The onset of pigment epithelial proliferation after retinal detachment.

The adult mammalian retinal pigment epithelium (RPE) is mitotically inactive, yet retains the capacity to proliferate under certain conditions. To determine the onset of RPE proliferation after retinal detachment, we examined experimentally detached cat retinas of 12, 24, 48, and 72 hr duration. An additional animal served as a nondetached, sham-operated control. 3H-thymidine was injected into the vitreous chamber and the eyes were processed for light microscopic autoradiography. Autoradiograms from both the control and the 12 hr detachment showed no evidence of labeled RPE nuclei; however, labeled nuclei were present at both 24 and 48 hr after detachment. Labeled nuclei per millimeter of RPE at 24 hr were 55% of the 48 hr rate. Mitotic figures were noted only at 48 and 72 hr after detachment. No labeled RPE nuclei appeared in autoradiograms that bordered the detachment zone. Electron micrographs showed that proliferating RPE cells assume several configurations, some of which have been reported in other species. The proliferative response of the RPE occurs much sooner than had previously been thought. It appears to be a local effect that does not involve retinal regions beyond the detachment boundaries, and it may have potentially adverse effects when the retina and RPE are reapposed after retinal reattachment surgery.

Intraretinal proliferation induced by retinal detachment.

Cellular proliferation after retinal detachment was studied by 3H-thymidine light microscopic autoradiography in cats that had experimental detachments of 0.5-180 days duration. The animals underwent labeling 2 hr before death with an intraocular injection of 200 microCi of 3H-thymidine. The number of labeled nuclei were counted in 1-micron thick tissue sections in regions of detachment, in regions of the experimental eyes that remained attached, and in control eyes that had no detachments. In the normal eye, in one that had only the lens and vitreous removed, and in the eyes with 0.5- and 1-day detachments, the number of labeled nuclei ranged from 0/mm (0.5-day detachment) to 0.38/mm (lens and vitreous removed only). By 2 days postdetachment, the number of labeled nuclei increased to 2.09/mm. The highest levels of labeling occurred in two animals with detachments of 3 (7.86/mm) and 4 (7.09/mm) days. Thereafter, the numbers declined steadily until near-baseline counts were obtained at 14 days. The number of labeled nuclei was slightly elevated in the attached regions of two animals with 3-day detachments. Labeled cell types included: Müller cells, astrocytes, pericytes, and endothelial cells of the retinal vasculature, and both resident (microglial cells) and invading macrophages. In an earlier study RPE cells were also shown to proliferate in response to detachment. Thus, these data show that proliferation is a rapid response to detachment, reaching a maximum within 4 days, and that virtually every nonneuronal cell type in the retina can participate in this response. The data suggest that events leading to such clinical manifestations as proliferative vitreoretinopathy and subretinal fibrosis may have their beginnings in this very early proliferative response.

Retinal detachment in the cat: the pigment epithelial-photoreceptor interface.

Twenty-six cat retinae were surgically detached by injecting fluid into the subretinal space (SRS). The retinae were then studied by light and electron microscopy at detachment intervals ranging from 1/2 hr to 14 months. Degenerative and proliferative changes occur at the retinal pigment epithelial (RPE)-photoreceptor interface very soon after detachment, and the severity of these changes depends upon both the duration and height of the detachment. The specialized apical RPE processes that ensheath the outer segments are replaced by a uniform fringe of short, undifferentiated processes. The apical RPE surface becomes mounded, and this mounding becomes more pronounced at longer detachment durations. Labeling experiments with 3H-thymidine showed that some cat RPE cells enter a phase of stimulated DNA synthesis 12-24 hrs after detachment; RPE mitotic figures are first apparent 48 hrs after detachment. In the cat, discrete regions of proliferated RPE cells usually appear in one of several configurations. A number of different cell types, including polymorphonuclear neutrophils, monocytes at various maturational stages, photoreceptor cells, Müller cells, and RPE cells, appear in the expanded SRS of detached retinae. Rod and cone outer segments degenerate rapidly and become membrane bound sacs by 3 days postdetachment; the assembly of new outer segment membrane apparently does not stop completely even at moderately long detachment intervals (ie, 2 months). Degenerative changes in the inner segments do not take place with the same rapidity as those in the outer segments. The changes that occur at the RPE-photoreceptor interface are rapid, progressive, and sometimes irreversible events that have significant implications for photoreceptor recovery following retinal reattachment surgery.

