Pharmacokinetics of genistein distribution in blood and retinas of diabetic and non-diabetic rats.

Genistein, a natural tyrosine kinase inhibitor, may act as an intraocular antiangiogenic agent. Its therapeutical use, however, is limited by its nonlinear pharmacokinetics. We aimed to determine genistein's kinetics and retinal tissue distributions in normal and diabetic rats. We developed an isocratic, reverse-phase C18 HPLC system to measure genistein concentration in blood and retinas of streptozotocin (65 mg/kg IV)-diabetic and non-diabetic rats receiving two types of genistein-rich diet (150 and 300 mg/kg) for ten days. Genistein's decay exhibited a two-compartmental open model. Half-lives of distribution and elimination were 2.09 and 71.79 min, with no difference between groups. Genistein steady-state concentration in blood for 150 and 300 mg/kg diet did not differ between diabetic (0.259 ± 0.07 and 0.26 ± 0.06 µg/ml) and non-diabetic rats (0.192 ± 0.05 and 0.183 ± 0.09 µg/ml). In retina, genistein concentration was significantly higher in diabetic rats (1.05 ± 0.47 and 0.997 ± 0.47 µg/gm wt. vs. 0.087 ± 0.11 and 0.314 ± 0.18 µg/gm wt., p < 0.05). The study determined that increasing genistein dose did not change its bioavailability, perhaps due to the poor aqueous solubility. The retina's increased genistein could be due to increased permeability of blood-retinal barrier that occurs early in diabetes.

[Does Stewart-Fencl improve the evaluation of acid-base status in stable patients on hemodiafiltration?]. / ¿Mejora la aproximación físico-química de Stewart-Fencl la valoración del equilibrio ácido-base en pacientes estables en hemodiafiltración?

INTRODUCTION: The traditional evaluation of acid-base status relies on the Henderson-Hasselbach equation. In 1983, an alternative approach, based on physical and chemical principles was proposed by P. Stewart. In this approach, plasma pH is determined by 3 independent variables: pCO2, Strong Ion Difference (SIDm), which is the difference between the strong cations (Na +, K +, Ca ++, Mg ++) and the strong anions (Cl-, lactate) and total plasma concentration of nonvolatile weak acids (ATot), mainly inorganic phosphate and albumin. Bicarbonate is considered a dependent variable. The aim of this study was to evaluate the acid-base status using both perspectives, physical chemical and traditional approach. MATERIAL AND METHODS: We studied 35 patients (24 M; 11F) on hemodiafiltration, mean age was 67,2+/-15,7, 8+/-19,2 kg. We analyzed plasma chemistry including pH, pCO2, HCO3-, base excess and Na+, K+, Cl-, Ca++, Mg++, lactate and SIDm. The SID estimated (SIDe) was calculated by Figge's formula (1000 x 2.46E-11 x pCO2 / (10-pH) + Album gr/dl x (0.123 x pH-0.631) + P in mmol/l x (0.309 x pH-0.469) and Gap of the SID as the difference SIDm-SIDe. RESULTS: pH preHD was 7,36+/-0,08 and pH posHD 7,44+/-0,08 (p < 0.001). There was no significant differences between pCO2 pre and pos-HD. HCO3 - and base excess increased during the session (p < 0.001). SIDm decreased from 46,2+/-2,9 preHD to 45+/-2,3 mEq/l postHD (p < 0.05). On the opposite, SIDe increased from 38,5+/-3,8 to 42,9+/-3,1 mEq/l (p < 0.001). The Gap Anion descended from 18,6+/-3,8 preHD to 12,8+/-2,8 mEq/l mEq/l postHD (p < 0.001) and the Gap of the SID 7,6+/-3 to 2,1+/-2 (p < 0.001). Anion Gap correlated with the Gap-SID so much pre-HDF as pos-HDF. Delta Base excess correlated only with Delta of the Gap SID. CONCLUSION: Stewart-Fencl's approach does not improve characterization of acid-base status in patients on chronic HDF. In presence of normocloremia the SIDm does not reflect the alkalinizing process of the session of hemodialysis. According this approach, hemodialysis therapy can be viewed as a withdrawal of inorganic anions, especially the sulphate. These anions are replaced by OH - and secondarily for HCO3-. The approach only improves the evaluation of unmeasured anions by the Gap of the SID, without the effect of albumin and phosphate.

