Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors

Structurally stable in vitro-model systems are indispensible to analyse neural development during embryogenesis, follow cellular differentiation and evaluate neurotoxicological or growth factor effects. Here we describe a three-dimensional, long-term in vitro-culture system of the embryonic chick retina which supports photoreceptor development. Retinal tissue was isolated from E6 chick eye, and cultured as explants by continuous orbital rotation to allow free floatation without any supporting materials. Young stage (E6) immature retinas were cultured for various time periods in order to follow the differentiation of cell types and plexiform layers by immunocytochemical methods. These explants could be cultured for at least 2–3 weeks with remarkable retention of retinal architecture. Interestingly, photoreceptors developed in the absence of pigment epithelium. Electron microscopic studies revealed formation of structures resembling photoreceptor outer segments, a feature not reported previously. Thus, the verification of photoreceptors, Müller cells, inner retinal cells and the inner plexiform layer described in our study establishes this explant culture as a valuable in vivo-like model system.     

L‐ and M‐cone input to 12Hz and 30Hz flicker ERGs across the human retina

We recorded L‐ and M‐cone isolating ERGs from human subjects using a silent substitution technique at temporal rates of 12 and 30 Hz. These frequencies isolate the activity of cone‐opponent and non‐opponent post‐receptoral mechanisms, respectively. ERGs were obtained using a sequence of stimuli with different spatial configurations comprising; (1) circular stimuli of different sizes which increased in 10° steps up to 70°diameter, or (2) annular stimuli with a 70° outer diameter but with different sized central ablations from 10° up to 60°. L‐ and M‐cone isolating ERGs were obtained from five colour normal subjects using a DTL fibre electrode. Fourier analysis of the ERGs was performed and we measured the amplitude of the first harmonic of the response. For 12 Hz ERGs the L:M cone response amplitude ratio (L:M_ERG) was close to unity and remained stable irrespective of the spatial configuration of the stimulus. The maintenance of this balanced ratio points to the existence of cone selective input across the human retina for the L‐M cone opponent mechanism. For 30 Hz the L:M_ERG ratio was greater than unity but varied depending upon which region of the retina was being stimulated. This variation we consider to be a consequence of the global response properties of M‐cone ERGs rather than representing a real variation in L:M cone ratios across the retina.     

Brain-derived neurotrophic factor enhances neurite regeneration from retinal ganglion cells in aged human retina in vitro

To investigate the capability of neurite regeneration from retinal ganglion cells (RGCs) in an adult human retina and to evaluate the effect of neurotrophin on the neurite regeneration, an in vitro model for retinal explants was developed. A human retina was obtained from a 70 year old patient with retrobulbar carcinoma. The retina was excised and the retinal explants were cultured in serum-free medium with or without brain-derived neurotrophic factor. The capability of neurite regeneration was evaluated by counting the numbers of outgrowing neurites outside the retinal explants. In culture without brain-derived neurotrophic factor (control), there was no neurite outgrowth from the retinal explants after 2 days. And at 3 days in culture, a small number of outgrowing neurites were first observed outside the retinal explants. In contrast, within 24 hr in culture with brain-derived neurotrophic factor, there were a considerable number of elongating neurites with spread growth cones from the retinal explants. Immunohistochemical analysis revealed that these neurites were derived from RGCs. The addition of brain-derived neurotrophic factor increased the number of outgrowing neurites approximately 10-fold compared to that of the control at 3 days in culture. The enhancement of neurite regeneration induced by brain-derived neurotrophic factor continued for longer than 1 week in culture. In conclusion, an aged human retina can regenerate neurites from RGCs in vitro and brain-derived neurotrophic factor significantly promotes the regeneration.     

