Resting state electroretinography: An innovative approach to intrinsic retinal function monitoring.

The electroretinogram (ERG) represents the biopotential evoked by the retina in response to a light stimulus. The flash evoked ERG (fERG) is the ERG modality most frequently used clinically to diagnose and monitor retinal disorders. We hereby present a new method to record spontaneous retinal activity, without the use of a flash stimulus, that we named the resting-state ERG (rsERG). The recordings were done in normal subjects under light- and dark-adaptation and with different background light conditions (i.e., variations of wavelength and intensity). Additionally, rsERG recordings were obtained in five patients with retinopathies. The signals were subsequently analyzed in the frequency domain, extracting both periodic (i.e., frequency peaks) and aperiodic (i.e., background trend) components of the signal. The later was further assessed through a multifractal analysis using Wavelet Leaders. Results show that, irrespective of the recording conditions used, the rsERG always includes the same 90 Hz component; a frequency component also present in the fERG response, suggesting a retinally-intrinsic origin. However, in addition, the fERGs also includes a low-frequency component which is absent in the rsERGs, a finding supporting a retinally-induced origin. Comparing rsERGs with fERGs in selected patients with various retinal disorders indicates that the two retinal signals are not always similarly affected (either as a result of underlying retinal pathology or otherwise), suggesting an added value in the assessment of retinal function. Thus, the rsERG could have a similar role in clinical visual electrophysiology as that of the resting-state EEG in neurology namely, to quantify changes in spontaneous activity that result from a given disease processes.

AAV-mediated PEX1 gene augmentation improves visual function in the PEX1-Gly844Asp mouse model for mild Zellweger spectrum disorder.

Patients with Zellweger spectrum disorder (ZSD) commonly present with vision loss due to mutations in PEX genes required for peroxisome assembly and function. Here, we evaluate PEX1 retinal gene augmentation therapy in a mouse model of mild ZSD bearing the murine equivalent (PEX1-p[Gly844Asp]) of the most common human mutation. Experimental adeno-associated virus 8.cytomegalovirus.human PEX1.hemagglutinin (AAV8.CMV.HsPEX1.HA) and control AAV8.CMV.EGFP vectors were administered by subretinal injection in contralateral eyes of early (5-week-old)- or later (9-week-old)-stage retinopathy cohorts. HsPEX1.HA protein was expressed in the retina with no gross histologic side effects. Peroxisomal metabolic functions, assessed by retinal C26:0 lysophosphatidylcholine (lyso-PC) levels, were partially normalized after therapeutic vector treatment. Full-field flash electroretinogram (ffERG) analyses at 8 weeks post-injection showed a 2-fold improved retinal response in the therapeutic relative to control vector-injected eyes. ffERG improved by 1.6- to 2.5-fold in the therapeutic vector-injected eyes when each cohort reached 25 weeks of age. At 32 weeks of age, the average ffERG response was double in the therapeutic relative to control vector-injected eyes in both cohorts. Optomotor reflex analyses trended toward improvement. These proof-of-concept studies represent the first application of gene augmentation therapy to treat peroxisome biogenesis disorders and support the potential for retinal gene delivery to improve vision in these patients.

Evidences Suggesting that Distinct Immunological and Cellular Responses to Light Damage Distinguishes Juvenile and Adult Rat Retinas.

To unravel the mechanisms behind the higher resistance to light damage of juvenile (JR) versus adult (AR) rats, Sprague Dawley rats were exposed to a bright luminous environment of 10, 000 lux. The light-induced retinopathy (LIR) was assessed with histology, electroretinography and immunohistochemistry (IHC). In JR, 2 days of exposure induced the typical LIR, while >3 days added little LIR. IHC revealed a subtle migration of microglia (Iba1 marker) from the inner to the outer retina after 3 days of exposure in JR contrasting with the stronger reaction seen after 1 day in AR. Similarly, in JR, the Müller cells expressed less intense GFAP, CNTF and FGF2 staining compared to AR. Our results suggest that in JR the degree of retinal damage is not proportional to the duration of light exposure (i.e., dose-independent retinopathy), contrasting with the dose-dependent LIR reported in AR. The immature immune system in JR may explain the delayed and/or weaker inflammatory response compared to AR, a finding that would also point to the devastating contribution of the immune system in generating the LIR phenotype, a claim also advanced to explain the pathophysiology of other retinal degenerative disorders such as Age-related Macular Degeneration, Diabetic Retinopathy and Retinitis Pigmentosa.

