Total Retinal Area Research Articles

Ultra-Wide–Field Imaging for Cytomegalovirus Retinitis

To compare the Optomap imaging system (Optos PLC, Dunfermline, Scotland) with conventional fundus photography in patients with cytomegalovirus (CMV) retinitis. Patients treated at Rush University for acquired immune deficiency syndrome and CMV retinitis were studied prospectively. Conventional nine-field photography and Optomap ultra-wide-field photography were performed on the same day. A patient satisfaction questionnaire was completed. The main outcome measures were total retinal area imaged, area of CMV retinitis imaged, and patient satisfaction. Twelve eyes met the inclusion criteria. Ultra-wide-field imaging captured 48.3% greater retinal area and 40.0% greater CMV retinitis area compared with standard photography. Standard photography missed peripheral CMV lesions in two eyes. The patient satisfaction survey indicated a preference for ultra-wide-field imaging based on increased comfort and decreased imaging time. The ultra-wide-field Optomap imaging system tended to capture greater areas of total retina and peripheral CMV retinitis lesions. Patients tended to prefer ultra-wide-field imaging because of the perceived time requirements and comfort of use.

The protective effect of 17 beta-estradiol on oxygen-induced retinopathy and its relation with the changes of malondialdehyde

Objective Retinopathy of prematurity is becoming obvious with the improvement of neonatal ambulance. However there is still not a good treatment. The present study is to observe the effect of 17 beta-estradiol (E2) on oxygen-induced retinopathy (OIR), and explore the relationship between the changes of avascular area and malondialdehyde (MDA) in retina. Methods Newborn oxygen-exposed mice underwent subcutaneous injections of different dose of E2 (0.1 μg, 1.0 μg, 10.0 μg), tamoxifen or phosphate buffered saline (PBS; controls) everyday from post-natal day (p)7 to p17. At p17, retinal flat mounts were scored for the percentage of avascular/total retinal area, and pathological changes during revascularization. The MDA concentration in the retina was determined also. In the most efficacious E2 group (10.0 μg), 100.0 μg tamoxifen was also administered, and the percentage of capillary-free/total retinal area determined, and the retinal malondialdehyde concentration assayed. Results The mean percentage of capillary-free area over total retinal area was 0(PBS, in room air), 34.197±1.301(PBS, in hyperoxia), 23.685±0.407 (0.1 μg E2), 14.648±0.355 (1.0 μg E2), 4.693±0.450 (10.0 μg E2) and 32.240±0.654 (10.0 μg E2 +100.0 μg tamoxifen). The difference was significant ( F = 2778.759, P < 0.01), and the difference between any two groups were also significant (all P value were less than 0.01). The predilection of tufts and clusters during revascularization was mainly aggregated in zones 2 and 3, but the difference of retinal neovascular clusters and tufts in fourth zone among different groups were significant [clusters ( F = 44.719, P < 0.01) vs tufts ( F = 39.997, P < 0.01)]. The mean MDA concentration were 0.711±0.037(PBS, in room air), 2.084±0.066 (PBS, in hyperoxia), 1.829±0.091(0.1 μg E2), 1.152±0.067(1.0 μg E2), 0.796±0.027(10.0 μg E2), 1.988±0.049(10.0 μg E2 +100.0 μg tamoxifen) ( F = 628.103, P < 0.01). The difference between any two groups were also significant (all P value were less than 0.05). The close relation between the percentage of avascular/total retinal area and MDA concentration was also verified ( r = 0.981, P < 0.01). Conclusion Oxidative stress responses play a pivotal role in OIR, by means of receptor pathway. E2 can alleviate oxidative stress reaction, and thus ameliorate the severity of oxygen induced retinopathy.