Influence of donor age,post‐mortem time and cold storage on metabolic profile of human cornea

Purpose: Limited knowledge exists about the influence of donor age and death‐to‐preservation interval (DPI) on the metabolic properties of the cornea. The aim of this study is to investigate the relationship between both factors and metabolite content of the cornea. Methods: Corneas from 15 human donors (age: 41–78 years) were obtained within 16 hrs post‐mortem and kept in cold storage for 8 days. The metabolic profiles of the samples were investigated using high‐resolution, magic angle spinning ¹H nuclear magnetic resonance spectroscopy before and after 8 days of preservation. Results: Twenty‐two metabolites were detected and assigned in the corneal spectra. The significant metabolic differences before and after hypothermic storage were revealed between younger and older donors. DPI‐related significant differences revealed before preservation of the corneas were not displayed after 8 days of cold storage. Conclusions: Age of donor as well as post‐mortem time influences the biochemical properties of the cornea. Cold storage decreases the metabolite differences between the tissues collected at different post‐mortem time.     

Cancer‐like metabolism of the mammalian retina

The retina, like many cancers, produces energy from glycolysis even in the presence of oxygen. This phenomenon is known as aerobic glycolysis and eponymously as the Warburg effect. In recent years, the Warburg effect has become an explosive area of study within the cancer research community. The expanding knowledge about the molecular mechanisms underpinning the Warburg effect in cancer promises to provide a greater understanding of mammalian retinal metabolism and has motivated cancer researchers to target the Warburg effect as a novel treatment strategy for cancer. However, if the molecular mechanisms underlying the Warburg effect are shared by the retina and cancer, treatments targeting the Warburg effect may have serious adverse effects on retinal metabolism. Herein, we provide an updated understanding of the Warburg effect in mammalian retina.     

VIP,PACAP‐38, BDNF and ADNP in NMDA‐induced excitotoxicity in the rat retina

Purpose:  To evaluate the effect of intravitreal injection of N‐methyl‐D‐aspartate (NMDA) on brain‐derived neurotrophic factor (BDNF), pituitary adenylate cyclase‐activating peptide‐38 (PACAP‐38), vasoactive intestinal peptide (VIP) and the VIP‐associated glial protein activity‐dependent neuroprotective protein (ADNP) in the rat retina. These elements have well‐documented neuroprotective properties and may thus be integrated in endogenous neuroprotective mechanisms in the retina which break down in NMDA excitotoxicity. Methods:  A volume of 2 μl of 100 nmol NMDA was intravitreally injected into one eye of rats, the untreated eye served as a control. Time‐dependent effects of NMDA on VIP, PACAP‐38 and BDNF were detected by radioimmunoassay and ELISA, and the effect on the expression of VIP, PACAP‐38 and ADNP was evaluated by quantitative RT‐PCR 20 days after NMDA injection. Topical flunarizine served to find out whether the effect of NMDA is counteracted. Results:  Compared to PACAP‐38, VIP levels significantly decreased on days 1, 7, 14, 28 and 56 after NMDA injection indicating that VIPergic cells are more vulnerable than PACAP‐38‐expressing cells. The expression of VIP and ADNP but not of PACAP‐38 was found to be reduced, and application of topical flunarizine counteracted the decrease of VIP. BDNF levels significantly increased after days 1 and 3. Conclusion:  The early upregulation of BDNF seems to act neuroprotectively and leads to a delay of ganglion cell loss. Although there is no direct evidence, the decrease of VIP and ADNP – the consequence of the presence of NMDA receptors on these peptide‐expressing cells – might contribute to the breakdown of endogenous neuroprotective mechanisms given that the decrease of the VIP‐related ADNP runs in parallel with the decrease of VIP. Activating and maintaining these mechanisms must be the primary aim in the therapy of diseases with retinal neuronal degeneration.     

