Blindness Foundation (RBF) and now by the Carter Center. OEPA was conceived as a means of maintaining a regional initiative to eliminate what is otherwise a low priority disease. Since its inception in 1993, the OEPA has provided more than US$ 2 million in financial, managerial, and technical assistance to stimulate and/or support programs in Brazil, Colombia, Ecuador, Guatemala, Mexico, and Venezuela, so as to take full advantage of the Merck donation. Now halfway into a five-year, US$ 4 million grant provided through the Inter-American Development Bank, the OEPA's capacity to support the regional initiative is assured through 1999.

The onchocerciasis elimination program for the Americas: a history of partnership

The decision in 1987 by the pharmaceutical firm Merck & Co. to provide Mectizan (ivermectin) free of charge to river blindness control programs has challenged the international public health community to find effective ways to distribute the drug to rural populations most affected by onchocerciasis. In the Americas, PAHO responded to that challenge by calling for the elimination of all morbidity from onchocerciasis from the Region by the year 2007 through mass distribution of ivermectin. Since 1991, a multinational, multiagency partnership (consisting of PAHO, the endemic countries, nongovernmental development organizations, the Centers for Disease Control and Prevention in Atlanta, Georgia, as well as academic institutions and funding agencies) has developed the political, financial, and technical support needed to move toward the realization of that goal. This partnership is embodied in the Onchocerciasis Elimination Program for the Americas (OEPA), which is supported by the River Blindness Foundation (RBF) and now by the Carter Center. OEPA was conceived as a means of maintaining a regional initiative to eliminate what is otherwise a low priority disease. Since its inception in 1993, the OEPA has provided more than US$2 million in financial, managerial, and technical assistance to stimulate and/or support programs in Brazil, Colombia, Ecuador, Guatemala, Mexico and Venezuela, so as to take full advantage of the Merck donation. Now halfway into a five-year, US$ 4 million grant provided through the Inter-American Development Bank, the OEPA's capacity to support the regional initiative is assured through 1999

The Onchocerciasis elimination program for the Americas: a history of partnership

The decision in 1987 by the pharmaceutical firm Merck & Co. to provide Mectizan (ivermectin) free of charge to river blindness control programs has challenged the international public health community to find effective ways to distribute the drug to rural populations most affected by onchocerciasis. In the Americas, PAHO responded to that challenge by calling for the elimination all morbidity from onchocerciasis from the region by the year 2007 through mass distribution of ivermectin. Since 1991, a multinational, multiagency partnership (consisting of PAHO, the endemic countries, nongovernmental development organizations, the Centers for Disease Control and Prevention in Atlanta, Georgia, as well as academic institutions and funding agencies) has developed the political, financial, and technical support needed to move toward the realization of that goal. This partnership is embodied in the Onchocerciasis Elimination Program for the Americas (OEPA), which is supported by the River Blindness Foundation (RBF) and now by the Carter Center, OEPA was conceived as a means of maintaining a regional initiative to eliminate what is otherwise a low priority disease. Since its inception in 1993, the OEPA has provided more than US$ 2 million in financial, managerial, and technical assistance to stimulate and/or support programs in Brazil, Colombia, Ecuador, Guatemala, Mexico, and Venezuela, so as to take full advantage of the Merck donation, Now halfways into a five-year, US$ 4 million grant provided through the Inter-American Development Bank, the OEPA's capacity to support the regional initiative is assured through 1999 (AU)

The onchocerciasis elimination program for the Americas: a history of partnership / Programa de Eliminación de la Oncocercosis para las Américas: historia de solidaridad