A longitudinal study of retinopathy in the PEX1-Gly844Asp mouse model for mild Zellweger Spectrum Disorder.

Zellweger Spectrum Disorder (ZSD) is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly. Retinopathy leading to blindness is one of the major untreatable handicaps faced by patients with ZSD but is not well characterized, and the requirement for peroxisomes in retinal health is unknown. To address this, we examined the progression of retinopathy from 2 to 32 weeks of age in our murine model for the common human PEX1-p.Gly843Asp allele (PEX1-p.Gly844Asp) using electrophysiology, histology, immunohistochemistry, electron microscopy, biochemistry, and visual function tests. We found that retinopathy in male and female PEX1-G844D mice was marked by an attenuated cone function and abnormal cone morphology early in life, with gradually decreasing rod function. Structural defects at the inner retina occurred later in the form of bipolar cell degradation (between 13 and 32 weeks). Inner segment disorganization and enlarged mitochondria were seen at 32 weeks, while other inner retinal cells appeared preserved. Visual acuity was diminished by 11 weeks of age, while signal transmission from the retina to the brain was relatively intact from 7 to 32 weeks of age. Molecular analyses showed that PEX1-G844D is a subfunctional but stable protein, contrary to human PEX1-G843D. Finally, C26:0 lysophosphatidylcholine was elevated in the PEX1-G844D retina, while phopshoethanolamine plasmalogen lipids were present at normal levels. These characterization studies identify therapeutic endpoints for future preclinical trials, including improving or preserving the electroretinogram response, improving visual acuity, and/or preventing loss of bipolar cells.

Evaluating the neuroprotective effect of 17ß-estradiol in rodent models of oxidative retinopathies.

PURPOSE: To determine the neuroprotective effect of estrogen on the structure and function of the retina exposed to an oxidative stress. METHODS: Male Sprague-Dawley rat pups were exposed to either hyperoxia (O2E: from P8 to P14) or bright light (LE: from P14 to P28) with or without 17 ß-estradiol (ßE2) treatment. Retinal structure (histology) and function (ERG) were assessed at selected time points. RESULTS: In the O2E model, ßE2 injections caused a significant reduction of the ERG and a significantly thinner OPL compared to untreated oxygen-exposed group (O2-exposed) rats. In contrast, in the LE model ßE2, treatment was beneficial to the retinal structure (thicker ONL) and function (better preserved ERG amplitudes) compared to untreated light-exposed group (light-exposed rats). CONCLUSION: Our results show that in conditions where the primary target of the oxidative stress is the outer retina (i.e., the photoreceptors) estrogen can protect the retina, while in situations where the inner retina (or retinal vasculature) is the main site of oxidative damage, estrogen may potentiate the detrimental effect of oxidative stress on the retina.

Light-Induced Retinopathy: Young Age Protects more than Ocular Pigmentation.

