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We have previously shown that the amplitude of the mfERG response obtained to a single (large) hexagon is significantly smaller than that obtained when summating all the mfERG responses evoked to an array of 7–61 hexagons covering the same retinal area. The purpose of this study was to confirm our initial findings in normal subjects of different ages and in selected patients. Binocular mfERGs (1, 7, 19, 37 and 61 hexagon arrays; Espion V6.0.54 Diagnosys LLC) were recorded from 40 normal subjects (25 aged 18–25, and 15 aged 3–12). Individual mfERG waveforms evoked in response to the multi-hexagon arrays (7, 19, 37 and 61) were summated, and the amplitude of the resulting composite mfERG waveform was compared to that measured in the response evoked to the single (large) hexagon stimulus to yield the amplitude ratio (i.e., 7:1 X100, 19:1X100, etc.). In normal subjects, the 7:1 ratio was 119.5 ± 9.2%, a value that gradually decreased to reach 109.4 ± 20.6% with the 61:1 ratio and a finding that was similar across all ages. The present study indicates a significant enhancement in amplitude of the summed mfERG composite waveform evoked to the 7 hexagon stimulus array (and to a lesser extent to the 19, 37 and 61 stimuli) compared to the 1 hexagon array, possibly mediated through the retinal lateral pathway (horizontal or amacrine cells), a claim that awaits confirmation. Preliminary results obtained from patients treated with Plaquenil suggest that this new method of mfERG analysis might probe a feature of macular function different from that investigated with the more usual method of mfERG ring ratio.

Retinopathy of prematurity (ROP), the most common cause of blindness in premature infants, has long been associated with inner retinal alterations. However, recent studies reveal outer retinal dysfunctions in patients formerly afflicted with ROP. We have recently demonstrated that choroidal involution occurs early in retinopathy. Herein, we investigated the mechanisms underlying the choroidal involution and its long-term impact on retinal function. An oxygen-induced retinopathy (OIR) model was used. In vitro and ex vivo assays were applied to evaluate cytotoxic effects of IL-1β on choroidal endothelium. Electroretinogram was used to evaluate visual function. We found that proinflammatory IL-1β was markedly increased in retinal pigment epithelium (RPE)/choroid and positively correlated with choroidal degeneration in the early stages of retinopathy. IL-1β was found to be cytotoxic to choroid in vitro, ex vivo, and in vivo. Long-term effects on choroidal involution included a hypoxic outer neuroretina, associated with a progressive loss of RPE and photoreceptors, and visual deterioration. Early inhibition of IL-1β receptor preserved choroid, decreased subretinal hypoxia, and prevented RPE/photoreceptor death, resulting in life-long improved visual function in IL-1 receptor antagonist-treated OIR animals. Together, these findings suggest a critical role for IL-1β-induced choroidal degeneration in outer retinal dysfunction. Neonatal therapy using IL-1 receptor antagonist preserves choroid and prevents protracted outer neuroretinal anomalies in OIR, suggesting IL-1β as a potential therapeutic target in ROP.

Although a great deal of emphasis has been placed on the vasculopathy that is associated with oxygen-induced retinopathy (OIR), our studies also revealed significant and irreversible structural (retinal histology) and functional (scotopic and photopic electroretinograms) impairments that were significantly more severe in pigmented Long-Evans rats compared to the more commonly used albino Sprague Dawley rats. In the following pages, we will highlight what we have learned about the retinal pathophysiological processes of OIR taking place in strains of both rats with the hope that this will trigger investigations into new therapeutic strategies to complement those geared at preventing the vasculopathy.

The neonatal rat is born with its eyes closed and an immature visual system, that some say is equivalent to that of a human fetus at 26 weeks of gestation. From birth, the visual system of the newborn rat will gradually mature, the first manifestation of that being the opening of the eye which usually take place at postnatal day 14. Complete maturation of the retina and visual pathways is normally reached at the end of the first month of life. The neonatal rat model thus represents a unique paradigm to study the normal and abnormal maturation of the primary visual pathways that normally occurs in utero in human subjects. Our laboratory has, over the past decade, developed two animal models of postnatally induced retinopathy, namely the Oxygen-Induced Retinopathy (OIR) that share several common features with the human Retinopathy of Prematurity (ROP) and the Light-Induced Retinopathy that is viewed by some as a valid model of some forms of Retinitis Pigmentosa (RP). The following pages review what is known of the pathophysiological processes taking place and suggest possible therapeutic avenues that could be explored in order to halt the degenerative process.