The decision in 1987 by the pharmaceutical firm Merck & Co. to provide Mectizan (ivermectin) free of charge to river blindness control programs has challenged the international public health community to find effective ways to distribute the drug to rural populations most affected by onchocerciasis. In the Americas, PAHO responded to that challenge by calling for the elimination of all morbidity from onchocerciasis from the Region by the year 2007 through mass distribution of ivermectin. Since 1991, a multinational, multiagency partnership (consisting of PAHO, the endemic countries, nongovernmental development organizations, the Centers for Disease Control and Prevention in Atlanta, Georgia, as well as academic institutions and funding agencies) has developed the political, financial, and technical support needed to move toward the realization of that goal. This partnership is embodied in the Onchocerciasis Elimination Program for the Americas (OEPA), which is supported by the River Blindness Foundation (RBF) and now by the Carter Center. OEPA was conceived as a means of maintaining a regional initiative to eliminate what is otherwise a low priority disease. Since its inception in 1993, the OEPA has provided more than US$2 million in financial, managerial, and technical assistance to stimulate and/or support programs in Brazil, Colombia, Ecuador, Guatemala, Mexico and Venezuela, so as to take full advantage of the Merck donation. Now halfway into a five-year, US$ 4 million grant provided through the Inter-American Development Bank, the OEPA's capacity to support the regional initiative is assured through 1999

La decisión tomada en 1987 por la Merck & Co., fabricante de productos farmacéuticos, de proveer Mectizan® (ivermectina) gratuitamente a los programas de control de la oncocercosis ha obligado a la comunidad sanitaria internacional a buscar formas de distribuir el medicamento a las poblaciones rurales que se ven más afectadas por la enfermedad. En las Américas, la OPS respondió al reto con un llamado a eliminar de la Región toda morbilidad por oncocercosis para el año 2007 mediante la distribución de ivermectina al público. Desde 1991, una alianza multinacional de diversas entidades (la OPS, países con oncocercosis endémica, agencias de desarrollo no gubernamentales, los Centros para el Control y la Prevención de Enfermedades en Atlanta, Georgia, instituciones académicas y agencias de financiamiento) ha generado el apoyo político, económico y técnico necesario para tratar de alcanzar esa meta. Esta alianza está representada por el Programa de Eliminación de la Oncocercosis en las Américas (OEPA), subvencionado por la Fundación Ceguera de los Ríos y actualmente por el Centro Carter. El OEPA se creó como iniciativa de alcance regional destinada a eliminar una enfermedad que no merece atención prioritaria. Desde su aparición en 1993, el OEPA ha aportado más de US$ 2 millones en ayuda económica, administrativa y técnica para fomentar y subvencionar programas en Brasil, Colombia, Ecuador, Guatemala, México y Venezuela, logrando así aprovechar al máximo la donación de la Merck & Co. Ahora que hemos llegado a la mitad de una subvención de 5 años y US$ 4 millones aportada por el Banco Interamericano de Desarrollo, se sabe que el OEPA tiene la capacidad para apoyar la iniciativa regional hasta fines de 1999

Stimulation of P-selectin glycoprotein ligand-1 on mouse neutrophils activates beta 2-integrin mediated cell attachment to ICAM-1.

The entry of neutrophils into inflamed tissues is initiated by cell rolling on the blood vessel wall followed by arrest and transendothelial migration. Rolling is mediated by the selectins, while the two subsequent steps require activated beta 2-integrins. We have investigated whether the binding of P-selectin to mouse neutrophils could trigger the activation of beta 2-integrins. We show that cross-linking of P-selectin glycoprotein ligand-1 (PSGL-1) on mouse neutrophils with an antibody-like recombinant form of P-selectin or with monoclonal antibodies stimulated the production of reactive oxygen intermediates and enhanced neutrophil attachment to intercellular adhesion molecule 1 (ICAM-1)-expressing CHO cells. This effect was independent of whether complete antibodies or F(ab')2 fragments were used. The adhesion-stimulating effect of P-selectin could be blocked by monoclonal antibodies against PSGL-1. Increase of cell attachment was dependent on lymphocyte function-associated antigen 1 (LFA-1) and on Mac-1, since it could be blocked with antibodies against both respective integrin alpha-chains. Moreover, cell surface expression of Mac-1 increased upon cross-linking of PSGL-1. In agreement with published data, treatment of human neutrophils with P-selectin-IgG did not enhance attachment to ICAM-1. Our data suggest that ligation of PSGL-1 on mouse neutrophils, but not on human neutrophils, activates beta 2-integrin mediated cell attachment to ICAM-1.

Migration and synaptogenesis of cone photoreceptors in the developing mouse retina.