Retinal detachment in the cat: the outer nuclear and outer plexiform layers.

The retinae of cats were surgically detached for 1/2 hr to 14 months, and the outer nuclear (ONL) and outer plexiform layers (OPL) were studied by light and electron microscopy. The longer the duration or the greater the height of detachment the more likely was the occurrence of cell death. Histologic signs of degeneration were present 1 hr after detachment. The number of photoreceptor nuclei in the ONL decreased significantly by 1 month. Loss of cells in the ONL occurred by necrosis and by the migration of photoreceptor cell bodies into the subretinal space. The OPL degenerated by the necrosis of cell processes and synaptic terminals and by the retraction of the synaptic terminals. By 2 weeks most synaptic terminals were necrotic or in the process of retracting. Photoreceptor synaptic contact with second order neurons was diminished by 30 days and was essentially absent by 50 days. Müller cells proliferated and hypertrophied; their nuclei and cell processes filled the intraretinal spaces left by the degenerating photoreceptors. In addition, Müller cells protruded into the subretinal space and formed multiple layers of cell bodies and processes between the retina and retinal pigment epithelium. By 14 months these subretinal Müller cell processes covered the entire detached retina, and appeared morphologically like an astroglial scar. Similar changes in human retinal detachments may significantly influence the degree of visual recovery after retinal reattachment, especially in retinae detached for more than a few days.

Morphological recovery in the reattached retina.

After experimental retinal detachment in the cat, a number of morphological changes take place in retinal and RPE cells. Following reattachment, the ultrastructural relationship between the photoreceptors and the RPE is re-established, but it does not return to the predetachment state even after short detachment episodes coupled with prolonged recovery periods. All of the reattached retinae show some degree of abnormality, ranging from subtle changes in photoreceptor ultrastructure to dramatic degenerative effects in the outer retina. Abrupt transitions in morphology from one reattached area to an adjacent area are not unusual. Photoreceptor recovery varies widely between animals, and between adjacent regions within the same retina. Ensheathment of outer segments by RPE apical processes is abnormal. In some reattached areas rod outer segment dimensions and disc structure are near normal as is the displacement rate of rod outer segment discs. In others, especially in areas of RPE or Müller cell proliferation and hypertrophy, the outer segments are shortened or absent completely, and there is a reduction of cell bodies in the outer nuclear layer. In some retinae, recovery in cones is inferior to that in rods. At short detachment durations (less than 1 wk) morphological recovery in the reattached retina is optimal while at long intervals (greater than 1 month) recovery is poor. The changes at the photoreceptor-RPE interface identified in the reattached cat retina probably have adverse effects on visual recovery when they occur within the human macula.

Clearance and localization of intravitreal liposomes in the aphakic vitrectomized eye.

The authors have examined the fate of intravitreally injected liposomes in the aphakic, vitrectomized eye of the rabbit. Liposomes labelled with 125[I]-p-hydroxybenzimidylphosphatidylethanolamine were eliminated rapidly from the intraocular fluid. Nonetheless, a significant fraction of these liposomes were found to bind to various ocular tissues including the retina, iris, sclera, and cornea. Ultrastructural studies with gold colloid-loaded liposomes revealed that retinal bound liposomes were attached to the inner limiting lamina but did not penetrate to the internal cells of the retina. Epiretinal cells bound and internalized gold colloid-loaded liposomes suggesting that these cells may be very sensitive to liposome mediated drug delivery.