PURPOSE: The purpose of this study was to compare the efficacy that ocular melanin confers in protecting the retina of juvenile and adult rats exposed to a bright luminous environment. METHODS: Juvenile (JLE) and adult (ALE) Long-Evans pigmented rats were thus exposed to a bright cyclic light (10,000lux; white light) from postnatal day 14-28 or for 6 consecutive days, respectively. Flash electroretinograms (ERG) and retinal histology were performed at different predetermined ages, post-light exposure. RESULTS: Despite a significant reduction in ERG responses immediately following light exposure, with time, retinal function fully recovered in JLE compared to a 54% recovery for the ALE. In ALE, we noted a region of the supero-temporal quadrant that was highly vulnerable to light damage. This region was also devoid of melanin granules prior to the light exposure. This melanin-free zone increased in size in the days that followed the end of exposure, a process that was accompanied by the gradual degeneration of the thus uncovered photoreceptors. In contrast, melanin and photoreceptor losses were minimal in JLE. CONCLUSION: Our results suggest that the light-induced photoreceptor degeneration in ALE would be secondary to the initial destruction of the RPE and ensuing loss of melanin protection. In contrast, the melanin granules of JLE appear to be significantly more resistant to light damage, a characteristic that would explain the higher resistance of JLE photoreceptors to light damage. Our results would thus suggest that the efficacy of ocular melanin protection against light damage declines with age.

Characterizing the Retinal Function of Psammomys obesus: A Diurnal Rodent Model to Study Human Retinal Function.

PURPOSE: To compare the retinal function of a diurnal murid rodent, Psammomys obesus, with that of Wistar albino rat and human subjects. MATERIALS AND METHODS: Adult Psammomys obesus were captured and transferred to the animal facilities where they were maintained at 25°C with standard light/dark cycles and natural halophilic plants, rich in water and mineral salts. Standard full-field photopic and scotopic electroretinograms were obtained. RESULTS: The right eye of all animals displayed well detectable and reproducible scotopic and photopic electroretinogram (ERG) responses. Results were compared with those obtained from human subjects and Wistar rats. ERG measurement showed that the amplitudes of scotopic responses in Psammomys obesus are quite similar to those of human subjects. The amplitude of the photopic a-wave was comparable to that of humans and six times higher than that of the albino rat. The amplitudes of photopic b-wave, photopic oscillatory potentials (OPs), and 30 Hz flicker were all markedly larger in Psammomys obesus compared to those obtained from human subjects and Wistar rats. Furthermore, like the human photopic ERG, the photopic ERG of Psammomys obesus also includes prominent post b-wave components (i.e. i- and d-waves) while the ERG of Wistar rats does not. CONCLUSIONS: Our results suggest that the retinal function of Psammomys obesus, especially the cone-mediated function, shares several features with that of human subjects. We believe that Psammomys obesus represents an interesting alternative to study the structure and function of the normal and diseased retina in a human-like rodent model of retinal function.

Choroidal Involution Is Associated with a Progressive Degeneration of the Outer Retinal Function in a Model of Retinopathy of Prematurity: Early Role for IL-1ß.

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-1ß on choroidal endothelium. Electroretinogram was used to evaluate visual function. We found that proinflammatory IL-1ß was markedly increased in retinal pigment epithelium (RPE)/choroid and positively correlated with choroidal degeneration in the early stages of retinopathy. IL-1ß 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-1ß 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-1ß-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-1ß as a potential therapeutic target in ROP.

Strain Differences in Light-Induced Retinopathy.

The purpose of this study was to better understand the role of ocular pigmentation and genetics in light-induced retinal damage. Adult pigmented [Long Evans (LE) and Brown Norway (BN)] and albino [Sprague Dawley (SD) and Lewis (LW)] rats were exposed to a bright cyclic light for 6 consecutive days and where compared with juvenile animals exposed to the same bright light environment from postnatal age 14 to 28. Flash ERGs and retinal histology were performed at predetermined days (D) post-light exposure. At D1, ERGs were similar in all adult groups with no recordable a-waves and residual b-waves. A transient recovery was noticed at D30 in the LW and LE only [b-wave: 18% and 25% of their original amplitude respectively]. Histology revealed that BN retina was the most damaged, while LE retina was best preserved. SD and LW rats were almost as damaged as BN rats. In contrast, the retina of juvenile BN was almost as resistant to the bright light exposure as that of juvenile LE rats. Our results strongly suggest that, although ocular pigmentation and genetic background are important factors in regulating the severity of light-induced retinal damage, the age of the animal at the onset of light exposure appears to be the most important determining factor.

