Method for retinal gene repair in neonatal mouse.

Gene correction at the site of the mutation in the chromosome is the absolute way to really cure a genetic disease. The oligonucleotide (ODN)-mediated gene repair technology uses an ODN perfectly complementary to the genomic sequence except for a mismatch at the base that is mutated. The endogenous repair machinery of the targeted cell then mediates substitution of the desired base in the gene, resulting in a completely normal sequence. Theoretically, it avoids potential gene silencing or random integration associated with common viral gene augmentation approaches and allows an intact regulation of expression of the therapeutic protein. The eye is a particularly attractive target for gene repair because of its unique features (small organ, easily accessible, low diffusion into systemic circulation). Moreover therapeutic effects on visual impairment could be obtained with modest levels of repair. This chapter describes in details the optimized method to target active ODNs to the nuclei of photoreceptors in neonatal mouse using (1) an electric current application at the eye surface (saline transpalpebral iontophoresis), (2) combined with an intravitreous injection of ODNs, as well as the experimental methods for (3) the dissection of adult neural retinas, (4) their immuno-labelling, and (5) flat-mounting for direct observation of photoreceptor survival, a relevant criteria of treatment outcomes for retinal degeneration.

Retinal embolisation with localised retinal detachment following retrobulbar anaesthesia.

BACKGROUND: The aim of this communication is to describe an unusual and serious complication of retrobulbar anaesthesia for cataract surgery. HISTORY AND SIGNS: A 78-year-old female was referred for visual loss (light perception) 24 hours after apparently uneventful cataract surgery with retrobulbar anaesthesia in her left eye. Fundus examination revealed multiple arterial emboli and a localised retinal detachment. MRI revealed a retrobulbar hypersignal of the optic nerve associated with perineuritis. The cardiovascular examination was normal. We assumed this condition resulted from injection of the anaesthetic mixture into the optic nerve. THERAPY AND OUTCOME: In order to improve retinal circulation and oxygenation, the intraocular pressure was maximally lowered and anticalcic therapy administered, expecting optimal arterial dilatation. Methylprednisolone (1 g/day 3 days i. v., then rapidly tapered) was also added. The retina slowly reattached but visual acuity remained unchanged. CONCLUSIONS: Retrobulbar anaesthesia is routinely used for ocular surgery. Serious complications may still happen, however. This case adds to the previously reported spectrum of complications from retrobulbar anaesthesia.

Tearing and folding of the retinal internal limiting membrane associated with macular epiretinal membrane

PURPOSE: To describe the clinical and histologic features of a particular form of macular epiretinal membrane. METHODS: The charts of all patients operated for macular epiretinal membrane by a single surgeon (E.H.B.) between June 2001 and January 2005 were retrospectively reviewed. Patients with macular epiretinal membrane associated with tearing and folding of the internal limiting membrane (ILM) were identified and the following parameters were recorded when available: age, gender, best-corrected visual acuity before and after vitrectomy; optical coherence tomography; pre-, intra-, and postoperative macular status; intraoperative staining by indocyanine green; histology. RESULTS: Twenty-three of 268 eyes (8.6%) with macular epiretinal membrane were associated with tearing and folding of the ILM, forming a whitish prominent band on the surface of the retina. The mean age of the patients was 68.6 years with a significant female predominance (78.3%). The vitreous was completely detached in 21 eyes. After surgical peeling, the mean visual gain was 3.2 Early Treatment Diabetic Retinopathy Study lines. No recurrence was observed. CONCLUSION: Tearing and folding of the ILM was associated with macular epiretinal membranes in 8.6% of cases. The ILM was probably torn during posterior hyaloid detachment, but the pathogenesis has not been clearly elucidated. The surgeon should begin to peel the macular epiretinal membrane by grasping the folded ILM to ensure complete removal of the ILM together with the epiretinal membrane. The postoperative visual prognosis was good

Heritability of the retinal microcirculation in flemish families.