Fluorouracil therapy for proliferative vitreoretinopathy after vitrectomy.

Fluorouracil effectively inhibits epiretinal membrane formation and traction retinal detachment after vitrectomy surgery. When 0.5 mg of fluorouracil was administered intraocularly every 24 hours for seven days, traction retinal detachment two weeks after the intraocular injection of 200,000 cultured retinal pigment epithelial cells occurred in 12 of 12 control eyes but in only six of 14 eyes treated with fluorouracil (P less than .001). Four weeks after cell injection, eight of 12 eyes treated with fluorouracil had traction retinal detachments whereas 12 of 12 control eyes did (P less than .001). The height of the traction retinal detachment four weeks after intraocular injection of 200,000 cultured retinal pigment epithelial cells was reduced 50% in eyes treated with 0.5 mg of fluorouracil every 24 hours for seven days compared to control eyes (P less than .001). When the number of injected retinal pigment epithelial cells was increased to 400,000 cells and 1.25 mg of fluorouracil was administered intraocularly every 24 hours for seven days, traction retinal detachment two weeks after injection occurred in 15 of 15 eyes in the control group but in none of ten eyes in the treated group. Four weeks after cell injection, eight of eight eyes in the control group and five of five eyes in the fluorouracil-treated group had detachments and the mean height of the detachments in the two groups was equal. Autoradiography of the epiretinal membranes in eyes injected with 200,000 cultured retinal pigment epithelial cells and labeled for two hours with tritiated thymidine showed that 0.8% of the epiretinal cell nuclei were labeled two weeks after cell injection but that no labeled cells were present in the fluorouracil-treated eyes. Tritiated thymidine labeling of epiretinal cells in the fluorouracil-treated eyes was first noted three weeks after the cell injection. The presence of tritiated thymidine labeling in the fluorouracil-treated eyes correlated with an increase in the number of epiretinal cells and an increase in the incidence of traction retinal detachment.

Ocular toxicity of fluorouracil after vitrectomy.

The retinal and corneal toxicity of fluorouracil in the rabbit eye after lensectomy and vitrectomy depended on both the dosage and the frequency of intraocular injection and was reversible at certain dosages. All eyes in Group 1 (1.25 mg of fluorouracil every 12 hours for four days and then every 24 hours for three days) had opaque corneas by three days; these did not clear for four weeks. Histologic studies showed loss of photoreceptor outer segments and loss of ribosomes in all the retinal cells examined. The electroretinographic b-wave decreased to 0% of the baseline value (no b-wave), and did not recover after three weeks. In Group 2 eyes (1.25 mg of fluorouracil every 24 hours for seven days), corneal opacification increased to a maximum after two weeks and gradually decreased by four weeks. The electroretinographic b-wave diminished to 9.6% of the baseline value at two weeks but later recovered to 62.5% of the baseline value at three weeks. Histologic studies showed loss of photoreceptor outer segments and ribosomes at nine days; both returned to near normal after five weeks. Clinical, electrophysiologic, and histologic studies showed no toxicity in Group 3 eyes (0.5 mg of fluorouracil every 24 hours for seven days). This dosage of fluorouracil exerts a significant antiproliferative effect on injected retinal pigment epithelial cells and is well tolerated by the rabbit eye.

Epiretinal membrane formation after vitrectomy.

Our experimental model of epiretinal membrane formation in the rabbit eye after lensectomy and vitrectomy provides a way of studying pharmacologic and surgical approaches to inhibiting epiretinal cellular proliferation and contraction in the eye that has undergone vitrectomy. We injected 400,000 tissue-cultured retinal pigment epithelial cells onto the retinal surface of rabbit eyes that had undergone lensectomy, vitrectomy, and fluid-gas exchange. By one week, a funnel-shaped detachment of the medullary rays had occurred in 100% of the injected eyes. Histologically, the cells formed an epiretinal membrane by six hours after injection and caused major wrinkling of the inner retina after 24 to 48 hours. The percentage of tritiated-thymidine-labeled epiretinal cells increased dramatically 24 hours after injection and then declined. Cellular membranes bridging the optic nerve, followed by growth and contraction of the epiretinal cells on the detached internal limiting membrane, were responsible for the closed funnel appearance of the medullary rays.