Mouse retinal photoreceptor cell generation and morphogenesis take place in a well-characterized temporal sequence. Both rod and cone photoreceptor differentiation and synaptogenesis occur postnatally, but the relative timing of these events has been difficult to document due to the paucity of cell-specific markers. We have found that antibodies to neuron-specific enolase (NSE) preferentially label a subpopulation of photoreceptors in the outer nuclear layer (ONL) of the mouse retina in addition to labeling ganglion, amacrine, bipolar, and horizontal cells within the inner layers of the retina. The appearance of NSE immunoreactivity in the different classes of retinal neurons during development showed a close temporal relationship to the onset of expression of the synaptic vesicle-associated protein SV2 and clearly preceded the sequential development of synaptic connections in both inner and outer synaptic layers. The NSE-immunoreactive photoreceptors were identified as cones by dual labeling of their inner segments with the lectin peanut agglutinin or by colabeling with antisera to cone photopigments. Axonal extensions of NSE-labeled cone cells were shown to interact with those of differentiating horizontal cells as early as postnatal day 3 (P3). Colocalization of NSE with SV2 indicated that cone cells began to make synaptic contacts with horizontal cell processes several days prior to the development of rod synaptic terminals. Between P4 and P11, cone photoreceptor cell nuclei were observed to be scattered at various levels throughout the ONL and thus appeared to have become displaced from their previous position directly beneath the outer limiting membrane (OLM). By P12, the cone nuclei had migrated sclerad once again and were now observed to be neatly aligned adjacent to the OLM. In the rd mouse mutant, this migratory process was delayed, so that, at P12, positioning of the cone cell nuclei within the ONL was still quite irregular. Thus, we have identified a late migratory phase for cone photoreceptors during the second week after birth that correlates with the timing of maturation of the rod synaptic terminals just prior to eye opening. The types of cues used by maturing cone cells for their eventual sclerad location remain to be elucidated.

Lineage study of degenerating photoreceptor cells in the rd mouse retina.

PURPOSE: A retroviral marker was used to label daughter cells arising from individual neuroblasts in the rd mouse retina, in order to investigate the hypothesis that a clonal relationship exists among degenerating photoreceptor cells. METHODS: On the day of birth, a single injection of retrovirus with a lac Z (beta-galactosidase) reporter construct was injected into the retina in the vicinity of the subretinal space. Descendants of single neuroblasts were identified histochemically by examining the retinas at P14 (postnatal day 14). Light and electron microscopic studies were used to identify the retrovirally-induced marker beta-galactosidase using Bluo-gal dye. Double-labeling of degenerating cone cells was accomplished by taking 100 microns vibratome sections of retrovirally-injected eyes and using either FITC-PNA or HRP-PNA to visualize clusters of degenerating cones as well as Bluo-gal labeled clones of photoreceptor cells on the same tissue section. RESULTS: A relatively large number of clones of primarily photoreceptor cells were observed in the peripheral retinas of both normal and rd mice. In a few cases in the rd, photoreceptor cells in a given clone consisted of both PNA- and Bluo-gal-labeled cells as well as of only Bluo-gal-labeled cells. CONCLUSION: These results suggest that during the period of intense cell death in the rd retina, a single dying photoreceptor cell can be surrounded by photoreceptors (either rods or cones) from the same clone that appear morphologically normal without evidence of degeneration.

Aberrant expression of c-Fos accompanies photoreceptor cell death in the rd mouse.

Selective degeneration of rod photoreceptor cells in the retinal degenerative (rd) mouse prior to their complete maturation is thought to result from elevated cyclic guanosine monophosphate (cGMP) levels owing to the inherited defect in cGMP-phosphodiesterase. To investigate potential signaling pathways which might lead to apoptotic death of photoreceptors in the rd retina, the expression of immediate-early genes (IEG) of the activating protein-1 transcription factor (AP-1) family was examined. Increasing numbers of apoptotic photoreceptor nuclei were observed in the outer nuclear layer of the rd mouse beginning at postnatal day (P) 10. The peak incidence of apoptotic cells was observed at P13; by P16, almost the entire population of photoreceptors had been lost. Although c-Fos-like immunoreactivity was absent in photoreceptors of normal retinas, we observed that commencing at around P10, increasing numbers of rod photoreceptors in the rd retina exhibited nuclear staining for c-Fos protein. While no change in the distribution patterns of other members of the AP-1 family (c-Jun, JunB, and JunD) was observed in photoreceptors, Müller cell nuclei were transiently immunoreactive for c-Jun on P11. The incidence of c-Fos-positive photoreceptors peaked sharply at P12, 1 day earlier than the peak in apoptosis. Furthermore, the population of c-Fos-positive photoreceptors was distinct from apoptotic photoreceptors exhibiting chromatin condensation. The aberrant expression of c-Fos protein in rod photoreceptors immediately prior to their death in the rd mouse raises the possibility that c-Fos may be directly or indirectly involved in triggering the apoptotic cascade. Furthermore, the additional finding of c-Jun induction in Müller glia suggests that the IEG response to photoreceptor degeneration involves both intra- and intercellular signal transduction pathways.