Contains fulltext : 50306.pdf (Publisher’s version ) (Closed access) PURPOSE: To test human CRB1 heterozygotes for possible clinical or functional retinal changes and to evaluate whether a patient with Leber congenital amaurosis (LCA) with CRB1 mutations not consistent with previously described CRB1 phenotypes carried a modifier allele in another LCA gene. METHODS: Seven unrelated heterozygous carriers of CRB1 mutations underwent phenotyping by full eye examinations (indirect ophthalmoscopy and slit lamp biomicroscopy) and functional testing (standard full-field electroretinography [ERG] and multifocal ERG). For genotyping of the LCA patients and their parents, denaturing high-performance liquid chromatography (dHPLC) analyses were performed, followed by sequence analysis of CRB1, followed by sequence analysis of the AIPL1 and CRX genes to identify a putative modifier effect in a patient with an atypical CRB1 phenotype. RESULTS: Reduced full-field ERG b-wave amplitudes were observed with scotopic -2 dB flash (140 microV; P < 0.05), normal full-field cone ERGs, and significant regional retinal dysfunction on mfERG in five of seven carriers of CRB1 mutations. A known AIPL1 mutation (p. R302L) was identified as a potential modifier allele in a patient with LCA carrying two CRB1 mutations and with a prominent maculopathy. CONCLUSIONS: In human heterozygotes of CRB1 mutations (parents of offspring with LCA), distinctive regional retinal dysfunctions were found by multifocal ERG measurements that were consistent with the focal histologic abnormalities reported for the two CRB1 knockout mice models. This phenotypic finding may identify CRB1 carriers and point to the causal gene defect in affected LCA offspring, significantly facilitating the molecular diagnostic process. Evidence suggests a modifier allele in AIPL1 in a patient with LCA with prominent atrophic macular lesions and homozygous defects in CRB1.

International audience; Purpose: Retinopathy of prematurity (ROP) is a major cause of visual handicap in the pediatric population. To date, this disorder is thought to stem from deficient retinal vascularization. Intriguingly, functional electrophysiological studies in patients with mild or moderate ROP and in the oxygen-induced retinopathy (OIR) model in rats reveal central photoreceptor disruption that overlies modest retinal vessel loss; a paucity of retinal vasculature occurs predominantly at the periphery. Given that choroidal circulation is the major source of oxygen and nutrients to the photoreceptors, the authors set out to investigate whether the choroidal vasculature system may be affected in OIR.Methods: Rat models of OIR treating newborn animals with 80% or 50/10% alternated oxygen level for the first two postnatal weeks were used to mimic ROP in humans. Immunohistology staining and vascular corrosion casts were used to investigate the vessel layout of the eye. To investigate the effect of 15-deoxy-Δ12,14-PGJ(2) (15d-PGJ(2); a nonenzymatic product of prostaglandin D(2)) on endothelial cells, in vitro cell culture and ex vivo choroid explants were employed and intravitreal injections were performed in animals.Results: The authors herein demonstrate that deficient vascularity occurs not only in the retinal plexus but also in the choroid. This sustained, marked choroidal degeneration is specifically confined to central regions of the retina that present persistent photoreceptor loss and corresponding functional deficits. Moreover, the authors show that 15d-PGJ(2) is a prominent contributor to this choroidal decay.Conclusions: The authors demonstrate for the first time pronounced, sustained choroidal vascular involution during the development of ROP. Findings also suggest that effective therapeutic strategies to counter ROP should consider choroidal preservation.

