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Biomedical subjects

I Constable

Publications and source records attributed to I Constable.

At least 19 recordsLinked to original sources
PubMed1999

Evaluation of adeno-associated virus-mediated gene transfer into the rat retina by clinical fluorescence photography.

The purpose of this study was to evaluate recombinant adeno-associated virus (AAV) as an in vivo gene transfer vector for the retina and to explore the possibility of monitoring the expression of green fluorescent protein (GFP) using a noninvasive method. Rats were injected subretinally with rAAV-gfp or rAAV-lacZ. Strong expression of the reporter gene in a circular area surrounding the injection site was observed in retinal whole mounts and tissue sections. Higher magnification revealed that cells demonstrating high levels of green fluorescence were hexagonal in shape, indicating they were retinal pigment epithelium (RPE) cells. Histological observation of retinal sections demonstrated that recombinant AAV specifically transduced RPE cells. Ten animals were injected with rAAV-gfp for longitudinal studies and the fluorescence was monitored by retinal fluorescence photography. The GFP signal was detected in 100% of the animals as early as 2 weeks postinjection and remained present throughout the experimental period of 4 months. After 2 weeks, a gradual increase in the number of transduced cells occurred before reaching maximal levels of GFP expression at 8 weeks. This was followed by a small decrease over 4 weeks before reaching stable expression at 16 weeks. Our results demonstrated that rAAV efficiently transduces rat RPE cells and that retinal fluorescence photography is suitable for monitoring GFP expression. By using this noninvasive technique, we demonstrated that repetitive measurements of GFP expression in vivo in the rAAV-gfp-transduced retina are possible. This study demonstrated that retinal fluorescence photography is a potent tool for studying AAV-mediated gene delivery in the retina.

Animals↗
PubMed1998

Adeno-associated virus-mediated gene transfer into human retinal pigment epithelium cells.

PURPOSE: Adeno-associated virus (AAV) is emerging as a promising vector for gene therapy METHOD: To determine the ability of recombinant AAV (rAAV) to express and integrate exogenous DNA into human retinal pigment epithelium (RPE) cells, a rAAV-GFP vector containing the green fluorescent protein (gfp) and neomycin resistance (neo(r)) genes was constructed and used to transduce RPE 407A cell line. RESULTS: Fluorescent RPE cell clones were obtained and were confirmed to still be expressing GFP after 24 passages (3.5 months). CONCLUSION: Adeno-associated virus-based vectors are able to efficiently transduce and stably persist in RPE cells.

Cell Line↗
PubMed1997

Ocular gene therapy: the basic science and current state of research.

Recent technical advances have led to the demonstration of the molecular basis of many genetic eye diseases. Methods now in use have been able to both suppress and insert genes in vitro and in vivo in ocular cells. The combination of this new knowledge and these techniques offers the potential for a new therapeutic approach to diseases that currently have no treatments. The scientific basis of gene therapy is introduced and then the current state of research is discussed in relation to the eye and ocular tissues.

Adenoviruses, Human↗
PubMed1996

Targeted delivery of an antisense oligonucleotide in the retina: uptake, distribution, stability, and effect.

In this article, we describe the preliminary results of the development of an animal model that will enable us to study the effect of photoreceptor-derived debris accumulation on the normal function of the retina in vivo. An antisense oligonucleotide (Cat 5), saline, and two control oligonucleotides were injected into the vitreous of 7-week-old RCS-rdy+ rats. The uptake, distribution, and persistence of the antisense oligonucleotide in the retina was demonstrated by fluorescent confocal microscopy, and the stability of the oligonucleotide was shown by GeneScan analysis using a fluorescein-labeled derivative of Cat 5 (Cat 5F). The accumulation of photoreceptor-derived debris was monitored by the number of undigested phagosomes in the RPE layer by light microscopy. Following intravitreal injection of Cat 5F, penetration of the oligonucleotide was observed in the ganglion cell layer in 2 hours and in the photoreceptor and pigment epithelial layers 3 days later. However, at 7, 28, and 56 days postinjection, only the RPE layer had significant amounts of Cat 5F present. Using GeneScan analysis, it was demonstrated that the fluorescein-labeled oligonucleotide present in the RPE layer was not degraded and it retained its original 19-mer length. There was no statistically significant difference in the number of phagosomes found in the RPE layer of control uninjected, saline-injected, and two sense and two antisense oligonucleotides-injected animals at 7 and 28 days postinjection. In contrast, the number of phagosomes was significantly higher (p < 0.001) in the RPE layer of Cat 5 antisense oligonucleotide-injected animals at 7 and 28 days postinjection. This difference, however, disappeared by 56 days postinjection. The inner nuclear layers of the retina of control and experimental animals were not affected by the injections.

Adolescent↗
PubMed1996

Initiation of impaired outer segment degradation in vivo using an antisense oligonucleotide.