Switching from three times a week to short daily online hemodiafiltration: effects on acid-base balance.

AIMS: This study examines the effect of a change from the standard 4-5 hours 3 times a week of online hemodiafiltration (OL-HDF) to 2-2.5 hours daily (6 times a week) OL-HDF, on acid-base balance, and attempts assess the modifications of acid-base parameters, ionic concentration, and electrical charges of albumin and phosphate available for diffusion and convection mechanisms across the membrane and subsequent infusion. METHODS: In 18 patients on online HDF, blood gas, electrolytes (Na, K, Cl), lactate, phosphate, albumin, apparent strong ion difference (SIDa), effective strong ion difference (SIDe), strong ion gap (SIG), anion gap (AG), and bicarbonate and pH time-averaged concentration (TAC) and time-averaged deviation (TAD) variables were evaluated at baseline, and 1, 3, 6, 9, and 12 months after patients were switched to daily OL-HDF. Additionally, in 12 patients, the same parameters measured simultaneously at dialyzer inlet, outlet, and after reinfusion were studied. RESULTS: Throughout the study, weekly single-pool Kt/V, equilibrated Kt/V, and TAC urea remained constant. However, standard Kt/V increased and TAD urea decreased on daily OL-HDF. There were no statistical differences during the time span of 12 months in pH, cations (Na, K), anions (Cl, HCO3(-) AG, and lactate), or SIDa, SIDe, and SIG pre-HDF; while pH and HCO3(-) TAD decreased from 0.02 and 1.02 +/- 0.74 mEq/L, to 0.01 and 0.64 +/- 0.52 mEq/L, respectively (p<0.01). Net albumin charge and AG increased significantly at dialyzer outlet and decreased after reinfusion. CONCLUSIONS: We did not observe changes in the acid-base balance in patients who switched from 3 times a week to short daily OL-HDF. The main benefit observed was a lower pH and bicarbonate TAD. This shows a better physiology for daily OL-HDF.

Racial differences in normal values of proptosis.

Measurement of proptosis was made with a Luedde exophthalmometer by one experimenter in 402 black and 325 white adults without endocrine disease or obvious orbital pathologic conditions. Black subjects had significantly higher values of proptosis than white subjects. It is suggested that the following "upper limits of normal" be used when clinically estimating proptosis: 19 and 21 mm for white female and male patients, respectively; and 23 and 24 mm for black female and male patients, respectively.

Stimulation by the antioxidant thioproline of the lymphocyte functions of old mice.

Diet supplementation with thioproline (thiazolidine-4-carboxylic acid), an intracellular sulfhydryl antioxidant and free radical scavenger, may slow the aging process of metazoans and prolongs their life span. In the present experiment Swiss mice fed thioproline (0.07%, w/w) from 13 to 22 months of age were used. Six- and 22-month-old mice fed standard diet were used as controls. Two important functions of lymphocytes, the proliferative response to the mitogen Concanavalin A (Con A) and the mobility, both spontaneous and directed to a chemoattractant gradient (chemotaxis), were analyzed in lymphocytes from axillary nodes, spleen and thymus. Mobility and chemotaxis were studied by Boyden's technique, using filters of 3 microns pore diameter, 3 h of incubation and fmet-leu-phe at 10(-8) M as chemoattractant. The proliferative response was estimated as 3H-thymidine incorporation in lymphocytes incubated for 72 h in the presence of Con A (1 and 5 micrograms/ml). The results show a decrease in mobility, chemotaxis and lymphoproliferative response in old mice in comparison to adults. However, a significant increase in these functions was observed in old mice fed thioproline. The advantage of using this antioxidant for immunostimulation during aging, a stage of life characterized by a decreased immune response, is discussed.

Breakdown of the blood-retinal barrier in a model of retinal neovascularization.

