Intravitreal docosahexaenoic acid in a rabbit model: preclinical safety assessment

Purpose The purpose of the present study was to evaluate the retinal toxicity of a single dose of intravitreal docosahexaenoic acid (DHA) in rabbit eyes over a short-term period. Methods Sixteen New Zealand albino rabbits were selected for this pre-clinical study. Six concentrations of DHA (Brudy Laboratories, Barcelona, Spain) were prepared: 10 mg/50 µl, 5 mg/50 µl, 2'5 mg/50 µl, 50 µg/50 µl, 25 µg/50 µl, and 5 µg/50 µl. Each concentration was injected intravitreally in the right eye of two rabbits. As a control, the vehicle solution was injected in one eye of four animals. Retinal safety was studied by slit-lamp examination, and electroretinography. All the rabbits were euthanized one week after the intravitreal injection of DHA and the eyeballs were processed to morphologic and morphometric histological examination by light microscopy. At the same time aqueous and vitreous humor samples were taken to quantify the concentration of omega-3 acids by gas chromatography. Statistical analysis was performed by SPSS 21.0. Results Slit-lamp examination revealed an important inflammatory reaction on the anterior chamber of the rabbits injected with the higher concentrations of DHA (10 mg/50 µl, 5 mg/50 µl, 2'5 mg/50 µ) Lower concentrations showed no inflammation. Electroretinography and histological studies showed no significant difference between control and DHA-injected groups except for the group injected with 50 µg/50 µl. Conclusions Our results indicate that administration of intravitreal DHA is safe in the albino rabbit model up to the maximum tolerated dose of 25 µg/50 µl. Further studies should be performed in order to evaluate the effect of intravitreal injection of DHA as a treatment, alone or in combination, of different retinal diseases.

Puoliautomaattinen silmänpohjakuvien tulkintatyökalu

Tässä tutkimuksessa toteutettiin uusi versio aikaisemmin tuotetusta työkalusta merkintöjen tekemiseksi pääasiassa silmänpohjakuviin. Tarkoituksena oli toteuttaa kuvankäsittelyyn perustuvia aputoimintoja kuvien valaistuksenkorjaamiseksi, sekä korostaa lääkärille mahdollisia diabeettiseen retinopatiaan kuuluvia löydöksiä. Kuvien annotoinnin helpottamiseksi toteutettiin kaksi menetelmää valaistuksenkorjaamiseksi: yksiulotteinen käyrämenetelmä sekä värikanavien ominaisuuksia hyödyntävä menetelmä. Kuvien annotoinin helpottamiseksi toteutettiin kuvan vihreän kanavan jakaumaan perustuva aputoiminto, joka pyrkii korostamaan mahdollisia diabeettiseen retinopatiaan kuuluvia löydöksiä.

Eye Fundus Image Analysis for Automatic Detection of Diabetic Retinopathy

Diabetes is a rapidly increasing worldwide problem which is characterised by defective metabolism of glucose that causes long-term dysfunction and failure of various organs. The most common complication of diabetes is diabetic retinopathy (DR), which is one of the primary causes of blindness and visual impairment in adults. The rapid increase of diabetes pushes the limits of the current DR screening capabilities for which the digital imaging of the eye fundus (retinal imaging), and automatic or semi-automatic image analysis algorithms provide a potential solution. In this work, the use of colour in the detection of diabetic retinopathy is statistically studied using a supervised algorithm based on one-class classification and Gaussian mixture model estimation. The presented algorithm distinguishes a certain diabetic lesion type from all other possible objects in eye fundus images by only estimating the probability density function of that certain lesion type. For the training and ground truth estimation, the algorithm combines manual annotations of several experts for which the best practices were experimentally selected. By assessing the algorithm’s performance while conducting experiments with the colour space selection, both illuminance and colour correction, and background class information, the use of colour in the detection of diabetic retinopathy was quantitatively evaluated. Another contribution of this work is the benchmarking framework for eye fundus image analysis algorithms needed for the development of the automatic DR detection algorithms. The benchmarking framework provides guidelines on how to construct a benchmarking database that comprises true patient images, ground truth, and an evaluation protocol. The evaluation is based on the standard receiver operating characteristics analysis and it follows the medical practice in the decision making providing protocols for image- and pixel-based evaluations. During the work, two public medical image databases with ground truth were published: DIARETDB0 and DIARETDB1. The framework, DR databases and the final algorithm, are made public in the web to set the baseline results for automatic detection of diabetic retinopathy. Although deviating from the general context of the thesis, a simple and effective optic disc localisation method is presented. The optic disc localisation is discussed, since normal eye fundus structures are fundamental in the characterisation of DR.