BACKGROUND Few population studies have described the heritability and intrafamilial concordance of the retinal microvessels, or the genetic or environmental correlations of the phenotypes of these vessels. METHODS We randomly selected 413 participants from 70 families (mean age, 51.5 years; 50.1% women) from a Flemish population. We postprocessed retinal images using IVAN software to generate the central retinal arteriole equivalent (CRAE), central retinal venule equivalent (CRVE), and arteriole-to-venule-ratio (AVR) from these images. We used SAGE version 6.2 and SAS version 9.2 to compute multivariate-adjusted estimates of heritability and intrafamilial correlations of the CRAE, CRVE, and AVR of the retinal microvessels in the images. RESULTS Sex, age, mean arterial pressure, and smoking explained up to 12.7% of the variance of the phenotypes of the retinal microvessels of the study participants. With adjustments applied for these covariates, the heritability estimates of CRAE, CRVE, and AVR were 0.213 (P = 0.044), 0.339 (P = 0.010), and 0.272 (P = 0.004), respectively. The parent-offspring correlations for CRAE, CRVE, and AVR were 0.118 (NS), 0.225 (P < 0.01), and 0.215 (P < 0.05), respectively. The corresponding values were 0.222 (P < 0.05), 0.213 (P < 0.05), and 0.390 (P < 0.001) for sib-sib correlations, respectively. The genetic and environmental correlations between CRAE and CRVE were 0.360 and 0.545 (P < 0.001 for both). CONCLUSION Our study showed moderate heritability for CRAE, CRVE, and AVR, and a significant genetic correlation of CRAE with CRVE in the Flemish population of our study. These findings suggest that genetic factors influence the diameter of the retinal microvessels, and that CRAE and CRVE share some genetic determinants.

The vasodilatory effect of juxta-arteriolar microinjection of endothelinA receptor inhibitor in healthy and acute branch retinal vein occlusion minipig retinas.

Purpose. To investigate the effect of the endothelin(A) receptor inhibitor BQ-123 on the retinal arteriolar vasculature in minipig retinas in normal eyes and eyes with acute branch retinal vein occlusion (BRVO). Methods. Seven healthy eyes of seven minipigs and six eyes of six minipigs with experimental BRVO were evaluated under systemic anesthesia. An intravitreal juxta-arteriolar microinjection of 30 microL BQ-123 0.61 microg/mL (pH 7.4) was performed in all but one eye from each group, into which the physiologic saline vehicle alone was injected. Vessel-diameter changes were measured with a retinal vessel analyzer. Results. In healthy minipig retinas (n = 6), arteriolar diameter (+/-SD) increased 6.19% +/- 3.55% (P < 0.05), 25.98% +/- 2.37% (P < 0.001), 23.65% +/- 1.2% (P < 0.001), and 16.84% +/- 1.95% (P < 0.001), at 1, 5, 10, and 15 minutes, respectively, after BQ-123 microinjection. Two hours after experimental BRVO (n = 5), the retinal arteriolar diameter had decreased (13.07% +/- 5.7%; P < 0.01). One, 5, 10, and 15 minutes after BQ-123 microinjection, retinal arteriolar diameter had increased by 7.14% +/- 3.3% (P < 0.01), 26.74% +/- 7.63% (P < 0.001), 23.67% +/- 6.4% (P < 0.001), and 16.09% +/- 3.41% (P < 0.001), respectively. Vehicle only injection had no vasoactive effect on physiologic or BRVO retinas. Conclusions. A significant increase in retinal arteriolar diameter was demonstrated after juxta-arteriolar BQ-123 microinjection in healthy and in acute BRVO minipig retinas. The results suggest a role for endothelin-1 in maintaining retinal basal arteriolar tone. Reversing the BRVO-related vasoconstriction by juxta-arteriolar BQ-123 microinjection could bring a new perspective to the management of BRVO.

In vivo inhibition of lens regrowth by fibroblast growth factor 2-saporin.