Breakdown in the blood-retinal barrier occurs in retinal neovascularization in a number of diseases. To study the anatomic basis of this breakdown, we examined retinal neovascularization induced by injection of 250,000 homologous fibroblasts into the vitreous cavity of pigmented rabbits. Neovascularization is evident by electron microscopy in this model 3 days after fibroblast injection. Fluorescein angiography followed by intravenous horseradish peroxidase (HRP) injection was performed prior to enucleation on 2, 3, 5, 7, and 14 days after fibroblast injection. Fluorescein leakage from retinal vessels occurs early (at day 1) and persists as the neovascularization progresses. The leakage in the early stages is concentrated near puckers from the medullary wings. In the later stages, fluorescein leakage is most prominent in the developing tips of the new vessels. Horseradish peroxidase was not observed to leak from the lumen of new vessels. "Gaps" or separations in the endothelial cell junctions were not observed in developing vessels. The breakdown of the blood-retinal barrier in this model of retinal neovascularization is therefore selective, (ie, fluorescein leaks but not HRP) and it is not due to gaps or fenestrations between endothelial cells in developing vessels.

Basic fibroblast growth factor stimulates 3H-thymidine uptake in retinal venular and capillary endothelial cells in vivo.

Recent studies have found basic fibroblast growth factor (bFGF), an angiogenic peptide, in retina and have suggested that bFGF is responsible for retinal vascular proliferation. To test the hypothesis that bFGF stimulates 3H-thymidine uptake in retinal vascular cells in vivo, we injected bFGF (100 ng) into the vitreous cavity of six cats at 0 hr and again at 24 hr. Eight control eyes received boiled bFGF or no injection. After 46 hr, 3H-thymidine was injected into the vitreous cavity of all eyes and 2 hr later the eyes were enucleated. Intense 3H-thymidine uptake was seen in eyes with bFGF (56 +/- 20 SD positive cells per section) but not in control eyes (7-10 positive cells per section (P less than 0.001). Trypsin digest preparations showed that the thymidine uptake was predominantly in the venular (89%) and capillary (10%) endothelium and not in arterioles (1%) (P less than 0.001). The data suggest that retinal venular endothelial cells respond preferentially to exogenous bFGF, and in part may explain their prominent role in the neovascular process. In a second group of experiments to test the hypothesis that retinal ischemia releases a diffusable factor similar to bFGF that can cause 3H-thymidine uptake in retinal vascular cells, we created branch retinal vein occlusion in six cat eyes. The fellow eyes received no injections. In the eyes with branch vein occlusion there was an intense 3H-thymidine uptake within the distribution of the occluded vein (84 +/- 77 SD positive cells per section), but none in the areas outside the occluded vein (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Histopathology of experimental preretinal neovascularization.

Despite the morbidity resulting from abnormal retinal neovascularization, morphological events associated with its development have not been fully described. We therefore studied sequential morphologic events during preretinal neovascularization in an experimental model induced by injection of 250,000 homologous fibroblasts into the vitreous cavity of rabbits. Within 2 days following fibroblast injection, thickening of many venular and capillary endothelial cells resulted in partial obliteration of their lumina. 3H-thymidine incorporation occurred first in the nonvascular cells of the superficial medullary ray and thereafter in the preretinal vessels and extraretinal fibroblasts. Capillary budding was obvious within 3 days, with endothelial cells extending cytoplasmic processes into fragmented extracellular matrix (ECM). Endothelial cells, at the tips of budding vessels, and at more proximal sites in the parent vessel, incorporated 3H-thymidine and did not lose cell contact or migrate individually into the ECM. Lumina were present throughout the entire length of the buds and endothelial cells remained polarized. Neovascular events observed in this experimental model parallel those previously described in diabetic retinopathy and retinopathy of prematurity in humans.

Glio-vascular architecture in the rabbit retina.

Differences between the rabbit and human retinal circulation, and the use of the rabbit eye in a model of experimental retinal neovascularization, necessitates a complete description of the normal vascular structure in the rabbit and its relationship to adjacent tissue, particularly the glia. The gliovascular relationships in the rabbit were studied by scanning and transmission electron microscopy. Utilizing gas compression of the vitreous to clear the wings of overlying vitreous, the authors were able to make detailed observations of retinal surface by scanning electron microscopy. Glial sheaths surrounding a large number of medium size and smaller vessels were observed. The glial sheaths contained cells which were ultrastructurally similar to Müller cells. No isolated glial tufts were observed in avascular areas. Finally, small, smooth-surfaced cells were found adjacent to many vessels. The exact nature and function of these cells remains unknown.