Effect of Ethylene Diurea on Oxygen-induced Ischemic Retinopathy in the Neonatal Rat

This study investigated the effect of N[2(2-oxo-1-imidazolidinyl) ethyl]-N-phenylurea (ethylene diurea, EDU) on oxygen-induced ischemic retinopathy (OIR) in a neonatal rat model. OIR was induced by maintaining daily cycles of 80% oxygen (20.5 h), ambient air (0.5 h), and a progressive return to 80% oxygen (3 h) for 12 days (postnatal day : P12). The rats were treated intraperitoneally with EDU (30 mg / kg body weight) or distilled water (DW) from P6 to P17. At P18, the percentage of avascular areas in the total retinal area (%AVA) was measured, and retinal neovascularization (NV) was scored in ADPase-stained retinas. Retinal superoxide dismutase (SOD) activity in the retina was also determined by a chemiluminescence method. The mean %AVA in the EDU-treated group (9.3±1.7%, n= 16) was lower than in the DW group (18.2 ± 4.7%, n = 17). EDU did not signi cantly affect NV, but significantly increased SOD activity (1.36±0.13 units / mg protein, n = 4) compared to DW treatment (1.04±0.01 units / mg protein, n = 4, P= 0.032) at P18. These results suggest that EDU treatment decreased the %AVA, accompanied by an increase in normal retinal vascular growth and / or a decrease in vessel proliferation. The increased SOD activity observed in the present study is likely to involve the EDU-mediated effects.

Effects of Green Tea Fractions on Oxygen-Induced Retinal Neovascularization in the Neonatal Rat

This study aimed to investigate the preventive effects of green tea fractions (GTFs) on rat model of oxygen-induced retinopathy (OIR). Neonatal Sprague-Dawley rats were exposed to daily cycles of 80% oxygen (20.5 h), ambient air (0.5 h), and progressive return to 80% oxygen (3 h) until postnatal day 12 (P12), then the rats were placed in ambient air until P18. The green tea was fractionated by DM-A50, DM-W, M-B, and M-W. The rats were treated once daily from P6 to P17 by gastric gavage of GTFs (0.05 or 0.01 g/ml) or distilled water (DW) at 50 µl/10 g body weight. On P18, the rats were sacrificed and the retinal samples were collected. The retinal neovascularization (NV) was scored and avascular areas (AVAs) were measured as a % of total retinal area (%AVAs) in ADPase stained retinas. The NV scores in 0.01 g/ml M-W were significantly lower than those in DW. The %AVAs in 0.05 g/ml DM-A50 and in 0.05 g/ml and 0.01 g/ml M-W were significantly lower than those in DW. There were less catechins, and less caffeine in M-W fraction compared with other GTFs, suggesting components of green tea except for catechins and caffeine might suppress the neovascularization in rat model of OIR.

Defective Angiogenesis, Endothelial Migration and MAPK Signaling in Rap1b−/− Mice.

The small GTPase rap1 regulates multiple cellular processes. Murine rap1b-deficiency is associated with up to 85% lethality due to embryonic hemorrhage, occurring at about E13.5. Surviving rap1b-null mice have a mild platelet aggregation defect (Chrzanowska-Wodnicka et al., JCI, 115:680), but a minimal symptomatic bleeding. Because of the discrepancy between the severity of embryonic and adult phenotypes and because vascular defects are a more likely cause of embryonic hemorrhage we examined vascular patterning in rap1b−/− mice. PECAM1 whole-mount staining at E10.5, and PECAM1 and smooth muscle actin staining of multiple organs at E13.5 was normal in rap1b−/ − mice. Angiogenesis is the main mechanism through which primary vascular plexus is remodeled in embryos after mid-gestation and after birth. In the neonatal retinal model of angiogenesis, blood vessels form as a superficial primary vascular plexus originating at the optic nerve head and spreading over the inner surface of the retina. The measurement of the fluorescently-labeled isolectin-stained vascularized area of the fixed retinas provides a quantitative assessment of angiogenesis. Primary vascular plexus covered 74.3 ± 5.6% of the total retinal area of P7 rap1b +/− mice. In contrast, the vascularized area in rap1b −/ − littermates was decreased to 57.3 ± 7.6% of the total area (data from 7 litters). There was no significant reduction in the vascularized area of rap1b+/− mice when compared to wild-type (WT) littermates (73.63 ± 6.24% and 75.17 ± 4.67%, respectively; data from 9 litters). To further explore the impact of rap1b-deficiency on ex-vivo angiogenesis, we performed aortic ring assay in which isolated aortas were sectioned, embedded in Matrigel and subjected to angiogenic factors. We found a significant decrease in the average number of sprouts from rap1b−/ − rings compared to WT controls in response to bFGF (18.5 ± 7.3 in WT vs. 2.2 ± 3.5 in rap1b−/ − aortas), VEGF (4.94 ± 0.57 in WT vs. 0.86 ± 0.52 in rap1b−/ − aortas), and sphingosine-1− phosphate (3.45 ± 1.23 in WT and 0.88 ± 1.13 in rap1b−/ −). To establish if defective migration may be responsible for the defective angiogenesis in rap1b−/ − mice, we wounded monolayer cultures of lung endothelial cells isolated from rap1b−/ − and WT mice with a pipette tip and subjected them to VEGF or bFGF stimulation. Cells at the wound edge migrated into the wound, closing it over time. The progress of wound closure, a measure of the migration rate, was significantly delayed in rap1b−/ − cells compared to WT cells in response to either VEGF or bFGF (P values were 0.0234 and 0.0476, respectively; α=0.025 in a two-tailed paired t-test, n=9, cells isolated from 4 sets of mice). To begin to elucidate the molecular mechanism responsible for the defective angiogenic response of rap1b−/ − endothelial cells we analyzed signaling downstream from VEGF and bFGF receptors. We found that phosphorylation of p38 MAP kinase, a key regulator of migration, was decreased in rap1b−/ − cells in response to either angiogenic factor. Phosphorylation of ERK, another MAP kinase and a key regulator of cellular proliferation, was decreased in rap1b−/ − cells in response to VEGF treatment. As phosphorylation of these kinases is indicative of their activation, we conclude that rap1b deficiency in endothelial cells leads to defective p38 and ERK signaling downstream from the angiogenic receptors and may provide a molecular explanation for the observed defective in vivo and ex vivo angiogenesis in rap1b−/ − mice.