Perioperative blood loss: the effect of valproate.

The purpose of this investigation was to determine the effect of the use of valproate (VPA) on bleeding and requirement for replacement blood products in patients undergoing major surgical procedures. One hundred thirty-nine patients had posterior spinal fusion performed by 1 of 3 surgeons at our institution from 1987 to 1993. The clinical status of the patient, pre- and postoperative laboratory values, type and extent of instrumentation, surgeon performing the procedure, and medications (including VPA) were variables considered. The outcome measures were intra- and postoperative blood loss and number of blood products used. Intraoperative blood loss was correlated with the method of instrumentation, platelet count, and the surgeon performing the procedure. Postoperative blood loss was correlated with the diagnosis of cerebral palsy. By hierachical stepwise regression analysis, the only outcome measure correlated with VPA was the number of blood products used.

Free fatty acid and triglyceride levels in neonates receiving triple mix hyperalimentation.

Total nutrient admixture (TNA) combines amino acids, lipids, and glucose in a single bottle for continuous parenteral use. This cost-effective and easily administered solution is now available for use in neonates. The present study was performed to assess the metabolism of fat administered as TNA in sick neonates as reflected by serum free fatty acid (FFA) and triglyceride (TG) levels. During a 6-month period, TG and FFA levels were monitored in all infants receiving TNA. Levels were measured within 24 hours of a change in lipid dose and then weekly when maximum intake (about 2 g/kg/day) was achieved. Sixty-nine TG and 58 FFA levels were obtained from 42 neonates who at the time of sampling were receiving 2 g/kg/day or more of parenteral lipid. Ninety-one percent of TG levels were less than or equal to 200 mg/dL. Ninety-six percent of FFA levels were less than or equal to 2000 mumol/L. A weak but statistically significant correlation was noted between TG and FFA levels with a correlation coefficient of 0.54. In conclusion, although the range of FFA and TG levels obtained from sick neonates on TNA therapy is relatively wide, these levels are comparable to those reported in the literature for infants receiving standard intravenous lipid infusions.

Tritiated uridine labeling of the retina: variations among retinal quadrants, and between right and left eyes.

We have determined the pattern of RNA labeling (uridine incorporation) in the normal retina of the domestic cat. One eye in each of eight cats was labeled by injecting [3H]uridine into the vitreous cavity. Two of the labeled eyes had the lens and vitreous removed 10 days before labeling. Three additional animals received intravenous (i.v.) injections of [3H]uridine. All animals were injected 4 hr into the light period and fixed 24 hr later; then the retinas were divided into quadrants (ST = superior temporal, SN superior nasal, IT = inferior temporal, and IN = inferior nasal). The ST quadrant contains the area centralis and the SN quadrant the optic nerve head. Autoradiograms were prepared from plastic sections 1 micron thick taken near the centre of each quadrant. In animals receiving intravitreal [3H]uridine, the ganglion cells and the inner and outer nuclear layers (INL; ONL) were heavily labeled; the synaptic layers and the retinal pigment epithelium (RPE) were very lightly labeled. Amacrines were the heaviest labeled cells in the INL; cones were more heavily labeled than rods in the ONL. This finding indicates that amacrines and cone photoreceptors may be synthesizing RNA more actively than other retinal neurons. In animals receiving intravenous [3H]uridine the pattern of labeling was the same as above except that the RPE was heavily labeled. Because cells in the ST quadrant appeared to be more heavily labeled than the same cell types in the other retinal quadrants, silver grains over the ONL in each quadrant were counted as grains micron -2 or grains per rod nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Glial fibrillary acidic protein (GFAP) immunoreactivity in rabbit retina: effect of fixation.