Blood-retinal barrier breakdown investigated by real-time magnetic resonance imaging after gadolinium-diethylenetriaminepentaacetic acid injection.

Recent magnetic resonance imaging (MRI) studies show that gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) entry into the vitreous space can be used as a qualitative marker of blood-retinal barrier (BRB) disruption. To determine if a more quantitative measurement of BRB breakdown could be obtained, the utility of acquiring real-time, T1-weighted proton images was studied after Gd-DTPA injection. Two days before the MRI experiment, panretinal photocoagulation was done. The mean signal intensity over selected regions-of-interest (ROI) in the vitreous and anterior chamber was followed before and after (0, 10, 20, 30, 45, and 60 min) Gd-DTPA injection (1.0 mmol/kg, intravenously). At every laser power setting used in this study (0, 200, 400, 600, and 800 mW), the change in the mean signal intensity could be approximated by a simple exponential equation. However, the time constants determined for these curves were too imprecise to be useful as correlates between laser power and BRB breakdown. The slope of the line fit to the data in the first 20 min postinjection (ie, an initial-rate analysis) was a more precise correlate between BRB breakdown and laser power. This slope represented the rate of change in mean signal intensity in the ROI as a result of the entry of Gd-DTPA, and it was called the "leakiness" parameter. The leakiness parameter reflected changes in the permeability surface area product of the BRB if the blood flow and the Gd-DTPA arterial concentration immediately after injection were approximately the same between animals.

In vivo imaging of breakdown of the inner and outer blood-retinal barriers.

Real-time contrast-enhanced magnetic resonance imaging (MRI) was used to distinguish between experimentally induced breakdown of the vascular (inner) and retinal pigment epithelial (RPE; outer) blood-retinal barrier (BRB) in vivo. Pigmented rabbits were treated with intravenous sodium iodate 30 mg/kg, (a specific RPE cell poison), intravitreal N-ethylcarboxamidoadenosine (NECA) 10(-3) mol/l (which specifically disrupts the vascular BRB), or retinal diode laser photocoagulation. Coronal T1-weighted proton images were acquired in a timed sequence after intravenous injection of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). Images were analyzed to localize leakage of Gd-DTPA and determine the permeability surface area product normalized per unit area (PS). The pattern of enhancement observed in eyes treated with sodium iodate differed clearly from that in eyes treated with NECA. PS' values were significantly higher in eyes treated with sodium iodate than with NECA. Simultaneous leakage from the outer and inner BRB in eyes treated with dense retinal laser photocoagulation could be localized and quantitated independently.

Genistein, a protein tyrosine kinase inhibitor, ameliorates retinal degeneration after ischemia-reperfusion injury in rat.

PURPOSE: The authors sought to determine the effect of genistein, a naturally occurring protein tyrosine kinase inhibitor, in a model of ischemia-reperfusion injury in the rat retina. METHODS: Ischemia-reperfusion injury was induced by temporary optic nerve ligation. A dose of 0.034 mg, 0.34 mg, and 3.4 mg of genistein or dimethyl sulfoxide (DMSO) alone was injected intraperitoneally 1 hour before the ligation of the optic nerve and just after the start of reperfusion. After 48 hours of reperfusion, the effect of genistein on overall protein tyrosine phosphorylation in the retina was studied using Western blot analysis. After 168 hours, the effect of increasing doses of genistein on retinal degeneration was examined by quantitative morphometric analysis of histologic sections of the retina. RESULTS: The authors found that tyrosine phosphorylation was increased after 48 hours of reperfusion in the ischemia-reperfusion-injured eyes treated with DMSO alone. A severe inner retinal degeneration was observed in the animals treated with DMSO alone after 168 hours of reperfusion. The treatment with 3.4 mg genistein inhibited the increase in tyrosine phosphorylation and protected the eyes significantly from the induced ischemic retinal degeneration by morphometric analysis of the mean thickness of the inner limiting membrane to the outer limiting membrane, the inner plexiform layer, and the inner nuclear layer (P < 0.02). Treatments with lower amounts of genistein (0.034 mg and 0.34 mg) did not show a significant protection of retinal degeneration after the injury. CONCLUSIONS: Systemic administration of high dose of genistein, a dietary-derived isoflavone, can ameliorate an ischemia-reperfusion-induced retinal degeneration. Genistein may be useful to prevent neuronal degeneration in the inner retina as a result of ischemic injury.