Inhibition of retinal neovascularization by soluble EphA2 receptor

Eph receptor tyrosine kinases (RTKs) and their ligands, known as ephrins, play an important role in vascular remodeling during embryogenesis, but their functions in adult angiogenesis are just beginning to be investigated. In this report, we investigated the effect of blocking EphA receptor activation on VEGF-induced angiogenic responses of cultured retinal endothelial cells and on retinal neovascularization in a rodent model of retinopathy of prematurity (ROP). Soluble EphA2-Fc receptors inhibited ephrin-A1 ligand or VEGF-induced BRMEC migration and tube formation without affecting proliferation in vitro. Since EphA2-Fc receptors can inhibit activation of multiple EphA receptors, the specific role of EphA2 receptor in angiogenesis was further investigated in EphA2-deficient endothelial cells. Loss of EphA2 in endothelial cells leads to defective cell migration and assembly in response to either ephrin-A1 or VEGF. Finally, a significant reduction in the severity of abnormal retinal neovascularization was observed in the eyes treated with soluble EphA2-Fc receptors, yet the normal total retinal vascular area was not significantly changed. Because soluble Eph receptor significantly inhibited pathologic retinal angiogenesis without affecting normal intraretinal vessels, it may be a promising agent for treatment of retinal angiogenesis in a number of human ocular diseases.

Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model

Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model, however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains. IOP was raised in one eye of Wistar ( N=5) and Brown-Norway(B-N)( N=7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5–4.5 additional months. B–N rats ( N=7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined. Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. The group of normal B–N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B–N experimental eyes had at least 30% fewer RGCs than contralateral control eyes. Total retinal area in B–N experimental eyes was higher compared to contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (>30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones. The above results indicate that RGC loss in both Wistar and B–N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP-stressed RGCs. Identification and enhancement of such factors could prove useful for glaucoma therapy.

Strain-Dependent Differences in Oxygen-Induced Retinopathy in the Inbred Rat

To examine the susceptibilities of different rat strains to oxygen-induced retinopathy, a model of human retinopathy of prematurity. Litters of newborn rats of five inbred strains (Fischer 344 [F344], Dark Agouti [DA], Sprague-Dawley [SD], Wistar-Furth [WF], Lewis [LEW]) and one outbred strain (Hooded Wistar [HW]) were maintained in room air or were exposed to alternating 24-hour cycles of hyperoxia (80% oxygen in air) and normoxia (21% oxygen in air) for 14 days and were killed for analysis, either immediately (postnatal day 14, [P14]) or after 4 days in room air (P18). The fluorophore-conjugated isolectin GS-IB4 was used to label the endothelial cells of wholemounted retinas, and digital images were analyzed for avascular area and for morphologic abnormalities. Exposure to cyclic hyperoxia inhibited retinal vascularization in all strains relative to age-matched room air control animals. Total retinal avascular area at P14 after cyclic hyperoxia varied significantly among strains (P < 0.001). Avascular areas were smallest for the albino F344, WF, and LEW strains; larger for the albino SD strain; and largest for the pigmented DA and HW strains. Susceptibility to hyperoxic vascular attenuation was associated with ocular pigmentation, but neither with body mass nor with natural variation in litter size. Room air exposure for 4 days after cyclic hyperoxia was also associated with strain-related differences in retinal vascularization and with abnormalities in vascular morphology (P < 0.05). For all strains, the size of the avascular retinal area at P14 was predictive of the severity of morphologic abnormality at P18. Marked and consistent variations in the response of different inbred rat strains to cyclic hyperoxia were observed, suggestive of a genetic component to oxygen-induced retinopathy.