P-selectin glycoprotein ligand-1 mediates rolling of mouse bone marrow-derived mast cells on P-selectin but not efficiently on E-selectin.

It has been shown recently that mast cells play an essential role as a source of tumor necrosis factor-alpha production during neutrophil recruitment to sites of bacterial infection. Increased numbers of mast cells are indeed noted at sites of wound healing and inflammation. These cells are either recruited from the bone marrow or proliferate locally under cytokine stimulation. Little is known about how mast cell progenitors extravasate into tissue. Using antibody-like fusion proteins of mouse E-selectin and P-selectin, we have analyzed the ability of immature mouse bone marrow-derived mast cells (BMMC) to interact with the endothelial selectins. The P-selectin glycoprotein ligand-1 (PSGL-1) was affinity-isolated from detergent extracts of surface biotinylated BMMC with both selectin-IgG fusion proteins. However, only P-selectin-IgG, but not E-selectin-IgG showed significant interaction with intact BMMC as tested by flow cytometry and cell attachment assays with the immobilized fusion proteins under flow and non-flow conditions at physiological shear stress. Thus, in spite of carrying the necessary carbohydrate modifications which enable solubilized PSGL-1 to bind avidly to E-selectin, PSGL-1 on the surface of BMMC is presented in a way that prevents it from interacting efficiently with E-selectin. Affinity-purified rabbit antibodies against mouse PSGL-1 almost completely blocked the interaction of BMMC with P-selectin-IgG in flow cytometry as well as in cell adhesion assays under static and under flow conditions. Our data reveal that PSGL-1 is the major binding site for P-selectin on mouse BMMC progenitors, but does not support efficient interactions with E-selectin.

L-selectin from human, but not from mouse neutrophils binds directly to E-selectin.

L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin-Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin-Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin-Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin-Ig fusion protein was not observed. In agreement with the biochemical data, the anti-L-selectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin-Ig but not on P-selectin-Ig. No such inhibitory effect was seen with the anti-mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin-binding carbohydrate moieties are attached to different protein scaffolds in different species.

Retinal light damage in rats with altered levels of rod outer segment docosahexaenoate.

PURPOSE: To compare retinal light damage in rats with either normal or reduced levels of rod outer segment (ROS) docosahexaenoic acid. METHODS: Weanling male albino rats were maintained in a weak cyclic light environment and fed either a nonpurified control diet or a purified diet deficient in the linolenic acid precursor of docosahexaenoic acid (DHA). Half the rats on the deficient diet were given linseed oil, containing more than 50 mol% linolenic acid, once a week to maintain ROS DHA at near normal levels. Diets and linseed oil supplementation were continued for 7 to 12 weeks. To replenish DHA in their ROS, some 10-week-old rats on the deficient diet were given linseed oil three times a week for up to 3 additional weeks. Groups of animals were killed at various times for ROS fatty acid determinations or were exposed to intense green light using intermittent or hyperthermic light treatments. The extent of retinal light damage was determined biochemically by rhodopsin or photoreceptor cell DNA measurements 2 weeks after exposure and morphologically by light and electron microscopy at various times after light treatment. RESULTS: Rats maintained for 7 to 12 weeks on the linolenic acid-deficient diet had significantly lower levels of DHA and significantly higher levels of n-6 docosapentaenoic acid (22:5n-6) in their ROS than deficient-diet animals supplemented once a week with linseed oil or those fed the nonpurified control diet. As determined by rhodopsin levels and photoreceptor cell DNA measurements, deficient diet rats exhibited protection against retinal damage from either intermittent or hyperthermic light exposure. However, the unsaturated fatty acid content of ROS from all three dietary groups was the same and greater than 60 mol%. In 10 week-old deficient-diet rats given linseed oil three times a week, ROS DHA was unchanged for the first 10 days, whereas 22:5n-6 levels declined by 50%. After 3 weeks of treatment with linseed oil, ROS DHA and 22:5n-6 were nearly the same as in rats supplemented with linseed oil from weaning. The time course of susceptibility to retinal light damage, however, was different. Hyperthermic light damage in rats given linseed oil for only 2 days was the same as for rats always fed the deficient diet. Six days after the start of linseed oil treatment, retinal light damage was the same as in rats given the linseed oil supplement from weaning. Morphologic alterations in ROS of linseed oil-supplemented rats immediately after intermittent light exposure were more extensive than in either the deficient-diet animals or those fed the control diet. The deficient-diet rats also exhibited better preservation of photoreceptor cell nuclei and structure 2 weeks after exposure. CONCLUSIONS: Rats fed a diet deficient in the linolenic acid precursor of DHA are protected against experimental retinal light damage. The relationship between retinal light damage and ROS lipids does not depend on the total unsaturated fatty acid content of ROS; the damage appears to be related to the relative levels of DHA and 22:5n-6.