The binding of monoclonal and polyclonal antibodies to glial fibrillary acidic protein (GFAP) antigenic sites in the rabbit retina was shown to be sensitive to aldehyde fixation. In chemically unfixed retina, the polyclonal anti-GFAP labeled Müller cells, astrocytes, and unidentified profiles in the outer plexiform layer; the monoclonal anti-GFAP labeled Müller cell endfeet and astrocytes only. The outer plexiform layer label with the polyclonal antibody was lost after fixation for 1 hr in 1% paraformaldehyde; elsewhere, the label was reduced. Fixation also reduced labeling by the monoclonal antibody. Such fixation sensitivity may underlie the different patterns reported for retinal GFAP immunoreactivity in the literature.

Epidemiological basis of tuberculosis eradication. 10. Longitudinal studies on the risk of tuberculosis in the general population of a low-prevalence area.

The introduction of chemotherapy dramatically changed the epidemiology of tuberculosis as the risk of infection was thereby nearly eliminated. The present paper illustrates the risk of disease under these conditions. A large and representative segment of the Danish population, a total of over 626 000 persons aged 15-44 years, was examined by a standardized technique in 1950-52 and has now been followed for 12 years. It has been possible by means of simple parameters such as infection and vaccination status, X-ray lesion and age to divide the population into groups with widely different incidence rates. The time trend in disease rates among vaccinated persons and natural reactors suggests that post-primary tuberculosis is of great significance in the present tuberculosis situation. Three-quarters of all cases stem from the natural reactors. It would have been of great practical significance to identify high-risk groups which yielded a great part of the patients. This was not possible since the majority of cases developed among reactors whose distinctive feature was that they were infected at time of examination.

Tritiated uridine labeling of the retina: changes after retinal detachment.

As part of a study designed to examine the response of photoreceptor cells to outer segment injury (retinal detachment), the pattern of RNA labeling ([3H]uridine incorporation) has been determined in detached cat retinas. Retinas were experimentally detached from the adjacent cellular layer (the retinal pigment epithelium:RPE) by injecting fluid into the extracellular space between the retina and RPE. Twenty-four hours before the animals were killed they received intravitreal injections of [3H]uridine. Autoradiograms were prepared from plastic sections 1.0 micron thick taken from detached retinal regions and, because the detachments do not encompass all of the retina, from nearby attached retinal regions. Twenty-four hours after retinal detachment there is a decrease in labeling intensity of the photoreceptors and Müller's glia in the region of detachment (compared to cells in nearby attached regions). Seventy-two hours after retinal separation, the same result is obtained in the photoreceptors, but labeling intensity is greatly increased in both the nuclei and cytoplasm of Müller's glia. The decrease in [3H]uridine labeling of the photoreceptors correlates with a decreased staining intensity of the cytoplasm and ultrastructural signs of necrosis. The striking change in the pattern and intensity of labeling of the Müller cells precedes extensive hypertrophy of these cells and the appearance within their cytoplasm of numerous 10-nm diameter filaments. Two weeks, and also 1 month, after detachment the pattern and labeling levels are similar to those observed 1 day after retinal separation. These data suggest a highly localized change in metabolism because the change in RNA labeling is restricted to the region of detached retina.

An immunocytochemical comparison of Müller cells and astrocytes in the cat retina.

Immunocytochemical localization, at the light and electron microscopic levels, of five different known glial proteins was used to compare Müller cells with astrocytes in the adult cat retina. Retina from two different areas of the eye was examined. A region of retina on the border of the optic nerve was used because of its large population of astrocytes, and a region away from the optic nerve was used to examine Müller cells (astrocytes are sparse in this region). Antibodies to cellular retinaldehyde binding protein and glutamine synthetase labeled the Müller cells but not the astrocytes, while labeling with anti-carbonic anhydrase C, anti-alpha crystallin and anti-glial fibrillary acidic protein was found in both Müller cells and astrocytes.

