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Neurotoxic Effects of Tumor Necrosis Factor Alpha in Primary Human Neuronal Cultures are Mediated by Activation of the Glutamate AMPA Receptor Subtype: Implications for AIDS Neuropathogenesis

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system is characterized by neuronal loss in discrete areas of the central nervous system. We have previously demonstrated that HIV-infected monocytes in culture with astroglial cells produce high levels (> or = 200 pg/ml) of the cytokine tumor necrosis factor-alpha (TNF alpha). We now demonstrate that TNF alpha (> or = 200 pg/ml) is neurotoxic to cultured primary human fetal cortical neurons at both light and electron microscopic levels. Subtoxic doses of TNF alpha (50 pg/ml) are neurotoxic in combination with the glutamate (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) subtype receptor agonist AMPA (100 microM). The neurotoxic effects of TNF alpha (200 pg/ml) are blocked in part by the AMPA receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (10 microM). This suggests that TNF alpha may exert neurotoxic effects on human neurons by indirect activation of AMPA receptors, which may be important in the pathogenesis and treatment of HIV-mediated encephalopathy.

Intraretinal transplantation of fluorescently labeled retinal cell suspensions

Dissociated cell suspensions of neonatal neural retina, labeled with the fluorescent dyes Fast blue or Fluoro-gold, were transplanted into the retina of normal adult rats or of rats affected by late stage phototoxic retinopathy. Light microscopy showed good survival, differentiation, and integration of the transplants, as well as permanence of the label up to 100 days. The results indicate that the transplantation of dissociated, fluorescently labeled retinal cells has a number of advantages over the transplantation of solid fragments of retinal tissue, previously performed by ourselves and others. The following are some of the most immediate procedural advantages: the number of transplanted cells can be assessed, the transplanted cells are in a more intimate contact with the host tissue and therefore integrate better with the host, and the fluorescent tags permit precise determination of the survival and distribution of the transplanted cells.

Efficacy of nonfetal human RPE for photoreceptor rescue: a study in dystrophic RCS rats.

This study determines the efficacy of nonfetal human retinal pigment epithelium (RPE) for photoreceptor rescue utilizing the dystrophic RCS rat as an animal model. Eyes from 10- and 49-year-old donors were obtained through the Rochester Eye and Human Parts Bank. The RPE was isolated by enzymatic treatment of the choroid-RPE with 2% dispase for 30 min at 37 degrees C. Mechanically dissociated RPE cells were injected at the superior hemisphere into the subretinal space of dystrophic RCS rats during the fourth postnatal week. Rats receiving vehicle injection served as sham controls. The animals were immunosuppressed with daily cyclosporine injections (10 mg/kg) and sacrificed 30 days posttransplantation for histologic evaluation of the RPE graft and its effect on photoreceptor survival. Transplantation of adult human RPE promoted the survival of photoreceptors in the dystrophic RCS rat. Morphometric analysis of the grafted superior hemisphere demonstrated a threefold increase in photoreceptor cell density (149.2 +/- 50 SD) compared to sham controls (39.7 +/- 31 SD) and the untouched inferior hemisphere (52.8 +/- 28 SD). RPE from the 49-year-old donor was as effective as RPE from the 10-year-old donor in promoting photoreceptor survival. The results of this study in RCS rats suggests that RPE from adult human donors of varied ages is suitable for transplantation and retains the capability to promote survival of photoreceptor cells. This finding opens the possibility of using nonfetal RPE cells in human retinal transplantation.

Correlates of Photoreceptor Rescue by Transplantation of Human Fetal RPE in the RCS Rat

This study uses a water maze paradigm as a tool to assess posttransplantation changes in behavior associated with a visual stimulus. A set of dystrophic RCS rats received bilateral injections of freshly isolated human fetal RPE cells into the subretinal space of the superior equatorial hemisphere. Five age-matched control dystrophic RCS rats received subretinal injections of vehicle. All animals were immunosuppressed. At 2 months posttransplantation, each rat was tested in the water escape apparatus. The rat used a single light source, randomly located on the edge of the tank, to locate a submerged platform, placed directly in front of the light. Each rat was timed and videotaped during 10 consecutive trials. The swimming paths and times for all rats were recorded and statistically analyzed. Subsequent to the water escape trials, the eyes were embedded for histologic analysis which included quantitative assessment of photoreceptor cells in predefined retinal regions. The water escape data indicated the differences between the sham and experimental groups changed significantly over time (P = 0.0017). Over time, the transplanted animals learned to use light as a clue (P < 0.0001), whereas the sham animals did not (P = 0.73). Transplanted eyes had a significantly greater mean number of photoreceptors in the superior, grafted region than seen in the inferior region of the same eyes and compared with either region of sham-injected eyes (P = 0.0023). Statistical analyses demonstrated a functional advantage for visually guided behavior in RCS rats transplanted with human fetal RPE cells and a statistically significant PRC rescue effect at 2 months after transplantation.