Lens tropomodulin: developmental expression during differentiation.

Lens epithelial cells undergo a dramatic transformation during the process of differentiation into elongated fiber cells. The membrane-associated actin cytoskeleton is likely to play a critical role in the stabilization and maintenance of the highly elongated fiber cell shape. Tropomodulin is a tropomyosin-binding protein associated with actin filaments in a variety of terminally differentiated cell types where stable actin filament organization is required for cell function. We now present results of studies to determine the temporal expression of tropomodulin in the developing lens. In situ hybridization experiments detected expression of tropomodulin mRNA in the developing mouse lens in elongating cells with a pattern similar to that of the fiber specific beta- and gamma-crystallins. Tropomodulin mRNA expression first appeared around 11.5 days post-coitum in elongating cells in the posterior part of the lens vesicle. At later stages the signal for tropomodulin was present in the elongating cells at the lens equator and in cortical fiber cells; signal was absent from the epithelium. To investigate the possible link between tropomodulin expression and fiber differentiation we used a well-established lens epithelial explant culture system in which fiber differentiation is induced by fibroblast growth factor (FGF). Tropomodulin expression was only observed in FGF-treated explants in conjunction with morphologic changes characteristic of lens fiber cell differentiation. The appearance of tropomodulin during the process of fiber cell differentiation suggests that tropomodulin may be important for stabilization and/or determination of actin filament length.

Use of a retroviral vector with an internal opsin promoter to direct gene expression to retinal photoreceptor cells.

PURPOSE: Viral-mediated gene transfer to retina, as well as to other tissues, is evolving rapidly. We have evaluated the potential of a retroviral vector with an internal opsin promoter fragment to direct gene expression to retinal photoreceptor cells. METHODS: Two recombinant retroviral vectors were prepared; in each Vector, a 1.4 kb fragment of the mouse opsin promoter was placed downstream from the neoR gene in the Moloney murine leukemia virus-based vector G1Na. The opsin promoter fragment was linked either to the cDNA for mouse rod photoreceptor phosphodiesterase (PDE) beta-subunit or to the bacterial lacZ reporter gene. These vectors were tested for their ability to direct gene expression after transduction of 3T3 and Y79 cells, or of dissociated retinal cell cultures or retinal explants from neonatal mice. RESULTS: As expected, PDE beta-subunit and beta-galactosidase mRNAs were expressed only at low levels in 3T3 fibroblasts and Y79 retinoblastoma cells. Northern blot analysis indicated that expression was derived from the viral long terminal repeat (LTR) promoter. Infection of primary retinal cell cultures or explants from neonatal mice with BAG retrovirus, in which beta-galactosidase is driven by the viral LTR, resulted in expression in many cell types, while the opsin-lacZ vector mediated the expression of the lacZ reporter gene specifically in photoreceptor cells. CONCLUSIONS: The internal opsin promoter fragment appears capable of selectively directing gene expression to photoreceptor cells after retroviral-mediated gene transfer. These findings serve as a basis for future studies using the opsin promoter-beta PDE retroviral vector to rescue photoreceptor cells in the rd mutant mouse, in which the beta-PDE gene is mutated resulting in degeneration of photoreceptor cells during the early postnatal period.

Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes in GCL.

Detailed analyses of neuronal and astrocyte cell numbers in the ganglion cell layer (GCL) of whole-mounted peripheral retinas from 16 Alzheimer's disease (AD) and 11 control eyes (11 and 9 cases, respectively) demonstrate extensive neuronal loss throughout the entire retina in AD as compared to control eyes. The observed neuronal loss is most pronounced in the superior and inferior quadrants, ranging between 40 and 49% throughout the midperipheral regions, and reaching 50-59% in the far peripheral inferior retina, while the overall neuronal loss throughout the entire retina amounts to 36.4% (p < 0.004). Although the 16% increase in astrocyte numbers is not significant, the ratio of astrocytes to neurons is significantly higher (82%; p < 0.0008) in AD as compared to normal retina (0.238 +/- 0.070 vs. 0.131 +/- 0.042). These results are strengthened by the close agreement (within +/- 15% of respective means) found between fellow eyes. Analysis of glial fibrillary acidic protein immunoreactivity (GFAP-ir) in sections of retinas from an additional 12 AD and 19 control cases show increased GFAP-ir with more extensive labeling of astrocytes in the GCL as well as increased labeling of Müller cell end-feet and radial processes in AD as compared to control retinas. The extensive loss of neurons documented in these retinas, accompanied by an increased astrocyte/neuron ratio, provides further support for the substantial involvement of the retina in AD.

Retinal pathology in Alzheimer's disease. I. Ganglion cell loss in foveal/parafoveal retina.

Morphometric analysis of the numbers of neurons in the ganglion cell layer (GCL) of the central retina (fovea/foveola/parafoveal retina) in eyes from 9 Alzheimer's disease (AD) and 11 age-matched control cases revealed an overall decrease of 25% in total numbers of neurons in AD as compared with control eyes. Detailed analyses of GCL neurons at various eccentricities from the foveola showed that the greatest decrease in neuronal density (43% decrease) occurred in the central 0-0.5 mm (foveal region), while at 0.5-1 mm and at 1-1.5 mm eccentricities, neuronal loss amounted to 24 and 26%, respectively. The temporal region of the central retina appeared most severely affected, with up to 52% decrease in neuronal density near the foveola (central 0-0.5 mm eccentricity). There was close agreement between fellow eyes analyzed separately for three AD and three control cases. Analysis of neuronal sizes showed that all sizes of neurons were similarly affected in AD. In the GCL of control retinas, neurons decreased with age (coefficient of correlation = -0.67), while in AD retinas no such relationship was evident. Since in the central 0-2 mm region of the retina 97% of neurons in the GCL are ganglion cells (while the remaining 3% consist of displaced amacrine cells), these results demonstrate extensive ganglion cell loss in the central retina in AD.

Light-induced acceleration of photoreceptor degeneration in transgenic mice expressing mutant rhodopsin.

PURPOSE: Mutations at various loci on the rhodopsin gene have been shown to cause autosomal dominant retinitis pigmentosa (ADRP). One of the most common is a point mutation (P23H) near the N-terminus of the protein. The authors have studied the effects of light deprivation on the rate of degeneration in pigmented transgenic mice expressing the P23H mutation as well as two additional mutations near the N-terminus of opsin (V20G, P27L). METHODS: Transgenic and normal littermates were reared in darkness or in cyclic light (approximately 7 foot-candle) for periods of 2, 4, or 6 months. Retinal structure and function were evaluated by electroretinography, retinal densitometry, light microscopy, and TUNEL labeling. RESULTS: Retinas of normal animals, whether reared in darkness or in cyclic light, had no structural or functional abnormalities. The rate of photoreceptor degeneration in dark-reared transgenic mice was significantly slower than in transgenic mice raised under cyclic light conditions. Differences between the two groups of animals were evident in the retinal histology, the electroretinographically determined sensitivity to photic stimulation, and the rhodopsin levels in the retina. TUNEL labeling of retinal wholemounts showed that cyclic light-reared animals had a threefold higher incidence of photoreceptor cell death than their dark-reared counterparts; the density of apoptotic cells was greatest in the inferior retina, the region most severely affected in patients with the P23H mutation. In comparison, photoreceptor cell death was more uniformly distributed across the retina in dark-reared transgenic mice. CONCLUSIONS: These findings suggest that light activation of rhodopsin contributes to the severity of the degenerative disease resulting from the P23H opsin mutation, and they raise the possibility that minimizing exposure to light may help to prolong useful vision of patients with this form of retinitis pigmentosa.