Retinotopic Distribution of Structural and Functional Damages following Bright Light Exposure of Juvenile Rats.

In the present study, we aimed at better understanding the short (acute) and long term (chronic) degenerative processes characterizing the juvenile rat model of light-induced retinopathy. Electroretinograms, visual evoked potentials (VEP), retinal histology and western blots were obtained from juvenile albino Sprague-Dawley rats at preselected postnatal ages (from P30 to P400) following exposure to 10,000 lux from P14 to P28. Our results show that while immediately following the cessation of exposure, photoreceptor degeneration was concentrated within a well delineated area of the superior retina (i.e. the photoreceptor hole), with time, this hole continued to expand to form an almost photoreceptor-free region covering most of superior-inferior axis. By the end of the first year of life, only few photoreceptors remained in the far periphery of the superior hemiretina. Interestingly, despite a significant impairment of the outer retinal function, the retinal output (VEP) was maintained in the early phase of this retinopathy. Our findings thus suggest that postnatal exposure to a bright luminous environment triggers a degenerative process that continues to impair the retinal structure and function (mostly at the photoreceptor level) long after the cessation of the exposure regimen (more than 1 year documented herein). Given the slow degenerative process triggered following postnatal bright light exposure, we believe that our model represents an attractive alternative (to other more genetic models) to study the pathophysiology of photoreceptor-induced retinal degeneration as well as therapeutic strategies to counteract it.

Differences in Retinal Structure and Function between Aging Male and Female Sprague-Dawley Rats are Strongly Influenced by the Estrus Cycle.

PURPOSE: Biological sex and age are considered as two important factors that may influence the function and structure of the retina, an effect that might be governed by sexual hormones such as estrogen. The purpose of this study was to delineate the influence that biological sex and age exert on the retinal function and structure of rodents and also clarify the effect that the estrus cycle might exert on the retinal function of female rats. METHOD: The retinal function of 50 normal male and female albino Sprague-Dawley (SD) rats was investigated with the electroretinogram (ERG) at postnatal day (P) 30, 60, 100, 200, and 300 (n = 5-6 male and female rats/age). Following the ERG recording sessions, retinal histology was performed in both sexes. In parallel, the retinal function of premenopausal and menopausal female rats aged P540 were also compared. RESULTS: Sex and age-related changes in retinal structure and function were observed in our animal model. However, irrespective of age, no significant difference was observed in ERG and retinal histology obtained from male and female rats. Notwithstanding the above we did however notice that between P60 and P200 there was a gradual increase in ERG amplitudes of female rats compared to males. Furthermore, the ERG of premenopausal female rats aged 18 months old (P540) was larger compared to age-matched menopausal female rats as well as that of male rats. CONCLUSION: Our results showed that biological sex and age can influence the retinal function and structure of albino SD rats. Furthermore, we showed that cycled female rats have better retinal function compared to the menopausal female rats suggesting a beneficial effect of the estrus cycle on the retinal function.

Visual Impairments Following Term Neonatal Encephalopathy: Do Retinal Impairments Also Play a Role?

PURPOSE: We investigated the effects of term neonatal encephalopathy on retinal function and structure. METHODS: A rat model of term neonatal hypoxic-ischemic (HI) encephalopathy (Vannucci model) was used. Hypoxia-ischemia was induced by a left common carotid ligation followed by a 2-hour period of hypoxia (8% oxygen) in Long-Evans rat pups at postnatal day 10 (P10). Sham operated rats served as controls.. Retinal function was assessed at P30 and P60 by electroretinograms (ERGs), after which retinal histology was performed. Retinocortical function was assessed with visual evoked potentials (VEPs) at P60 and subsequently brain histology was performed. RESULTS: The ERGs of the HI animals at P30 and P60 demonstrated a significant reduction in the scotopic and photopic b-wave amplitudes, but a preserved a-wave amplitude. The retinal histology of the HI animals confirmed that the photoreceptor layer remained intact, whereas the inner layers of the retina were damaged. The HI animals also showed reduced VEP P100 amplitudes, which correlated with reduced left cerebral hemisphere surfaces. There was no correlation between the severities of retinal versus cerebral injuries. CONCLUSIONS: Our findings suggest that term neonatal encephalopathy resulting from HI induces functional and structural damages to the inner retina, while relatively sparing the photoreceptors. These findings raise the possibility that retinal injuries may contribute to visual impairments with or without the presence of brain injury in term asphyxiated newborns and, thus, warrant further studies with humans and animals to better understand the disease process.