Purpose. Postnatal exposure to hyperoxia destroys the plexiform layers of the neonatal rat retina, resulting in significant electroretinographic anomalies. The purpose of this study was to identify the mechanisms at the origin of this loss. Methods. Sprague-Dawley (SD) and Long Evans (LE) rats were exposed to hyperoxia from birth to postnatal day (P) 6 or P14 and from P6 to P14, after which rats were euthanatized at P6, P14, or P60. Results. At P60, synaptophysin staining confirmed the lack of functional synaptic terminals in SD (outer plexiform layer [OPL]) and LE (OPL and inner plexiform layer [IPL]) rats. Uneven staining of ON-bipolar cell terminals with mGluR6 suggests that their loss could play a role in OPL thinning. Protein kinase C(PKC)-α and recoverin (rod and cone ON-bipolar cells, respectively) showed a lack of dendritic terminals in the OPL with disorganized axonal projections in the IPL. Although photoreceptor nuclei appeared intact, a decrease in bassoon staining (synaptic ribbon terminals) suggests limited communication to the inner retina. Findings were significantly more pronounced in LE rats. An increase in TUNEL-positive cells was observed in LE (inner nuclear layer [INL] and outer nuclear layer [ONL]) and SD (INL) rats after P0 to P14 exposure (425.3%, 102.2%, and 146.3% greater than control, respectively [P < 0.05]). Conclusions. Results suggest that cell death and synaptic retraction are at the root of OPL thinning. Increased TUNEL-positive cells in the INL confirm that cells die, at least in part, because of apoptosis. These findings propose a previously undescribed mechanism of cell death and synaptic retraction that are likely at the origin of the functional consequences of hyperoxia. Supported by Canadian Institutes of Health Research Grant MOP-13383; FRSQ-Réseau Vision; McGill University-Montreal Children's Hospital Research Institute; Ministerio de Ciencia e Innovación Grant BFU2009-07793/BFI; RETICS Grant RD07/0062/0012; Fundaluce; ONCE; Fundación Médica Mutua Madrileña (NC); and FIS Grant PS09/01854 (IP).

In a previous study we showed that juvenile rats exposed, for various durations of time, to a bright luminous environment between P14 (eye opening) and P34 developed a light-induced retinopathy (LIR), the severity of which depending on the duration of exposure as well as the age of the rat at the onset of exposure. Our study also revealed that the severity of the LIR increased as the time elapsed between the cessation of exposure and the structural/functional evaluation increased, suggesting that the LIR degenerative process proceeded in two distinct steps namely, an initial (rapid) acute phase that was followed by a (slower) chronic phase. In view of the above, the purpose of the present study was to reinvestigate previous claims suggesting that exposure to bright light prior to eyelid opening had no measurable consequences on the retinal structure and function; the claim being that despite a non-detectable acute phase, bright light exposure prior to eyelid opening could nonetheless yield a significant retinopathy during the chronic phase of development of LIR. In order to test our hypothesis, neonatal rats were raised in a bright luminous environment from birth to P14. At P30, analysis of the results obtained from rats exposed between P0-P14 did not reveal, as previously acknowledged by others, significant LIR damages. However, results obtained at P60 disclosed significant functional anomalies with relative sparing of the retinal ultrastructure. Our results confirm that, in spite of closed eyelids, postnatal exposure to bright environment did trigger a slow degenerative process.

Purpose Previous studies have shown that newborn rats exposed to hyperoxia within the first 2 weeks of life develop vasculopathy in addition to permanent changes in retinal structure and function. It has also been suggested that free radicals may be the source of these pathologic effects. Trolox C, a water-soluble analogue of vitamin E, was previously shown to limit the vascular consequences of exposure to postnatal hyperoxia. The aim of this study was to investigate whether trolox C could also help prevent the functional (electroretinography) and structural (retinal histology) consequences associated with oxygen-induced retinopathy (OIR). Methods Newborn albino Sprague-Dawley rats exposed or not exposed to hyperoxia received daily injections of trolox C in doses of 300, 600, and 900 microg/kg (total volume, 50 microL). The effect of treatment was evaluated through electroretinography and retinal histology. Results Although trolox C tended to have a retinoactive effect on the normal retina, normalization of the hyperoxia-treated group to hyperoxic control and of the normoxia-treated group to normoxic control revealed that the a-wave remained relatively unaffected by hyperoxia exposure and by treatment with trolox C, the efficacy of trolox C at doses of 600 and 900 microg/kg largely outweighed the retinoactive effect, and the oscillatory potentials (OPs) benefited to the greatest extent from trolox C treatment. Furthermore, trolox C was able to limit the reduction in outer plexiform layer thickness but not the concomitant reduction of the horizontal cell count, each of which is associated with OIR. Conclusions These results show that, as had been previously demonstrated with retinal vasculature, trolox C limited the retinal functional and structural damages inherent in the rat model of OIR. However, despite treatment, there were still signs (albeit less severe) indicative of OIR. This suggests, as previously advanced, that the pathophysiology of OIR is not solely caused by the action of free radicals or that trolox C is inadequate in treating all aspects of OIR.