CFH Y402H polymorphism and response to intravitreal Ranibizumab in brazilian patients with neovascular age-related macular degeneration

Objective: To investigate the association between CFH gene polymorphism and response to ranibizumab in Brazilian patients with neovascular age-related macular degeneration (AMD).Methods: 95 patients were genotyped for the CFH rs1061170 (Y402H) single nucleotide polymorphism. Patients with neovascular AMD initially received intravitreal ranibizumab injections for three months and were retreated as needed. Visual acuity (VA) and central retinal thickness (CRT) were measured before treatment and at 1, 3, 6, and 12 months post-treatment.Results: For patients with the TT and TC genotypes, paired comparisons of VA showed a statistically significant improvement when the data obtained at all visits were compared with baseline. Patients homozygous for the risk genotype (CC) did not show a statistically significant improvement when VA obtained at visits 1, 3, 6 and 12 were compared with baseline. For all genotypes, paired comparisons of CRT showed a statistically significant improvement when the data obtained at visits 1, 3, 6 and 12 were compared with baseline.Conclusion: Patients with the CC genotype showed poorer long-term functional response to intravitreal ranibizumab.

Comparison of full-field electroretinogram in diabetic and non diabetic dogs with cataracts

Being the commonest ocular disorder, dense cataracts disable fundoscopic examination and the diagnosis of retinal disorders, which dogs may be predisposed. The aim of this study was to compare the electroretinographic responses recorded according to the International Society for Clinical Electrophysiology of Vision human protocol to evaluate retinal function of diabetic and non diabetic dogs, both presenting mature or hypermature cataracts. Full-field electroretinogram was recorded from 66 dogs, with ages varying from 6 to 15 years old allocated into two groups: (1) CG, non diabetic cataractous dogs, and (2) DG, diabetic cataractous dogs. Mean peak-to-peak amplitude (microvolts) and b-wave implicit time (milliseconds) were determined for each of the five standard full-field ERG responses (rod response, maximal response, oscillatory potentials, single-flash cone response and 30 Hz flicker). Comparing CG to DG, ERGs recorded from diabetic dogs presented lower amplitude and prolonged b-wave implicit time in all ERG responses. Prolonged b-wave implicit time was statistically significant (p< 0.05) at 30 Hz flicker (24.0 ms versus 22.4 ms). These data suggests full-field ERG is capable to record sensible alterations, such as flicker's implicit time, being useful to investigate retinal dysfunction in diabetic dogs.

Distribution of AMPA-type glutamate receptor subunits in the chick visual system

Several glutamate receptor (GluR) subunits have been characterized during the past few years. In the present study, subunit-specific antisera were used to determine the distribution of the AMPA-type glutamate receptor subunits GluR1-4 in retinorecipient areas of the chick brain. Six white leghorn chicks (Gallus gallus, 7-15 days old, unknown sex) were deeply anesthetized and perfused with 4% buffered paraformaldehyde and brain sections were stained using immunoperoxidase techniques. The AMPA-type glutamate receptor subunits GluR1, GluR2/3 and GluR4 were present in several retinorecipient areas, with varying degrees of colocalization. For example, perikarya in layers 2, 3, and 5 of the optic tectum contained GluR1, whereas GluR2/3 subunits appeared mainly in neurons of layer 13. The GluR4 subunit was only detected in a few cells of the tectal layer 13. GluR1 and GluR2/3 were observed in neurons of the nucleus geniculatus lateralis ventralis, whereas GluR4 was only present in its neuropil. Somata in the accessory optic nucleus appeared to contain GluR2/3 and GluR4, whereas GluR1 was the dominant subunit in the neuropil of this nucleus. These results suggest that different subpopulations of visual neurons might express different combinations of AMPA-type GluR subunits, which in turn might generate different synaptic responses to glutamate derived from retinal ganglion cell axons

Electrophysiological measurements of spectral sensitivities: a review

Spectral sensitivities of visual systems are specified as the reciprocals of the intensities of light (quantum fluxes) needed at each wavelength to elicit the same criterion amplitude of responses. This review primarily considers the methods that have been developed for electrophysiological determinations of criterion amplitudes of slow-wave responses from single retinal cells. Traditional flash methods can require tedious dark adaptations and may yield erroneous spectral sensitivity curves which are not seen in such modifications as ramp methods. Linear response methods involve interferometry, while constant response methods involve manual or automatic adjustments of continuous illumination to keep response amplitudes constant during spectral scans. In DC or AC computerized constant response methods, feedback to determine intensities at each wavelength is derived from the response amplitudes themselves. Although all but traditional flash methods have greater or lesser abilities to provide on-line determinations of spectral sensitivities, computerized constant response methods are the most satisfactory due to flexibility, speed and maintenance of a constant adaptation level