The influences of age, retinal topography, and gender on retinal degeneration in the Fischer 344 rat

The Fischer 344 (F344) rat is presently the animal of choice for age-related research. The existence of an age-related retinal degeneration was reported previously in the males of this strain, but a gender comparison has not been performed. In this study, histological and morphometric measurements of the retina related to age, retinal topography, and gender were made on 3- to 24-month-old animals. The thicknesses of the outer nuclear layer (ONL) and the photoreceptor layer (PRL) were measured from sagittal sections at six loci. Retinas of both sexes showed steady decline with age in the thicknesses of the ONL and PRL at all locations. An important finding was the presence, after 12 months of age, of a drastically accelerated rate of peripheral retinal degeneration seen only in male subjects. Females showed a less dramatic rate of peripheral degeneration which did not begin until after 18 months of age. In addition, two other forms of retinal degeneration were found--cystoid degeneration was found earlier and more frequently in the male, while a paving-stone type of degeneration was found in both sexes. These two types of lesions were preferentially, but not exclusively found in the peripheral retina. In conclusion, the F344 rat offers a convenient model to study a pattern of retinal degeneration affected by the combination of gender, regional and age-related factors.

Cyclosporine Treatment Promotes Survival of Human Fetal Neural Retina Transplanted to the Subretinal Space of the Light-Damaged Fischer 344 Rat

Abstract We have reported that xenografts of human fetal neural retina survive in the subretinal space of cyclosporine-immunosuppressed rats. In view of the current controversy regarding the role of cyclosporine, we wished to determine if cyclosporine immunosuppression was an absolute requirement for retinal xenograft survival. Neural retinas from human fetal eyes obtained within 1 h of termination of pregnancy were stored in Optisol medium (Chiron Vision, Irvine, CA) at 4°C for 2 and 7 days. Retinas were then transplanted to the subretinal space of either cyclosporine-treated (10 mg/kg/day) light-damaged Fischer 344 rat eyes (17 animals, 28 eyes) or to the subretinal space of light-damaged Fischer 344 rat eyes (9 animals, 15 eyes) receiving no cyclosporine treatment. Grafted eyes were observed clinically at 10, 20, and 30 days posttransplantation. At 30 days, the animals were sacrificed and the grafts observed histologically. Human fetal retina xenografted to the subretinal space of immunosuppressed rats survived (9/17 animals, 12/28 eyes), showed good integration with the host retina and initial photoreceptor differentiation. Tissue xenografted to the subretinal space of non-cyclosporine-treated rats was not observed to survive (0/9 animals, 0/15 eyes). A low level cellular reaction was seen around three of the injection sites within the nonimmunosuppressed rats. We conclude that immunosuppression is necessary for the survival of human fetal neural retina xenografted to the subretinal space.

A new procedure for fundus photography and fluorescein angiography in small laboratory animal eyes

Increasing interest in retinal research demands continuous improvement of experimental techniques and interpretation. Thus, the purpose of our research was to devise a new method for funduscopic photography and fluorescein angiography in the normal or diseased retina of the small laboratory animal that would produce results comparable in optical quality and field coverage to those obtained in human clinical practice. To enhance the view of the small eye, a 2.2 Volk Panretinal lens was held in apposition to the lens of a clinical fundus camera, the Topcon TRC 50FT, by means of a custom made metal sleeve. Albino mice, albino rats, and pigmented rats were photographed. Fluorescein angiography was performed on pigmented rats. Fluorescein was administered intravenously via the jugular vein at a dose of 5 mg/kg. Various speeds of film and flash settings were used depending on the light source and the pigmentation of the animal. Attachment of the 2.2 Panretinal lens to the clinical fundus camera allowed for more clearly defined fundus photographs of the small laboratory animal, as well as an enlarged field of observation over conventional techniques. Consequently, angiography fields and stages documented in the small laboratory animal approximated those obtained in human clinical practice. This technique facilitates the visualization of small fundi and it allows for a fuller documentation of experimental retinal models.