Clinician versus Reading Center Assessment of Cytomegalovirus Retinitis Lesion Size

To compare clinician and fundus photograph reading center assessments of the cytomegalovirus (CMV) retinitis area and change in the CMV retinitis area over time, and to investigate how these assessments correlate with the visual field (VF) of eyes with CMV retinitis. Analysis of pooled data from 2 multicenter randomized clinical trials and 1 prospective multicenter epidemiologic study. Ninety-five eyes of 79 patients. At baseline, each eye had CMV retinitis restricted to zone 1 and/or zone 2 (approximately the photographable postequatorial retina), as assessed by the evaluating clinician. Comparison of CMV retinitis area, change in area over time as assessed by clinicians and a fundus photograph reading center, and correlation of these assessments with VF measurement. Cytomegalovirus retinitis area, change in CMV retinitis area over time, and VF score. Baseline assessments of the mean retinitis area were, by clinicians, 12.8% of the total retinal area and, by the reading center, 6.3% of the total retinal area (P<0.001). There was a positive correlation between clinician and reading center assessments of retinitis area at baseline (rho = 0.77 and P<0.0001 by Pearson correlation and rho = 0.54 and P<0.001 by concordance). Both clinician and reading center size measures correlated negatively with VF (Spearman correlation rhos = -0.38 and -0.52, respectively; P<0.001 each). Mean changes in area over a 3-month interval were, by clinicians, +1.2% and, by the reading center, +1.1% (P = 0.68). Regression analysis showed a positive concordance (rho = 0.42, P<0.001). Change in VF over a 3-month interval did not correlate with change in retinitis area as assessed by clinicians or the reading center. Awareness of the similarities and differences between clinician and reading center assessments of CMV retinitis area should permit clinicians to apply research data to clinical practice more effectively. Clinician assessment of retinitis area correlates negatively with VF, a clinically meaningful visual outcome in patients with CMV retinitis.

Clinical study of laser-induced chorioretinal venous anastomosis

To test the effectiveness and safety of laser-induced chorioretinal venous anastomosis in the treatment of nonischemic central retinal vein occlusion (CRVO), and also the effectiveness of the preventative measures for complications associated with the treatment. Laser-induced chorioretinal venous anastomosis was performed on 34 cases, 35 eyes with nonischemic CRVO. During the treatment, preventative measures for complications were taken as follows: (1) Patients with total retinal papillary nonperfusion area of less than 5 DA were selected to receive the treatment. (2) Treatment sites must be at least 3 PD away from the optic disc and 4 PD away from the macula. (3) At most 2 argon laser shots were attempted over each treatment site, to avoid severe laser damage. (4) Scattered argon laser photocoagulation was done over the drainage area of the vein distal to the treatment site, in order to prevent neovascular complications in case distal vein occlusion occur. During the 3 approximately 12 months (average 6 months) follow-up, successful anastomosis formed in 28 eyes out of 35 eyes (80%) with a mean of 2.4 attempts to create an anastomosis in each eye. With successful anastomosis, retinal hemorrhage started to be absorbed gradually 2 weeks after the treatment, and almost entirely absorbed 1 approximately 2 months later. Retinal edema also alleviated. Visual acuity improved in 20 out of 28 eyes (71%), and remained unchanged in the rest of the patients with successful anastomosis. In comparison with the natural course of the disease, our treatment has improved both the visual prognosis and the retinal damage caused by the disease. No serious complications occurred in these patients with successful anastomosis. Anastomosis failed to form in 7 cases (7 eyes), among which, 2 eyes developed serious neovascular complications. CRVA treatment is effective in the treatment of non-ischaemic CRVO. With proper preventative measures for complications associated with the treatment, the safety of the treatment can be increased prominently, but serious neovascular complications was not entirely prevented.