Glial fibrillary acidic protein and its mRNA: ultrastructural detection and determination of changes after CNS injury.

We have previously demonstrated that glial fibrillary acidic protein (GFAP) containing intermediate filaments in retinal Müller cells undergo both quantitative induction and subcellular reorganization as a response to long-term retinal detachment (an induced CNS degeneration wherein the Müller cells form a multicellular scar). This study demonstrates by RNA blotting analysis that normal retina expresses a low basal level of GFAP mRNA, which is induced approximately 500% within 3 days of retinal detachment. At the cellular level, electron microscopic in situ hybridization analysis readily detects GFAP mRNA in Müller cells of detached retinas, but not in normal retinas. On the other hand, GFAP mRNA was readily detected in retinal astrocytes (which appear to express GFAP mRNA at high, constitutive levels). In both cell types, the ultrastructural localization of GFAP mRNA was the same. In the nuclei, the GFAP mRNA was associated with amorphous, electron-dense regions within the euchromatin. In the cytoplasm, the GFAP mRNA was associated with intermediate filaments near the nuclear pores, along the filaments when no other structures were apparent, and when the filaments appeared to be associated with ribosomes and polysomes. The ultrastructural location of the GFAP mRNA (especially along the intermediate filaments) may be unique to this mRNA or may represent a more generalized mRNA phenomenon.

Opsin distribution and protein incorporation in photoreceptors after experimental retinal detachment.

The distribution of opsin was examined immunocytochemically after experimental retinal detachment in adult cats. Retinal detachments were produced by injecting fluid between the retinal pigment epithelium and neural retina. One to 60 days later the animals were killed. Tissue areas from detached and attached retinal regions from the eye with the detached retina, as well as normal (control) retinas, were processed for post-embedding light and electron microscopic immunocytochemistry. In normal and attached retinal regions, anti-opsin labeled the outer segments and Golgi apparatus most heavily, although the entire photoreceptor plasma membrane was labeled at a low level. Beginning at 2 days after retinal detachment, immunolabeling increased in the photoreceptor inner segment, cell body and synaptic terminal plasma membranes. This pattern of anti-opsin labeling continued at all intervals up through the 60-day detachment time-point. Injection of radiolabeled amino acid in detachments from 1 to 30 days show that radiolabeled protein is still transported to the truncated outer segments of the photoreceptor cells. In addition, these outer segment disks label with anti-opsin. These data imply that opsin continues to be transported and incorporated into the outer segments of photoreceptors showing severe degeneration as a result of long-term detachment from the RPE.

A potential role for immune complex pathogenesis in drusen formation.

Drusen are abnormal extracellular deposits that accumulate between the retinal pigmented epithelium and Bruch's membrane and are commonly associated with age-related macular degeneration. Our recent work has identified a number of plasma proteins as molecular components of drusen. Of interest is the fact that many of these drusen-associated molecules are acute phase reactant proteins and some have established roles in mediating immune responsiveness. As immune and inflammatory responses appear to play a role in the formation of other pathologic age-related deposits, we examined the distribution of immunoglobulin molecules and terminal complement complexes at sites of drusen deposition. Here, we report that concentrations of immunoglobulin G and terminal C5b-9 complement complexes are present in drusen. In addition, we observe that retinal pigmented epithelial cells overlying or directly adjacent to drusen, as well as some within apparently normal epithelia, exhibit cytoplasmic immunoreactivity for immunoglobulin and the C5 component of complement. Taken together, these results suggest that drusen biogenesis may be a byproduct of immune responsiveness, and they implicate immune complex-mediated pathogenesis involving retinal pigmented epithelial cells as an initiating event in drusen formation.