Storage of human fetal retina in optisol prior to subretinal transplantation

We have previously reported that Optisol, a commercially available medium (Chiron Vision, Irvine, CA), is a favorable fluid for the storage of neural retina. We now characterize the cell survival and histologic integrity of the tissue following storage and then transplantation. Neural retinas from human fetal eyes obtained within 1 h or pregnancy termination were stored in Optisol-GS medium (containing TC-199 and MEM medium, 2.5% chondroitin sulfate, 1% dextran, HEPES buffer, gentamicin sulfate, 0.1 mmol/L nonessential amino acids, sodium bicarbonate, 1 mmol/L sodium pyruvate, and additional antioxidants), at 4 degrees C for the following time periods: 0, 1, 2, and 7 days, and then tested for viability. Retinas stored for 2 and 7 days were mechanically dissociated and grafted into the eyes of light-damaged Fischer 344 rats (17 animals, 28 eyes). Nontransplanted eyes received injections of vehicle only (5 animals, 6 eyes). All animals were immunosuppressed daily with cyclosporine (10 mg/kg). All experiments were conducted in strict accordance with institutional, federal, and ARVO guidelines. Cell viability averaged 94.8%, 90.2%, 83.2%, and 76.8% at 0, 1, 2, and 7 days storage, respectively. Light microscopy demonstrated that the fetal retina was preserved without evident changes for up to 48 h. Up to 7 days there was a good to very good preservation of the cells in the outer neuroblastic layer. One month after transplantation subretinal grafts of retinal tissue stored for 2 and 7 days in Optisol showed good integration with host retina and initial photoreceptor differentiation. Optisol is a widely used medium for the preservation of human corneas prior to transplantation. Our results show that this medium is also highly suitable for preserving human fetal retinas prior to transplantation.

An improved method of isolating fetal human retinal pigment epithelium

The purpose of this study was to develop an improved method of isolating fetal human retinal pigment epithelium (RPE) for tissue culture or transplantation. Fetal human eyes ranging from 8 to 20 wks of gestation were collected and stored in Optisol solution. Under a dissecting microscope, an incision was made behind the ora serrata and extended circumferentially to remove the anterior segment. The vitreous was withdrawn, and the neural retina was carefully detached from the RPE. The sclera then was teased away from the choroid-RPE. The choroid-RPE was treated with 2% dispase in DMEM + 20 mM HEPES at 37 degrees C for 25 min. While still in dispase, the RPE was separated from the choroid using a pair of fine tipped jewelers forceps under dark-field. An intact sheet of RPE could be separated from the choroid after treatment with dispase. No choroidal contamination was present as determined by light microscopy or cell culture. In vitro, the isolated RPE cells demonstrated classic cobblestone phenotype and expressed cytokeratin. This technique provides an easy and reliable method for isolating pure sheets of fetal human RPE. It also allows utilization of the neural retina of the same eye for other purposes, as the neural retina is not exposed to the enzymatic digestion. These features make this method especially useful for RPE and retinal transplantation; such an application is already underway.

Human neural xenografts: progress in developing an in-vivo model to study human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV) infection.

Human immunodeficiency virus type 1 (HIV-1) infection is highly specific for its human host. In order to study HIV-1 infection of the human nervous system, we have established a small animal model in which second-trimester (11-17.5 weeks) human fetal brain or neural retina is transplanted into the anterior chamber of the eye of immunosuppressed adult rats (Epstein et al., 1992; Cvetkovich et al., 1992), and more recently in immunodeficient (SCID) mice. The human xenografts survive for many months, vascularize and form a blood-brain barrier. Immunohistochemistry with PGP 9.5 identified neuronal cell bodies and neuritic processes. Electron microscopy revealed axonal growth cones and synaptic junctions. Infection of these xenografts with cell-free HIV-1 proved difficult, however co-engraftment with HIV-1-infected human monocytes resulted in characteristic pathological changes, including the formation of syncytial giant cells, neuronal loss, and astroglial proliferation, supporting the hypothesis that these cells can mediate neurotoxicity. In other studies, xenografts of human fetal retinal tissue were readily infected with cell-free human cytomegalovirus (HCMV) strain AD169. These grafts contained cells with intracytoplasmic and intranuclear inclusions typical of HCMV infection. Productive infection within these grafts was demonstrated by the presence of immediate early, and late (capsid) HCMV antigens, by recovery of HCMV on human fibroblast cultures, and by serial passage of virus to additional retinal xenografts (DiLoreto et al., 1994).(ABSTRACT TRUNCATED AT 250 WORDS)