A vitrectomy improves the transfection efficiency of adenoviral vector-mediated gene transfer to Müller cells.

The neural retina is a logical target of gene therapy for various ocular diseases. We developed a new gene delivery method to the neural retina using an adenoviral vector with a high degree of gene transfection efficiency and less functional damage. An adenoviral vector bearing the lacZ gene (AdCALacZ) was injected into the eyes of adult Wistar rats after an SF6 compression gas vitrectomy and left for 30 min followed by washing with balanced salt solution (BSS) (method A). Three other methods, comprising a simple intravitreal injection (method B), an intravitreal injection after an SF6 compression gas vitrectomy (method C) or a subretinal injection (method D), were also studied. The gene expression was examined 6 days after the AdCALacZ injection. An immunohistochemical study for antivimentin, antiglial fibrillary acidic protein and anti S100 protein antibodies showed the neural retinal cells (Müller cells) to be primarily transfected by methods A, B and C, while only a few cells were transfected by method D. The expression of beta-galactosidase was visualized by X-gal staining and the positive areas on each hemiflat mount specimen were measured by an image analyzer and then were adopted as a value of gene transfer efficiency. The highest degree of gene expression was obtained by methods A (23.2% of total retinal area) and C (19.8%), while the lowest degree was obtained by method B (8.9%). The inflammation was observed in all eyes and the value of inflammation was quantified as the average inflammatory cell number in each microscopic field (cells per fields). A moderate degree of inflammation was induced by methods B (28.3 cells per field) and C (27.5 cells per field) and a minimal degree of inflammation was induced by method A (11.2 cells per field). We evaluated the retinal function by measuring an electroretinogram (ERG). The amplitudes of the ERG were depressed in all eyes treated with AdCALacZ. This depression was manifested most by methods B and C, and least by method A. The deterioration in the ERG findings seemed to correlate with the intensity of inflammation. Our study showed that an intravitreal injection with an adenoviral vector can transfer the genes to the neural retinal cells and therefore a vitrectomy and the subsequent removal of the adenoviral vector, can thus significantly improve the transfection efficiency and also reduce the degree of functional damage.

Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.

We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

SHORT COMMUNICATION: The effect of raised inspired carbon dioxide on normal retinal vascular development in the neonatal rat

PURPOSE. Raised arterial carbon dioxide levels have been suggested as a risk factor for retinopathy of prematurity (ROP). We investigated the effect of raised inspired CO 2 on normal postnatal vasculogenesis in the neonatal rat retina. METHODS. One hundred fifty newborn rat pups were divided among 15 mothers (n = 10 for each litter). Five litters were exposed to low CO 2 (0.2%), 5 litters exposed to 6%, and 5 litters to 10% CO 2. On day 7 of life the rats were sacrificed and the total retinal and vascularized retinal areas analyzed. RESULTS. The vascularized retinal area and ratio of vascularized to total retinal area were reduced in rats exposed to 6% and 10% CO 2. CONCLUSIONS. Raised inspired CO 2 was associated with retardation of normal retinal vascular development and increased peripheral avascular area in neonatal rats. Raised CO 2 may be a risk factor for the development of abnormal neovascularization such as in ROP.

The effect of postnatal growth retardation on abnormal neovascularization in the oxygen exposed neonatal rat.

Severe retinopathy of prematurity (ROP) occurs in the smallest and sickest of premature infants. We hypothesized that, in a rat model of oxygen induced retinopathy, abnormal neovascularization would occur more frequently in larger litters where the pups are subject to postnatal growth retardation. Four litters of newborn Sprague-Dawley rats were studied; rats were randomly mixed to form two large litters (n = 25 each) and two small litters (n = 10 each). All litters were exposed to 7 days cyclic hyperoxia and hypoxia followed by 5 days in room air. ADPase stained retinae were evaluated in a masked manner for the presence and severity of abnormal neovascularization. Fluorescein perfused retinae were digitized and the ratios of vascularized:total retinal area were calculated using computer assisted image analysis. As expected, final weight in the large litters was less than in the small litters (15.3 +/- 3.8g vs. 23.4 +/- 2.1g, p < 0.001). Neovascularization occurred in 53% of rats in the large litters vs. 15% in the small litters (p = 0.009). Rats with retinae demonstrating neovascularization were smaller than those without (16.2 +/- 4.7g vs. 19.6 +/- 5.0g, p = 0.016). The severity of neovascularization in clock h was inversely correlated with final weight (rs = -0.35, p = 0.01) and ratio of vascularized:total retina area (rs = -0.46, p < 0.001). Smaller rat pups raised in larger litters, with resultant growth retardation, develop more frequent and more severe abnormal retinal neovascularization. Our results correlate with clinical experience in the premature infant.