Activation of protein tyrosine phosphorylation after retinal branch vein occlusion in cats.

PURPOSE: The authors examine the effect of retinal branch vein occlusion (BVO), a common retinal vascular disorder, on protein tyrosine phosphorylation, production of angiogenic growth factors, and activation of signal proteins in the tyrosine kinase pathways in the retina. METHODS: Retinal branch vein occlusion was induced in cat retina by coagulation of retinal veins with diathermy. At 2 days, 1, 3, and 6 weeks after induction of BVO, the retina was divided into three parts: a part within the distribution of the occluded vein (BVO[IN]) or a part outside the distribution of the occluded vein (BVO[OUT]). Each part of the retina was prepared for Western blot analysis of tyrosine-phosphorylated proteins, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and four signal proteins in the tyrosine kinase pathways, which were phospholipase C gamma (PLC gamma), C-Src, SH2-containing protein (SHC), and mitogen-activated protein kinase (MAPK). RESULTS: Overall, tyrosine-phosphorylated proteins were increased after BVO, especially in BVO(IN) at 2 days and 1 week. The VEGF and bFGF also were increased in BVO(IN) at 1 week and 2 days, respectively. The PLC gamma and MAPK were activated at these time points. The C-Src and SHC were not activated in the retina after BVO. CONCLUSIONS: The BVO increased overall protein tyrosine phosphorylation in the cat retina in association with increase of angiogenic growth factors (VEGF and bFGF) and activation of two signal proteins (PLC gamma and MAPK) in the tyrosine kinase pathways. These results suggest that the protein tyrosine phosphorylation may in part play an important role in mitogenesis of vascular endothelial cells and other retinal responses after BVO.

Increase of protein tyrosine phosphorylation in rat retina after ischemia-reperfusion injury.

PURPOSE: This study was conducted to examine the effect of retinal ischemia-reperfusion injury on protein tyrosine phosphorylation, the production of angiogenic growth factors, and the activation of signal proteins in tyrosine kinase pathways. METHODS: Ischemia-reperfusion injury was induced in rats by compression of the optic nerve for 2 hours. The rats were killed, and the retinas were collected at 0, 1, 6, 24, 48, 96, or 168 hours of reperfusion. Tyrosine phosphorylation of proteins in the retina was examined by Western blot analysis and immunohistochemistry. Angiogenic growth factors and their receptors, such as basic fibroblast growth factor (bFGF) and Flg, vascular endothelial growth factor (VEGF) and Flk-1, platelet-derived growth factor (PDGF)-B chain and PDGF-beta receptor, and five intracellular signal proteins (phosphatidylinositol 3-kinase [PI3K], phospholipase C gamma [PLC gamma], C-Src, SHC, and mitogen-activated protein kinase [MAPK]) were examined by Western blot analysis. RESULTS: Protein tyrosine phosphorylation increased after ischemia-reperfusion injury, reaching a peak at 48 hours of reperfusion. Increased staining of tyrosine-phosphorylated proteins in the inner retina were evident on immunohistochemical examination. The amount of bFGF decreased after injury, but the amounts of VEGF and PDGF-B chain increased. Tyrosine phosphorylation of PLC gamma, SHC, and MAPK was increased at 48 hours of reperfusion, and tyrosine phosphorylation of PDGF-beta receptor and PI3K was increased at 168 hours of reperfusion. CONCLUSIONS: Ischemia-reperfusion injury in the rat retina leads to activation of the tyrosine kinase pathway, increasing the amounts of angiogenic growth factors. The resultant activation of signal proteins PLC gamma, SHC, MAPK, PI3K, and PDGF-beta receptor may play an important role in ischemia-induced retinal changes such as cell proliferation.

Primary culture of human retinal glia.