Copper and zinc distribution in the human retina: relationship to cadmium accumulation, age, and gender

The essential metals copper and zinc play vital roles in retinal cell survival and are crucial for the normal functioning of antioxidant enzymes. Retinal zinc deficiencies and decreased cellular antioxidative capacity have been linked to human retinal diseases including age-related macular degeneration (AMD). We recently reported that cadmium (a toxic metal with no known physiological function that interferes with copper and zinc metabolism) accumulates in human retinal tissues during aging. Moreover, cadmium content was higher in specific retinal tissues of aged women compared to men. Since cadmium, zinc and copper bind to similar proteins, we hypothesized that Cu and Zn content of human retinal tissues change as functions of cadmium accumulation during aging. Thus, we assessed the distribution of zinc and copper in the neural retina, retinal pigment epithelium (RPE) and choroid (Bruch's membrane-choroid; BMC) in male and female donors aged 1.5-87 years. Two independent methods, graphite furnace atomic absorption spectrometry and inductively-coupled plasma mass spectrometry, were used to measure Cd, Zn, and Cu in retinal tissues in human eyes from donors aged 1.5 to 87 years and the resulting values were normalized to protein concentration. Zn levels were ∼5 times higher than Cu levels in the same tissues. The relative tissue distributions of these metals were: BMC > RPE > neural retina (Zn) and BMC > RPE = neural retina (Cu). In the choroid, mean Cu and Zn levels were higher in aged donors (≥55 years old) than young donors (<55 years) and levels of these metals were strongly correlated with each other (r = 0.90). In the neural retina, Cu and Zn both significantly decreased as a function of age. Several sex-related differences were found in the RPE. Specifically, copper levels were significantly higher in males than in females. In addition, both Zn and Cu levels in males were positively correlated with cadmium content, whereas this association did not occur in females. The results are consistent with co-regulation of zinc and copper stores in retinal tissues and suggest that the balance of these metals is associated with cadmium accumulation and gender. Thus, the roles of cadmium and gender differences in retinal metal balance warrant further investigation as factors in age-related retinal disease.     

Two separate Ni2+‐sensitive voltage‐gated Ca2+channels modulate transretinal signalling in the isolated murine retina

Purpose: Light‐evoked responses from vertebrate retinas were recorded as an electroretinogram (ERG). The b‐wave is the most prominent component of the ERG, and in the bovine retina its NiCl₂‐sensitive component was attributed to reciprocal signalling by pharmacoresistant R‐type voltage‐gated Ca²⁺ channels, which similar to other voltage‐dependent Ca²⁺ channels trigger and control neurotransmitter release. The murine retina has the great advantage that the effect of gene inactivation for Ni²⁺‐sensitive Ca²⁺ channels can be analysed to prove or disprove that any of these Ca²⁺ channels is involved in retinal signalling. Methods: Superfused retinas from different murine genotypes lacking either one or both highly Ni²⁺‐sensitive voltage‐gated Ca²⁺ channels were used to record their ex vivo ERGs. Results: The isolated retinas from mice lacking Ca_v2.3 R‐type or Ca_v3.2 T‐type or both voltage‐gated Ca²⁺ channels were superfused with a NiCl₂ (15 μm ) containing nutrient solution. The change in the b‐wave amplitude and implicit time, caused by NiCl₂, was calculated as a difference spectrum and compared to data from control animals. From the results, it can be deduced that Ca_v2.3 contributes rather to a later component in the b‐wave response, while in the absence of Ca_v3.2 the gain of Ni²⁺‐mediated increase in the b‐wave amplitude is significantly increased, probably due to a loss of reciprocal inhibition to photoreceptors. Thus, each of the Ni²⁺‐sensitive Ca²⁺ channels contributes to specific features of the b‐wave response. Conclusion: Both high‐affinity Ni²⁺‐sensitive Ca²⁺ channels contribute to transretinal signalling. Based on the results from the double knockout mice, additional targets for NiCl₂ must contribute to transretinal signalling, which will be most important for the structurally similar physiologically more important heavy metal cation Zn²⁺.     