Systemic inflammation perturbs developmental retinal angiogenesis and neuroretinal function.

PURPOSE: Perinatal inflammatory stress in preterm babies is associated with increased rates of severe retinopathy of prematurity (ROP) and adverse neurological dysfunction. In this study, we set out to determine the consequences of severe systemic inflammatory stress on developmental retinal vascularization and evaluate the subsequent outcome on retinal function in later life. METHODS: Systemic inflammatory stress was induced in C57BL/6J mouse pups by an intraperitoneal injection of lipopolysaccharide (LPS; 1 mg/kg) at postnatal day 4. In response to LPS, retinal inflammation was confirmed by quantitative RT-PCR analysis of diverse inflammatory markers. A detailed and systematic analysis of retinal microglial infiltration, retinal vascular morphology, density, and growth rate was performed at key time points throughout retinal vascularization. Retinal function in adult life was assessed by using electroretinography at 6 weeks postinjection. RESULTS: As early as 48 hours after intraperitoneal administration of LPS, a significant increase in retinal vascular density was noted throughout the retina. A pronounced increase in the number of activated microglial cell was observed in the retinal ganglion cell layer and in the outer plexiform layer just prior to their vascularization; direct physical contact between activated microglia and sprouting vessels suggested that microglia partake in promoting the aberrant retinal vascularization. With maturity, animals subjected to perinatal inflammatory stress displayed depleted retinal vascular beds and had significantly decreased retinal function as determined by electroretinography. CONCLUSIONS: Our data reveal that early severe postnatal inflammatory stress leads to abnormal retinal vascular development and increased vessel anastomosis and, ultimately, permanently compromises retinal function. The aberrant and initially exaggerated retinal vascularization observed is associated with microglial activation, providing a cellular mechanism by which perinatal sepsis predisposes to ROP.

Choroidal involution is a key component of oxygen-induced retinopathy.

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.

Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.

The failure of blood vessels to revascularize ischemic neural tissue represents a significant challenge for vascular biology. Examples include proliferative retinopathies (PRs) such as retinopathy of prematurity and proliferative diabetic retinopathy, which are the leading causes of blindness in children and working-age adults. PRs are characterized by initial microvascular degeneration, followed by a compensatory albeit pathologic hypervascularization mounted by the hypoxic retina attempting to reinstate metabolic equilibrium. Paradoxically, this secondary revascularization fails to grow into the most ischemic regions of the retina. Instead, the new vessels are misdirected toward the vitreous, suggesting that vasorepulsive forces operate in the avascular hypoxic retina. In the present study, we demonstrate that the neuronal guidance cue semaphorin 3A (Sema3A) is secreted by hypoxic neurons in the avascular retina in response to the proinflammatory cytokine IL-1ß. Sema3A contributes to vascular decay and later forms a chemical barrier that repels neo-vessels toward the vitreous. Conversely, silencing Sema3A expression enhances normal vascular regeneration within the ischemic retina, thereby diminishing aberrant neovascularization and preserving neuroretinal function. Overcoming the chemical barrier (Sema3A) released by ischemic neurons accelerates the vascular regeneration of neural tissues, which restores metabolic supply and improves retinal function. Our findings may be applicable to other neurovascular ischemic conditions such as stroke.

Functional and structural changes resulting from strain differences in the rat model of oxygen-induced retinopathy.

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 Long-Evans (LE) rats to verify this finding. METHODS: Newborn rats were exposed to 80% O(2) 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, hyperoxia-induced 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.