Inhibition of carbachol-induced inositol phosphate accumulation in the embryonic retina promoted by kainate and veratridine

In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 µM carbachol was used, the estimated IC50 value for kainate was 0.2 µM and the maximal inhibition of ~50% was obtained with 1 µM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 µM veratridine, but not 50 mM KCl, inhibited ~65% of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 ± 38.0 to 2044.5 ± 299.9 cpm/mg protein, retinal response decreased to 861.6 ± 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.

Nuclear exclusion of transcription factors associated with apoptosis in developing nervous tissue

Programmed cell death in the form of apoptosis involves a network of metabolic events and may be triggered by a variety of stimuli in distinct cells. The nervous system contains several neuron and glial cell types, and developmental events are strongly dependent on selective cell interactions. Retinal explants have been used as a model to investigate apoptosis in nervous tissue. This preparation maintains the structural complexity and cell interactions similar to the retina in situ, and contains cells in all stages of development. We review the finding of nuclear exclusion of several transcription factors during apoptosis in retinal cells. The data reviewed in this paper suggest a link between apoptosis and a failure in the nucleo-cytoplasmic partition of transcription factors. It is argued that the nuclear exclusion of transcription factors may be an integral component of apoptosis both in the nervous system and in other types of cells and tissues.

Treatment of cytomegalovirus retinitis with an intraocular sustained-release ganciclovir implant

The objective of this prospective study was to evaluate the efficacy and complications of the use of an intraocular sustained-release ganciclovir implant for the treatment of active cytomegalovirus (CMV) retinitis in AIDS patients. Thirty-nine eyes of 26 patients were submitted to ocular surgery. All patients underwent complete ocular examination before and after surgery. The surgical procedure was always done under local anesthesia using the same technique. The mean time for the surgical procedure was 20 min (range, 15 to 30 min). The average follow-up period was 3.7 months. Of all patient, only 4 presented recurrence of retinitis after 8, 8, 9 and 2 months, respectively. Three of them received a successful second implant. All 39 eyes of the 26 patients presented healing of retinitis as shown by clinical improvement evaluated by indirect binocular ophthalmoscopy and retinography. Retinitis healed within a period of 4 to 6 weeks in all patients, with clinical regression signs from the third week on. Six (15.4%) eyes developed retinal detachment. None of the patients developed CMV retinitis in the contralateral eye. The intraocular implant proved to be effective in controlling the progression of retinitis for a period of up to 8 months even in patients for whom systemic therapy with either ganciclovir or foscarnet or both had failed. The intraocular sustained-release ganciclovir implant proved to be a safe new procedure for the treatment of CMV retinitis, avoiding the systemic side effects caused by the intravenous medications and improving the quality of life of the patients.

Trabeculectomy and optic nerve head topography

The objective of the present study was to evaluate changes in optic nerve head parameters, measured by confocal laser tomography, before and after trabeculectomy in order to identify outcome measures for the management of glaucoma. The optic nerve head of 22 eyes (22 patients) was analyzed by confocal laser tomography with the Heidelberg retinal tomogram (HRT) before and after trabeculectomy. The median time between the first HRT and surgery was 4.6 months (mean: 7.7 ± 8.3) and the median time between surgery and the second HRT was 10.8 months (mean: 12.0 ± 6.8). The patients were divided into two groups, i.e., those with the highest (group A) and lowest (group B) intraocular pressure (IOP) change after surgery. Differences in the 12 standard topographic parameters before and after surgery for each group were evaluated by the Wilcoxon signed rank test and the differences in these parameters between the two groups were compared by the Mann-Whitney rank sum test. Multiple regression analysis was used to evaluate the influence of the change in IOP (deltaIOP and deltaIOP%) and the changes in the other parameters. There were significant differences in the HRT measures before and after surgery in group A only for cup volume. In group B, no parameter was statistically different. The changes in group A were not significantly different than those in group B for any parameter (P > 0.004, Bonferroni correction for multiple comparisons). deltaIOP and deltaIOP% had a statistically significant effect on delta cup disk area, delta cup volume and delta mean cup depth. Changes in cup shape size were influenced significantly only by deltaIOP. Some optic disc parameters measured by HRT presented a significant improvement after filtering surgery, depending on the amount of IOP reduction. Long-term studies are needed to determine the usefulness of these findings as outcome measures in the management of glaucoma.

