Sébastien Bonnel

Quantitative analysis of intravitreal injections in the rat

Intravitreal injections are currently used in the rat to introduce a therapeutic factor in the eye, especially for experimental treatments of retinal degenerations. The injected volume and its location can influence the quantification of results. We have investigated the quantitative effect of a single intravitreal injection in rats at different ages and for different volumes. Albinos rats aged three weeks or two months received intravitreal injections of 1, 3, 5 or 10 µl China ink. Animals were sacrificed immediately after injection, eyes were enucleated, fixated, embedded in paraffin and microtomy was performed in a sagittal plane. Regularly spaced sections were analyzed to reconstruct the vitreous and injected dye volumes. The measured vitreous volume was 6.76 ± 0.37 mm 3 in three weeks old rats and 13.36 ± 0.64 mm 3 in two months old rats. Mean intravitreal ink volumes immediately after injection were 0.8 mm 3 for 1 µl injections, 2 mm 3 for 3 µl, 2.3 to 2.6 mm 3 for 5 µl and 3.2 mm 3 for 10 µl. The percentage of vitreous volume involved by the injection ranged from 4.4% to 33.2%. The injected volume is limited by the large lens size of the rat. Extraocular loss of injected solution increases for higher injected volumes, with larger standard deviations. In this model, the dye tends to localize behind the lens. A 3 or 5 µl volume appears to have the best reproducibility with minimum loss of solution.

Ocular Cell Transfection with the Human Basic Fibroblast Growth Factor Gene Delays Photoreceptor Cell Degeneration in RCS Rats

Based on the K8/JTS-1-mediated transfection technique, we developed an in vivo protocol for an efficient transfer of plasmid DNA to ocular cells. As determined with condensed plasmids containing reporter genes for either beta-galactosidase (pcDNA-lacZ) or enhanced green fluorescent protein (pREP-EGFP), the immortalized human retinal epithelial cells RPE D407 and human embryonic kidney 293 cells can be transfected with typical efficiencies of 11 and 19%, respectively. Unlike 293 cells, RPE D407 cells had a reduced viability on transfection with both plasmids. In vivo, subretinal injections of DNA-K8/JTS-1 complexes revealed reporter gene expression in choroidal and RPE cells of normal pink-eyed Royal College of Surgeons (RCS) rats. The validity of this transfection technique in terms of retinal cell survival in RCS rats was then examined by using pREP-hFGF2 plasmid, which encodes the human basic fibroblast growth factor isoforms (hFGF2). Subretinal injection of pREP-hFGF2-K8/JTS-1 complexes into 3-week-old dystrophic RCS rat eyes reveals a delayed photoreceptor cell degeneration 60 days postinjection. In this case, the average analyzed field points with delayed cell dystrophy represent 14 to 17% of the retinal surface as compared with 2.6 and 4% in pREP5beta and vehicle-injected eyes, respectively. Peptide-mediated in oculo transfection thus appears to be a promising technique for the treatment of retinal cell and photoreceptor degenerations.

Epiretinal membrane recurrence: incidence, characteristics, evolution, and preventive and risk factors.

Background: To evaluate the incidence, evolution, clinical characteristics, possible risk factors or preventive factors, and visual outcomes of epiretinal membrane (ERM) recurrence. Methods: Retrospective study of 440 consecutive patients (440 eyes) who underwent pars plana vitrectomy for ERM. The internal limiting membrane (ILM) was peeled in 266 cases, with the help of indocyanine green in 27 cases and brilliant blue in 45 cases. Cases of symptomatic ERM recurrence were reoperated. Results: The incidence of ERM recurrence was 5% (22/440), and 2% of the patients were reoperated (9/440). Epiretinal membrane recurrence was symptomatic in 9 cases (41%) and asymptomatic in 13 cases (59%). ILM peeling was the only factor preventing ERM recurrence (adjusted odds ratio = 0.33, P = 0.026). The use of staining dyes did not prevent recurrence (adjusted odds ratio = 0.35, P = 0.338). In the case of ERM reproliferation, the absence of ILM peeling, the existence of ERM on the fellow eye, and poor visual acuity before surgery seemed to be associated with a high risk of symptomatic recurrence and reoperation. The mean duration for follow-up was 3.5 ± 1.7 years. Conclusion: ILM peeling not only reduces the likelihood of reproliferation of ERM but also seems to improve the visual prognosis of recurrent ERMs. The use of dyes did not reduce the rate of recurrence compared with when ILM was peeled without dyes.

Retinal cell type expression specificity of HIV-1-derived gene transfer vectors upon subretinal injection in the adult rat: influence of pseudotyping and promoter.

Gene therapy, and particularly gene restoration, is currently a great hope for non‐curable hereditary retinal degeneration. Clinical applications require a gene transfer vector capable of accurately targeting particular cell types in the retina. To develop such a vector, we compared the expression of a reporter gene after subretinal injections of lentiviral constructs of various pseudotypes and with the transgene expression driven by various promoters.