Somatostatin-immunoreactive neurons in the adult rabbit retina.

Somatostatin (SRIF) is a neuroactive peptide that is distributed throughout the nervous system, including the retina. This peptide has been localized to populations of amacrine cells in a variety of vertebrate species. In the rabbit retina, SRIF immunoreactivity is present in a sparse population of medium to large neurons (13.72 microns in diameter, or 147.84 mu 2) in the ganglion cell layer and in a small number of neurons in the inner nuclear layer. These cells display a preferential distribution to the inferior retina, with the highest density near the ventral and ventrolateral retinal margins (11.33 cells/mm2). SRIF-immunoreactive cells have two to five primary processes that arborize in the proximal inner plexiform layer (IPL). These give rise to a plexus of finer processes in the distal IPL. Occasional immunoreactive processes are also present in the outer plexiform layer. In the IPL, these laminar networks are present in all retinal regions. In addition, SRIF-immunoreactive cells often have a fine-caliber axonlike process that eminates from the soma or perisomal region. These processes travel for great distances across the retina in either the nerve fiber layer or in the distal IPL but are never seen to enter the optic nerve head. In addition, the number of SRIF-immunoreactive somata remains unchanged following transection of the optic nerve. Taken together, these data indicate that SRIF-immunoreactive neurons of the rabbit retina are displaced amacrine cells. Furthermore, the sparse distribution of SRIF-immunoreactive somata, the wide-ranging, asymmetric arborization of their cellular processes, and previous pharmacological studies suggest that these neurons mediate a broad modulatory role in retinal function.

Effects of Müller cell disruption on mouse photoreceptor cell development.

Müller cells have been proposed to play an important role in photoreceptor cell development during the final stages of retinal maturation. The effect of disrupting Müller cells during mouse retinal development was investigated using the specific glial cell toxin, DL-alpha-aminoadipic acid (AAA). By giving multiple systemic injections over several days, impairment of Müller cell function was maintained during the period of photoreceptor migration and differentiation. Following three consecutive days of AAA treatment [commencing on post-natal (P) day 3, 5, 7 or 9, and examined at P8-P14], clumps of photoreceptor nuclei were displaced through the inner segments, lying immediately beneath the retinal pigment epithelium (RPE). Apart from the scalloped appearance of the outer retina, the overall lamination pattern of the retina was relatively well preserved. Even when AAA treatment commenced as early as P3, several days prior to the formation of the outer nuclear layer, the majority of photoreceptors migrated to their correct position and formed inner and outer segments. Therefore, the signals for photoreceptor migration are either provided by the Müller cells prior to P3, or, alternatively, are derived from different intrinsic or extrinsic cues. Disruption of Müller cell function was evidenced by decreased glutamine synthetase activity as well as by increased glial fibrillary acidic protein (GFAP) and decreased cellular retinaldehyde-binding protein (CRALBP) immunoreactivity. Immunocytochemistry with an antibody to CD44, which labels the microvilli of Müller cells at the outer limiting membrane, coupled with electron microscopic analysis, demonstrated that the zonulae adherentes between Müller cells and photoreceptors were either irregular or absent in areas adjacent to displaced clumps of photoreceptors. Thus AAA treatment of early post-natal mice results in localized disruption of the contacts between Müller cells and photoreceptors. These pathologic changes persist into adulthood since at P28, while short stretches of photoreceptors appeared relatively normal with fully developed outer segments, periodic clumps of displaced photoreceptor nuclei were still present adjacent to the RPE. In conclusion, Müller cell processes at the outer limiting membrane appear to play a critical role in providing a barrier to aberrant photoreceptor migration into the subretinal space.