The effect of litter size on normal retinal vascular development in the neonatal rat.

Many animal models of retinal disease use the neonatal rat. Raising rat pups in large litters has been shown to result in postnatal growth retardation. We investigated the effect of litter size on the normal postnatal vascularization of the neonatal rat retina. Sixty-six newborn rat pups were divided among 5 nursing mothers into 3 small litters (n = 10) and 2 large litters (n = 18). On day 6 of life the rats were sacrificed and total retinal and vascularized retinal areas analyzed. The total retinal area was reduced in the rats raised in larger litters (28.6 mm2 vs. 25.9 mm2 p < 0.001) but there was a more pronounced reduction in vascularized retinal area (67% vascularized vs. 54% vascularized, p < 0.001). Postnatal vascularization of the normal rat retina may be influenced by litter size.

The effect of raised inspired carbon dioxide on developing rat retinal vasculature exposed to elevated oxygen.

Hyperoxia is a risk factor for retinopathy of prematurity (ROP), a blinding disease in infants. However, ROP develops in human infants without raised arterial oxygen levels, such as in cyanotic congenital heart disease. In these infants raised pCO2 may be a risk factor. We investigated the effect of inspired CO2 on oxygen induced retinopathy in the rat. 56 newborn Sprague-Dawley rats were exposed to high cyclical O2 for seven days. In a control group, 27 rats were exposed to negligible CO2 by the use of soda lime. In the high CO2 group, 29 rats were exposed to elevated CO2 by omitting soda lime from their chambers. Rats in both groups had a recovery period of three days in room air following cyclical O2 exposure. On the eleventh day all rats were sacrificed after intracardiac injections of fluorescein under deep anesthesia and the retinae were dissected and flat mounted for fluorescent microscopy. The ratio of vascularized:total retinal area was calculated using computer assisted image analysis. In the high CO2 group 62% +/- 7% SD of the retina was vascularized vs. 81% +/- 7% in low CO2 group (p < 0.001). Elevated inspired CO2 results in pronounced retardation of retinal vascular development in neonatal rats exposed to fluctuating raised oxygen.

The structural and neurochemical development of the fetal guinea pig retina and optic nerve in experimental growth retardation

In this study we have examined structural and neurochemical aspects of retinal and optic nerve development in experimentally growth-retarded fetal guinea pigs following maternal unilateral artery ligation. Eye weight (n = 4) and total retinal area (n = 6) at 62 days gestation (term ∼66 days) were both relatively spared when expressed as a percentage of body weight but in absolute terms were significantly reduced by 18% (P<0.001) and 13% (P<0.05) respectively when compared with age-matched controls.The numerical density of neurons in the ganglion cell layer was significantly higher at both 52 days (n = 4) and 62 days (n = 4) in growth-retarded fetuses compared with controls. However, there was no difference between the groups in the total number of neurons in this retinal layer at either age, since retinal areas are reduced in growth retardation. The area of neuronal somata in the ganglion and inner nuclear layers was significantly reduced in growth-retarded fetuses compared with controls. There was a concomitant reduction in the width of the cellular layers in the retina and also in the plexiform (synaptic) and photoreceptor layers. The growth of the outer segments of the photoreceptor layer was particularly affected in peripheral retina. The higher packing density of cells and the reduced growth of the plexiform layers suggests a reduction in the growth of the neuropile in growth-retarded fetuses compared with controls. The radial bundling of ganglion cell axons coursing across the retina to enter the optic nerve head was poorly defined in growth retardation. In addition myelination was delayed in the optic nerve with the numerical density of myelinated axons being significantly reduced (P<0.005) in growth-retarded fetuses compared with controls.There was a significant reduction (P < 0.01) in the number of amacrine cells in the inner plexiform layer expressing Substance P-like immunoreactivity in growth-retarded fetuses compared with controls. Glutamate-like immunoreactivity was most intense in the five laminae of the inner plexiform layer and in the outer plexiform layer and less pronounced in photoreceptors, ganglion cells and their axons. There was no qualitative difference in glutamate immunoreactivity between control and growth-retarded fetuses in any of these structures.Thus we have shown that intrauterine growth retardation has specific effects on the development of the fetal guinea pig retina, reducing the growth of several types of neurons and their processes and affecting the expression of the neuropeptide substance-P in amacrine cells.