PURPOSE. Results of studies that compared the racial incidence of retinopathy of prematurity (ROP) suggested that ocular pigmentation might offer protection against the development of severe ROP. The structural and functional consequences of postnatal hyperoxia (oxygen-induced retinopathy; OIR) were compared in albino Sprague-Dawley (SD) and pigmented LongEvans (LE) rats to verify this finding. METHODS. Newborn rats were exposed to 80% O2 during selected postnatal day intervals. The severity of the OIR was determined by examining retinal flatmounts (retinal vasculature assessment), protein level quantification and cellular localization of fibroblast growth factor (FGF)-2 and ciliary neurotrophic factor (CNTF; Western blot analysis and immunohistochemistry, respectively), retinal histology, and photopic and scotopic electroretinograms (ERGs). RESULTS. Irrespective of the parameter considered, structural and functional deficits resulting from postnatal hyperoxia were significantly more pronounced in LE rats. Although FGF-2 levels in LE rats had a tendency to increase after hyperoxia compared with normoxic littermates, it did not reach statistical significance. A similar finding was observed in SD rats. Of interest, however, baseline levels of FGF-2 were approximately four to five times higher in SD rats than in LE rats. There was a similar, hyperoxiainduced increase in CNTF levels between SD and LE rats. CONCLUSIONS. The findings suggest an increased susceptibility of newborn LE rats to postnatal hyperoxia in comparison with SD rats. Whether a pro-oxidant rather than antioxidant role of melanin or other genetic factors can explain these differences in oxygen susceptibility of the animal model of this retinopathy, remains to be determined. (Invest Ophthalmol Vis Sci. 2009;50:2436‐2450) DOI:10.1167/iovs.08-2297

PURPOSE. Postnatal hyperoxia in rats causes an arrest in growth of retinal blood vessels, along with severe changes in retinal ultrastructure and function. Previous studies focused on consequences of postnatal hyperoxia at time points substantially removed from the hyperoxic insult. In this study, the earliest manifestations of this retinopathy were examined. METHODS Newborn rats were exposed to 80% O 2 from birth to postnatal day 14. The retinas were collected for vascular assessment at postnatal days 6, 9, 12, and 14, and electroretinograms were recorded at postnatal days 15, 16, 17, 19, 24, 30, and 60, after which retinal histology was performed. RESULTS Hyperoxia significantly attenuated vascular development, especially after 6 and 9 days of exposure which resulted in 64% and 72% of normal coverage, respectively. Vascular growth continued despite hyperoxic exposure, reaching 87% of normal by postnatal day 14. Electroretinograms of hyperoxic rats retained very immature features throughout with nearly abolished b-waves and relatively preserved a-waves. Finally, while retinal structure was virtually complete in the control animals by postnatal day 15, hyperoxic rats always showed a significantly thinner outer plexiform layer (OPL) and lower horizontal cell count (P < 0.05), irrespective of the duration of exposure. CONCLUSION The findings confirm previous reports of reduced retinal vascular coverage that accompanies the earliest manifestation of postnatal hyperoxia in rats and suggest increased retinal susceptibility to hyperoxia within the first week of life. However, despite the fact that vasculature appears to repair itself, irreversible cytoarchitectural and functional changes occur, the consequences of which are documented immediately after the cessation of hyperoxia.

PURPOSE. To investigate the effect of chronic exposure to a bright, luminous environment, starting at the opening of the eyes, on the retinal structure and function of the suckling rat. METHODS. Juvenile Sprague-Dawley rats were exposed to 10,000 lux, for varying lengths of time between postnatal day (P)14 and P34. Results were compared with those obtained from adult rats exposed to the same light intensity and for the same duration. Electroretinograms (ERGs) were recorded at 1 month and 2 months of age, after which the retinas were harvested for histologic analysis. RESULTS. In juvenile rats, the severity of light-induced retinopathy (LIR) depended not only on the duration of the exposure but, more important, on the age of the rat at the onset of exposure. For example, in adult rats, 6-day exposure reduced the thickness of the outer nuclear layer (ONL) to less than 18% of normal, whereas in juvenile rats, 6-day exposure between P14 and P20 reduced it to 50% compared with 27% after exposure between P28 and P34. An adultlike effect could only be evidenced in rats exposed at the end of the first postnatal month (P28-P34). A similar age-dependent effect was also noted on the electroretinogram. CONCLUSIONS. These results show that, compared with the mature retina, the developing retina appears to be relatively preserved from the devastating consequences of exposure to bright light. This window of resistance to light damage gradually weakens as the juvenile rat approaches its 1-month anniversary.