Identification of a new human mtDNA polymorphism (A14290G) in the NADH dehydrogenase subunit 6 gene

Leber's hereditary optic neuropathy (LHON) is a maternally inherited form of retinal ganglion cell degeneration leading to optic atrophy in young adults. Several mutations in different genes can cause LHON (heterogeneity). The ND6 gene is one of the mitochondrial genes that encodes subunit 6 of complex I of the respiratory chain. This gene is a hot spot gene. Fourteen Persian LHON patients were analyzed with single-strand conformational polymorphism and DNA sequencing techniques. None of these patients had four primary mutations, G3460A, G11788A, T14484C, and G14459A, related to this disease. We identified twelve nucleotide substitutions, G13702C, T13879C, T14110C, C14167T, G14199T, A14233G, G14272C, A14290G, G14365C, G14368C, T14766C, and T14798C. Eleven of twelve nucleotide substitutions had already been reported as polymorphism. One of the nucleotide substitutions (A14290G) has not been reported. The A14290G nucleotide substitution does not change its amino acid (glutamic acid). We looked for base conservation using DNA star software (MEGALIGN program) as a criterion for pathogenic or nonpathogenic nucleotide substitution in A14290G. The results of ND6 gene alignment in humans and in other species (mouse, cow, elegans worm, and Neurospora crassa mold) revealed that the 14290th base was not conserved. Fifty normal controls were also investigated for this polymorphism in the Iranian population and two had A14290G polymorphism (4%). This study provides evidence that the mtDNA A14290G allele is a new nonpathogenic polymorphism. We suggest follow-up studies regarding this polymorphism in different populations.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

Can the fractal dimension be applied for the early diagnosis of non-proliferative diabetic retinopathy?

The fractal dimension has been employed as a useful parameter in the diagnosis of retinal disease. Avakian et al. (Curr Eye Res 2002; 24: 274-280), comparing the vascular pattern of normal patients with mild to moderate non-proliferative diabetic retinopathy (NPDR), found a significant difference between them only in the macular region. This significant difference in the box-counting fractal dimension of the macular region between normal and mild NPDR patients has been proposed as a method of precocious diagnosis of NPDR. The aim of the present study was to determine if fractal dimensions can really be used as a parameter for the early diagnosis of NPDR. Box-counting and information fractal dimensions were used to parameterize the vascular pattern of the human retina. The two methods were applied to the whole retina and to nine anatomical regions of the retina in 5 individuals with mild NPDR and in 28 diabetic but opthalmically normal individuals (controls), with age between 31 and 86 years. All images of retina were obtained from the Digital Retinal Images for Vessel Extraction (DRIVE) database. The results showed that the fractal dimension parameter was not sensitive enough to be of use for an early diagnosis of NPDR.

Protective mechanism of agmatine pretreatment on RGC-5 cells injured by oxidative stress

Agmatine has neuroprotective effects on retinal ganglion cells (RGCs) as well as cortical and spinal neurons. It protects RGCs from oxidative stress even when it is not present at the time of injury. As agmatine has high affinity for various cellular receptors, we assessed protective mechanisms of agmatine using transformed RGCs (RGC-5 cell line). Differentiated RGC-5 cells were pretreated with 100 μM agmatine and consecutively exposed to 1.0 mM hydrogen peroxide (H2O2). Cell viability was determined by measuring lactate dehydrogenase (LDH), and the effects of selective alpha 2-adrenergic receptor antagonist yohimbine (0-500 nM) and N-methyl-D-aspartic acid (NMDA) receptor agonist NMDA (0-100 µM) were evaluated. Agmatine’s protective effect was compared to a selective NMDA receptor antagonist MK-801. After a 16-h exposure to H2O2, the LDH assay showed cell loss greater than 50%, which was reduced to about 30% when agmatine was pretreated before injury. Yohimbine almost completely inhibited agmatine’s protective effect, but NMDA did not. In addition, MK-801 (0-100 µM) did not significantly attenuate the H2O2-induced cytotoxicity. Our results suggest that neuroprotective effects of agmatine on RGCs under oxidative stress may be mainly attributed to the alpha 2-adrenergic receptor signaling pathway.