PURPOSE: To investigate the ability of fibroblast growth factor (FGF) 2-saporin to prevent lens regrowth in the rabbit. METHODS: Chemically conjugated and genetically fused FGF2-saporin (made in Escherichia coli) were used. Extracapsular extraction of the lens was performed on the rabbit, and the cytotoxin either was injected directly into the capsule bag or was administered by FGF2-saporin-coated, heparin surface-modified (HSM) polymethylmethacrylate intraocular lenses. The potential of the conjugate was checked by slit lamp evaluation of capsular opacification and by measuring crystallin synthesis. Toxin diffusion and sites of toxin binding were assessed by immunohistochemistry. Possible toxicity was determined by histologic analysis of ocular tissues. RESULTS: FGF2-saporin effectively inhibited lens regrowth when it was injected directly into the capsular bag. However, high concentration of the toxin induced transient corneal edema and loss of pigment in the iris. Intraocular lenses coated with FGF2-saporin reduced lens regrowth and crystallin synthesis without any detectable clinical side effect. After implantation, FGF2-saporin was shown to have bound to the capsules and, to a lesser extent, to the iris; no histologic damage was found on ocular tissues as a result of implantation of drug-loaded HSM intraocular lenses. CONCLUSIONS: Chemically conjugated (FGF2-SAP) and genetically fused FGF2-saporin (rFGF2-SAP) bound to HSM intraocular lenses can prevent lens regrowth in the rabbit.

Autofluorescence Study In NR2E3 p.G56R-linked Autosomal Dominant Retinitis Pigmentosa In A Single Kindred

Purpose: To date, the genotype/phenotype correlation of p.G56R-linked autosomal dominant retinitis pigmentosa (ADRP) is limited to less than 10 kindred. The purpose of this study is to report an unusual appearance of fundus autofluorescence (AF) with NR2E3 p.G56R-linked ADRP in a single kindred.Methods: Patients were enrolled among three generations in a previously unreported family. Molecular diagnosis was performed on all exons of NR2E3 and a p.G56R mutation was identified in affected family members only. Examinations included fundus photography, visual fields, optical coherence tomography, AF, near-infrared AF and ISCEV-standard electrophysiology (ERG).Results: Among 10 examined family members, 5 were affected. The youngest and oldest patients were 16 and 65 years old, respectively. Fundus examination revealed a range of retinal disorder from normal to optic nerve pallor, attenuated arterial caliber and bone spicule-like pigment deposits. In all patients, AF showed a double hyperfluorescent ring; an inner paramacular ring which extension was comparable among patients and an outer ring along the vascular arcades which extended towards periphery in older patients and became hypofluorescent. Maximal scotopic ERGs when recordable showed an increased a/b wave ratio.Conclusions: A double hyperfluorescent ring on AF is an uncommon observation and might be a specific clinical finding in NR2E3 p.G56R-linked ADRP. The consistency of that finding in all affected members of our 3-generation family confirms a previous study. Further analysis is required to determine whether AF changes are associated with particular retinal layer abnormalities.

Role of the c-Jun N-terminal kinase pathway in retinal excitotoxicity, and neuroprotection by its inhibition.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06705.x Abstract Retinal excitotoxicity is associated with retinal ischemia, and with glaucomatous and traumatic optic neuropathy. The present study investigates the role of c-Jun N-terminal kinase (JNK) activation in NMDA-mediated retinal excitotoxicity and determines whether neuroprotection can be obtained with the JNK pathway inhibitor, d-form of JNK-inhibitor 1 (d-JNKI-1). Young adult rats received intravitreal injections of 20 nmol NMDA, which caused extensive neuronal death in the inner nuclear and ganglion cell layers. This excitotoxicity was associated with strong activation of calpain, as revealed by fodrin cleavage, and of JNK. The cell-permeable peptide d-JNKI-1 was used to inhibit JNK. Within 40 min of its intravitreal injection, FITC-labeled d-JNKI-1 spread through the retinal ganglion cell layer into the inner nuclear layer and interfered with the NMDA-induced phosphorylation of JNK. Injections of unlabeled d-JNKI-1 gave unprecedentedly strong neuroprotection against cell death in both layers, lasting for at least 10 days. The NMDA-induced calpain-specific fodrin cleavage was likewise strongly inhibited by d-JNKI-1. Moreover the electroretinogram was partially preserved by d-JNKI-1. Thus, the JNK pathway is involved in NMDA-mediated retinal excitotoxicity and JNK inhibition by d-JNKI-1 provides strong neuroprotection as shown morphologically, biochemically and physiologically.