Glial cells of the human retina participate in various pathologic processes characterized by cell migration, proliferation, and extracellular matrix production. To study these events in vitro, a procedure was developed to obtain primary cultures of human retinal glial cells. The cultures resulting from the processing of 130 globes contained cells with variable morphology including bipolar and multipolar or stellate cells. Most cells in the primary culture were labeled with antisera to the glial fibrillary acidic protein. The cultures were also examined with antibodies directed against factor VIII-related antigen and muscle-specific actin to determine the presence of endothelial cells and pericytes. A variable contamination of cells staining for the latter was found in these cultures (usually less than 10%). Together, these data indicated that the primary cultures arose principally from glial cells of the human retina but did not precisely identify the cell of origin.

Accurate and precise measurement of blood-retinal barrier breakdown using dynamic Gd-DTPA MRI.

Dynamic T1-weighted magnetic resonance imaging (MRI) after the injection of Gd-DTPA is a promising method for investigating breakdown of the blood-retinal barrier (BRB). Previously, the authors demonstrated that in a T1-weighted image, the initial rate of change in the vitreous water MRI signal as gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) enters the vitreous space strongly correlated with the extent of BRB breakdown. Here, a practical approach to measuring a more relevant physiologic parameter is presented: the permeability surface area product (PS). The theory is a development of earlier work used in investigating the breakdown of the blood-brain barrier. The accuracy and precision of this approach was investigated in rabbits pretreated with sodium iodate (30 mg/kg intravenously). The MRI-derived PS normalized to the area of leaky retina (5.65 +/- 0.25 x 10(-4) cm/min, mean +/- standard error of the mean; n = 6) was compared to a similarly normalized PS calculated using a classical physiologic method (4.12 +/- 0.73 x 10(-4) cm/min; n = 6). Good agreement between the two methods was found (P = 0.09). This result demonstrates that the MRI-derived PS is an accurate and precise measure of BRB breakdown under these conditions. The mathematical model of Gd-DTPA distribution in vivo also is validated. Based on these results, several potential sources of error are discussed, including the effect of back-flow of Gd-DTPA from the vitreous space to the plasma, the underlying vascular patency, and MRI slice selection.

Inhibition of choriocapillaris regeneration with genistein.

PURPOSE: To test the effects of genistein on choriocapillaris regeneration and retinal pigment epithelial (RPE) wound healing in a surgical model in the rabbit. METHODS: Intravitreal injections of either 0.1 ml of a 90-microM concentration of genistein, dimethyl sulfoxide (DMSO; negative control), or 2 microg cycloheximide (positive control) were given 24 hours before surgical debridement of RPE in rabbits. Scanning electron microscopy (EM) of choroidal vascular casts and the RPE wounds and histologic evaluation by light microscopy and EM of the disturbed areas were performed at days 1, 7, and 30 after surgery. Quantitative analysis of the area of the choriocapillaris bed and RPE was performed by automated image analysis, and the results were analyzed by paired Student's t-test. RESULTS: Loss of RPE caused a rapid initial atrophy followed by slower subsequent revascularization of the choriocapillaris, which paralleled the RPE wound healing. Choriocapillaris regeneration appeared nearly normal by day 30 in the DMSO group. Inhibition of choriocapillaris revascularization by genistein was significant at day 30 when compared with the DMSO-treated negative control (P = 0.013). There was a strong trend toward inhibition in the cycloheximide-treated positive control group (P = 0.062), which reached significance at day 7 compared with the DMSO group (P = 0.02). RPE covered the wound area by day 7 in all groups. CONCLUSIONS: Intravitreal injection of genistein was found to cause significant inhibition of choriocapillaris regeneration without apparent effect on RPE wound healing. Tyrosine kinase inhibitors such as genistein may be useful as a pharmacologic approach in the treatment of choroidal neovascularization.

Morphometric analysis of the extramacular retina from postmortem eyes with retinitis pigmentosa.

PURPOSE: To evaluate the degree of inner retinal preservation in the extramacular regions of postmortem retinitis pigmentosa (RP) eyes. METHODS: Eighteen RP retinas and 11 age-matched healthy retinas were sectioned for morphometric analysis by light microscopy. The 18 RP retinas were classified by disease severity and mode of inheritance. Cell nuclei in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) were counted in adjacent 125-microm segments from an area spanning the region between 4 mm and 10 mm from the fovea. RESULTS: A mixed-effects model showed a decrease in mean cell counts for each of the cell layers when the severity groups and inheritance types compared with those of control retinas. There was no statistically significant difference in the number of nuclei preserved in the INL and GCL in the moderate group compared with the severe group. Results from the INL counts for the different inheritance types of RP showed a higher overall mean percentage of cells was preserved for the autosomal dominant RP (ADRP) group when compared with the X-linked (XLRP) and simplex RP groups. Analysis of the GCL counts revealed significantly more counts only in the ADRP group compared with the XLRP group; the other group comparisons were not significant. CONCLUSIONS: Retinitis pigmentosa results in cell loss in all retinal layers, with the most profound loss in the ONL, followed by the GCL and then the INL. The preservation of the INL and GCL in the extramacular region is less than that previously reported for the macular region of the same retinas.