Quantitative analysis of subretinal injections in the rat

Abstract Background: Experimental therapeutic approaches to retinal degenerations often require the subretinal injection of a therapeutic agent. The injected volume and the age of the animal can influence the proportion of the retinal surface affected by the subretinal injection. We have investigated the quantitative effect of a single injection in the subretinal space. Methods: Normal and Royal College of Surgeons rats aged 1 week, 3 weeks or 2 months received subretinal transscleral injections of 1, 3, 5 or 10 µl China ink. After 24 h, animals were killed, injected eyes were enucleated and fixated, and the retinas flattened. An image analyzing program was used to measure the total retinal surface and the retinal surface affected by the dye. Results: The mean retinal surface affected by the injection ranged from 5.24±2.76 mm2 to 14.8±2.3 mm2, depending on ani- mal age and injected volume. The injection affected 8.79±0.89 to 36.9±8.13% of total retinal surface. There was no statistically significant difference between normal and Royal College of Surgeons rats. Intravitreal leakage of the dye was more frequent with increasing injection volumes. Conclusion: The retinal surface affected by a single subretinal injection increases with the injected volume, but this increase is not proportional. Higher volumes lead to a loss of injected solution, either in the vitreous body or through the sclero-tomy. In 2-month-old rats, a 3-µl subretinal injection appears to have the best reproducibility, with 20–30% of retinal surface covered by the injected dye.

Prominent beta-5 gene expression in the cardiovascular system and in the cartilaginous primordiae of the skeleton during mouse development.

The alpha v beta (αvβ5) heterodimer has been implicated in many biological functions, including angiogenesis. We report the β5 gene expression pattern in embryonic and foetal mouse tissues as determined by Northern blotting and in situ hybridization. During the earliest stages, β5 mRNA is widespread in the mesoderm. During later developmental stages, it remains mostly confined to tissues of mesodermal origin, although probable inductive effects trigger shifts of β5 gene expression from some mesenchymatous to epithelial structures. This was observed in the teeth, skin, kidneys, and gut. Of physiological importance is the β5 labeling in the developing cardiovascular and respiratory systems and cartilages. Furthermore, early β5 gene expression was observed within the intra- and extraembryonic sites of hematopoiesis. This suggests a major role for β5 in the hematopoietic and angiogenic stem cells and thus in the development of the vascular system. Later, the β5 gene was expressed in endothelial cells of the vessels developing both by angiogenesis and vasculogenesis in the lung, heart, and kidneys. Moreover, the β5 hybridization signal was detected in developing cartilages but not in ossified or ossifying bones. β5-Integrin is a key integrin involved in angiogenesis, vasculogenesis, hematopoiesis, and bone formation.

Epiretinal membrane surgery outcomes in highly myopic eyes without traction maculopathy: long-term results of a case-control study.

PURPOSE To evaluate the outcomes of epiretinal membrane (ERM) surgery in highly myopic eyes without traction maculopathy, and to compare them with those from non-highly myopic eyes. DESIGN Retrospective nested case-control study from a cohort of 509 consecutive patients (509 eyes) who underwent pars plana vitrectomy with ERM removal. METHODS Thirty-two highly myopic eyes (with a refractive error of more than -6.00 diopters [D]), which underwent surgery for isolated ERM, were included in the study. For each case studied, we selected from the same cohort 2 age-matched controls who had ERM surgery (n = 64 non-highly myopic eyes). The best-corrected visual acuity (BCVA), the central macular thickness (CMT), and the surgical complications were analyzed. RESULTS The mean follow-up duration was 3.2 ± 1.5 years for the study cases and 3.4 ± 1.6 years for the control group (P = .608). At the final follow-up examination, the mean logMAR BCVA had improved significantly, from 0.56 to 0.26 (P < .001) for the case group and from 0.54 to 0.22 (P < .001) for the control group. At the final optical coherence tomography (OCT), the mean CMT had improved significantly, from 433 to 314 μm (P < .001) for the case group and from 428 to 303 μm (P < .001) for the control group. There was no significant difference between the 2 groups as regards visual or CMT improvement (P = .526 and P = .483, respectively). The incidence of surgical complications was not significant between the 2 groups. CONCLUSIONS The results of ERM surgery were not different in terms of anatomic and visual outcomes and surgical complication between highly myopic and non-highly myopic eyes.

92. Efficient Trangene Expression in Central Nervous System through a Non-Integrative Lentiviral Vector

Lentiviral vectors are widely used in experimental gene transfer and are a promising tool for clinical applications, particularly for the treatment of neurodegenerative diseases. However, one of the most important limits to the application of this technology to humans is the risk of insertional mutagenesis through the integration of the viral genome into the host cell chromatin. This risk remains poorly evaluated and uncontrolled. In order to eliminate this risk, we have developed a lentiviral vector derived from the human immunodeficiency virus type 1 lacking integration property. After cell transduction, the vector genome persists in an extrachromosomic circular form in the nucleus and allows an effective and stable transgene expression in vitro and in vivo.