Neurofibromatosis 2 leads to higher incidence of strabismological and neuro‐ophthalmological disorders

Purpose: Ophthalmic features of neurofibromatosis 2 (NF2) include juvenile cataract, retinal hamartomas and tumours of the cranial nerves. We hypothesize that these tumours lead to strabismological and neuro‐ophthalmological symptoms, including palsies of cranial nerves III, IV and VI, nystagmus and gaze palsies. Methods: We carried out a retrospective review of 73 patients with known genotype. They underwent ophthalmic, neuro‐ophthalmological and strabismological examination. Statistical analysis was performed by calculating odds ratios and their 95% confidence intervals. Results: Mean best corrected visual acuity was 0.85. Strabismus was found in 38 of 73 patients (52%). A deviation based on a cranial nerve palsy was found in 16 patients (22%) and three had supranuclear palsies. Eleven of 73 patients had a nystagmus, mostly caused by peripheral–vestibular disturbance. Binocular single vision was normal in 41 (58%), subnormal in six (8%) and not present in 24 (34%) patients. The average refractive error was ? 0.57 D. Myopia of ≥ 0.5 D was present in 47 (33%) eyes and hyperopia of ≥ 2.0 D was measured in 11 (8%) eyes. In the subgroup analysis of NF2 mutation types, the relative risk for cranial nerve palsies and negative stereopsis was statistically significantly increased for the nonsense mutation group. The mosaicism group had a statistically significant decreased relative risk for concomitant squint, as did patients with unfound mutations for strabismus and poor stereopsis. Conclusions: The present study is, to our knowledge, the first to examine a larger collection of NF2 patients for strabismological and neuro‐ophthalmological lesions. Compared with the normal population, our sample showed a higher amount of strabismus, refractive errors and an increased incidence of vestibular nystagmus.     

A demonstration of a switch of cell type in human fetal eye tissues in vitro: pigmented cells of the iris or the retina can transdifferentiate into lens

Pigmented cells of iris or retina from two human fetuses at 12 weeks after conception were cultured in vitro. In early stages of culturing (up to about 20 days), all cultured cells lost pigment granules. “Lentoid bodies” appeared at about 45 days in primary cultures of iris cells. Primary cultures of retinal pigment cells were repigmented by 30 days. When a small number of repigmented cells was transferred into secondary cultures, colonies with “lentoid bodies” were formed by 40 days. The lens nature of “lentoid bodies” was confirmed by immunofluorescence and immunoelectrophoresis using anti-rat lens serum which crossreacted with human crystallins.     

Cadmium accumulation in the human retina: effects of age, gender, and cellular toxicity

Tobacco smoking and aging are among the few factors linked to age-related macular degeneration (AMD), a major cause of blindness in the elderly. Recent studies indicate that cadmium (Cd), an environmental toxic trace metal, is approximately four-fold higher in the retinas of smokers compared to non-smokers. In this study, we determined the effects of age and gender on Cd accumulation in human retinal tissues, specifically the neural retina, retinal pigment epithelium (RPE), and choroid. Cadmium levels in cultured RPE cells or retinal tissues isolated from frozen donor eyes were measured using inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrophotometry (GF-AAS). Cadmium uptake in cultured human RPE cells (ARPE-19) was also assessed using GF-AAS. Toxic effects of cadmium were determined from cell loss (measured as a decrease in cell density) and lactate dehydrogenase release (an indicator of membrane disruption). In "young" eyes (< 55 years) Cd was highest in the retinal pigment epithelium and lowest in the neural retina. Cd was higher in all tissues in aged eyes (>or=55 years) and was significantly higher in the neural retina and RPE in older females. Cultured RPE cells exposed to Cd showed altered cell morphology, decreased cell survival, elevated ROS levels and concentration-dependent disruption of membrane integrity. We conclude that cadmium is accumulated differently in the neural retinal and RPE of older men and women. The deleterious effects of Cd on RPE cells indicate that this environmental toxin is a potentially important factor in age-related retinal disease.