Photosensitization-induced retinopathy in the newborn beagle.

PURPOSE: Photosensitization is a mechanism by which oxygen and light may interact to generate free radicals capable of tissue injury. It has been proposed as a possible etiologic mechanism in the development of retinopathy of prematurity. The authors report the effects of light exposure and a photosensitizer, rose bengal (RB), on the developing retina of the beagle puppy. METHODS: Seven purebred beagle puppies (2 or 7 days old) were given RB (7.5 mg/ml) intravenously (0.9 ml bolus followed by a 74 microliters/min constant infusion), and one eye was exposed to filtered light delivered by a modified slit lamp at 30 mW/cm2 for 5, 15, 25, 35, and 45 minutes. The fellow eye was not irradiated and served as an RB-only control. Three beagles were exposed to light (one eye only) for 15 to 120 minutes in the absence of RB to provide light-only and no-treatment control eyes. Animals were followed clinically by indirect ophthalmoscopy and fluorescein angiography, and eyes were processed for light microscopy. RESULTS: Clinical and/or histologic abnormalities were found in all seven eyes exposed to light in the presence of rose bengal, as follows: vitreous hemorrhage in four eyes, incomplete peripheral retinal vascularization in four eyes, fibrovascular/fibrocellular proliferation with traction on the retina in two eyes (including preretinal neovascularization in one eye), total or partial retinal detachment in two eyes, dilated peripheral vessels compatible with shunt vessels in one eye, retinal dysplasia with loss of normal architecture and formation of rosettes in five eyes. All 13 control eyes showed normal, complete retinal vascularization. CONCLUSIONS: The failure to progress to the end-stage tractional retinal detachment seen in human infants is a shortcoming of existing animal models. Photosensitization injury to the developing retina in this animal model can produce a spectrum of retinal pathology that includes extraretinal vascularization and subsequent retinal detachment.

Human neural retinal transplantation.

PURPOSE: A pilot study of human neural retinal transplantation was undertaken to investigate three major issues: whether a safe surgical procedure could be devised for transplantation of neural retinal tissue into the subretinal space, whether the transplant would be accepted in the subretinal space, and whether an improvement in vision could be achieved. METHODS: Eight patients with bare light perception (LP) vision due to retinitis pigmentosa (RP) and one patient with bare LP vision due to advanced neovascular age-related macular degeneration (AMD) received subretinal transplants of human fetal retinal microaggregate suspensions without postoperative systemic immunosuppression. The patient with AMD also received a fetal retinal sheet transplant. The ages of the patients ranged from 31 to 94 years (median, 55 years). The pre- and postoperative evaluations included visual function testing, detailed fundus examinations, fundus photography, fluorescein angiography, macular perimetry using a scanning laser ophthalmoscope (SLO), and full field and focal electroretinograms (ERGs). RESULTS: Three of the eight RP patients demonstrated possible improved light sensitivity during the initial months of follow-up. However, visual improvement disappeared between 3 and 13 months of follow-up. After transplantation, no subject showed any changes in the ERG recordings or SLO macular perimetry relative to their preoperative baseline. No patient experienced a retinal detachment, infection, or extensive bleeding. None of the patients developed retinal vasculitis or intraocular inflammation. In one RP patient, fluorescein angiography and fundus photography documented the formation and maturation of new host retinal vessels in the area of the transplant. CONCLUSIONS: Transplantation of fetal retinal photoreceptor suspensions into the subretinal space was achieved safely in nine subjects. Although a definite positive effect on visual function could not be demonstrated, the apparent high tolerance for graft tissue is promising for future efforts in the field of neural retinal transplantation.