The Topographic Organization of the Retinal Ganglion Cell Layer of the Lizard Ctenophorus nuchalis.

The numbers of neurons of the ganglion cell layer (GCL) and their distribution in the retina of an Australian lizard Ctenophorus nuchalis were investigated. Retinal wholemounts and sections were prepared for light microscopic and optic nerves for electron microscopic study. Counts of cell numbers in the GCL from wholemounts varied from 200,000 to 380,000. Neurons in the GCL were non-uniformly distributed, forming a high cell density streak along the naso-temporal axis of the retina. Neurons of the GCL formed 2 to 9 layers in the visual streak and a single layer in the rest of the retina. The number of neurons of the GCL in this area was estimated at about 2,100,000. Although the visual streak represented only 16% of the total retinal surface area, it contained about 90% of all neurons of the GCL. Optic axon counts yielded 147,000 myelinated and 2,643,000 unmyelinated fibres. The estimated optic fibre number of 2,790,000 was 18.2% less than the total number of neurons counted from sections in the GCL of the same eye. The unexpected high number of neurons in the area of the visual streak indicates that cell numbers obtained only from wholemount preparations may vastly underestimate the total neuron numbers in the GCL of the lizard retina.

Origins of crossed and uncrossed retinal projections in pigmented and albino mice.

The extent of the binocular cortical field in albino mice, as revealed by recording from single cells, was almost normal; although the input from the ipsilateral eye was weaker than normal, most cells were driven from both eyes. By backfilling retinal ganglion cells from one optic tract with horseradish peroxidase we examined the origins of the retinofugal projections. Filled cells ipsilateral to the injected tract were concentrated in a crescent-shaped area bordering the inferior temperal retina. In black mice this area constituted 20% of the total retinal area, in albinos 17%. In black mice we counted nearly 1,000 labeled cells in the ipsilateral retina, or 2.6% of all cells filled in both eyes. Albinos had about one-third fewer filled cells ipsilaterally than black mice. Four percent of all ipsilaterally filled cells in black mice and 8% in albinos were scattered outside of the crescent region. The density of ipsilaterally projecting cells was uniform throughout the crescent region in black mice, but decreased toward the central retina in albinos. In retinas contralateral to the injection up to 39,000 cells were filled-about two-thirds of the cells in the ganglion-cell layer whose cytoplasm contained conspicuous Nissl substance. Depending on classification of unfilled cells as ganglion cells or interneurons, we estimated a total of 48,000 to 65,000 ganglion cells to exist in the retina. The size distribution of ipsilaterally projecting ganglion cells was similar in albinos and normals. Ipsilaterally projecting ganglion cells were on average 1.8-3 times larger in volume than contralaterally projecting ones in both types of mice. Displaced ganglion cells were relatively more common in ipsilateral retinofugal projections: 21% of all ipsilateral ganglion cells were displaced versus less than 1% of all the contralateral ganglion cells in black mice. In albinos only 13% of the ganglion cells in the ipsilateral retina were displaced. The overall reduction in ipsilaterally projecting cells in albinos was reflected twice as much in displaced ganglion cells as in normally placed ones.

Correlation of Histologic and Electroretinographic Changes in Peripheral Retinal Ablation in the Rhesus Monkey

We studied the effect of peripheral retinal ablation on the electroretinograms of five rhesus monkeys. An ablation procedure analogous to that commonly used in the treatment of diabetic retinopathy was carried out with an argon laser photocoagulator. Quantitative histologic measures showed retinal destruction of about 13% of total retinal area. This was associated with about a 50% reduction in scotopic electroretinographic amplitude.