This paper describes the first successful in vivo application of antisense DNA technology to induce the accumulation of photoreceptor outer segment derived debris in the retina. An antisense oligonucleotide (CatSC), which was previously demonstrated to be an effective tool to induce debris accumulation in vitro, was injected into the vitreous of pigmented and non-pigmented rats. The animals were euthanased 7 days after the injections. The number of inclusions significantly increased in the RPE layer of Long Evans and RCS-rdy + rats injected with 66 ug of CatSC to 96.2 +/- 13.6 (SD) (p < 0.0003) and 204.2 +/- 39.3 (SD) (p < 0.0001), respectively. The difference between the number of phagosome-like inclusions present in control saline, 6.6 ug of CatSC or 66 ug of sense oligonucleotide (S1) injected animals was not statistically significant. There were no abnormalities observed in the inner layers of the retina but the accumulation of phagosome-like inclusions was accompanied by disorganisation in the apices of outer segments. The large number of inclusions found in CatSC treated animals showed the characteristics of phagosomes containing stacks of undigested photoreceptor outer segment membranes which suggest that the lysosomal digestion process was halted or at least slowed down by the antisense oligonucleotide.

Animals↗
PubMed1995

Optimal conditions required for the creation of an iatrogenic chorioretinal venous anastomosis in the dog using argon green laser photocoagulation.

We have previously reported iatrogenic retinal to choroidal vein anastomosis, developed as a potential method of by-passing the site of obstruction to venous outflow in retinal venous occlusion in dogs (1), and in rats (2). In order to minimise tissue damage to the retina and choroid and increase the rate of success in these experiments, we investigated in the dog model the factors that would promote an anastomosis and compare the effects of three different power levels. A small spot size (50 microns) argon green laser beam of 514 nm at power levels of 0.5w, 1.5w and 2.5w were used. Spaced serial sections from each lesion were examined by light and by transmission electron microscopy. Morphometric measurements of the corresponding retinal pigment epithelium (RPE)/glial scar was computed using IBMPC digitising pad and sigma scan software and the extent of tissue damage at the different power levels assessed. At the lowest power level of 0.5w the damage to the retina was mild and there was an absence of anastomosis formation. At the 1.5w power level an anastomosis formed in 40% of the lesions. At the highest power level of 2.5w a 71% rate of success was obtained however, the damage to the retina tended to be severe. The results of this study also indicate that disruption of Bruch's membrane and vein rupture at the time of irradiation are essential for anastomosis formation, which may be further enhanced by necrotic tissue, retinal pigment epithelial and glial scar formation and inflammation. These findings are useful in establishing optimal conditions for the creation of a chorioretinal venous anastomosis, for consideration in human trials.

Anastomosis, Surgical↗
PubMed1992

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro.

The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4-week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7-year-old donor compared with those from a 47-year-old donor. Within both groups the BROS-challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S-labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS-challenged RPE cells of both bovine and human origin that did not appear in control or microsphere-phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.

Aging↗
PubMed1992

Induced chorioretinal venous anastomosis in experimental retinal branch vein occlusion.

Iatrogenic retinal vein to choroidal vein anastomoses were created using laser photocoagulation in six of seven dog eyes in which a partial branch retinal vein occlusion had previously been created photochemically. A similar attempt to create an anastomosis was made in six control eyes in which no branch vein occlusion was present. In the eyes in which a branch retinal vein had been created, a venous chorioretinal anastomosis appeared to be present by 3 to 6 weeks. In three control eyes similar venous anastomosis was created; however this took 6 to 8 weeks to develop and was of much smaller calibre than the one that developed in the presence of a partial branch retinal vein occlusion. No adverse complications were noted in the period of the study (3 months). This study demonstrates that chorioretinal venous anastomoses can be created and may be of use in the treatment of partial retinal vein occlusions that show signs of progression.

Anastomosis, Surgical↗
PubMed1992

HLA DQA tissue typing of cadaveric eye bank donor material with polymerase chain reaction.

It has been suggested that the provision of HLA matched tissue for corneal transplantation would be beneficial for graft survival especially in high risk patients. In this study we report the application of a polymerase chain reaction (PCR) based tissue typing procedure to type cadaveric donor material. Using beta-globin gene amplification as a test system we found that cornea, corneal rim, conjunctiva, sclera, rectus muscle, optic nerve and neural retina were all suitable for PCR amplification but DNA extracted from pigment epithelial cells and from the iris could not be amplified. HLA DQA typing results of 9 samples identified 5 alleles and 7 genotypes. In two cases the antigens detected by serology reflected the alleles detected by PCR. In a third case in which the class II serological typing was inconclusive we detected two DQA alleles by PCR. These alleles were consistent with those which would be predicted to be present on the basis of known linkage disequilibrium between HLA-Cw, B, DR and DQ. In this study we have shown that PCR based tissue typing is more sensitive and gives more detailed typing results than serology and would be suitable to type cadaveric donor material on a routine basis.

Alleles↗
PubMed1989

Non-allelic mutations in X-linked retinitis pigmentosa.

Using RFLP studies, the disease locus in two X-linked retinitis pigmentosa families was found to be centromeric to DXS7 in one family and telomeric to DXS7 in another, suggesting non-allelic heterogeneity.

Blotting, Southern↗
PubMed1989

Linkage heterogeneity between X-linked retinitis pigmentosa and a map of 10 RFLP loci.

In nine families in which X-linked retinitis pigmentosa (XLRP) is segregating, the lod scores of XLRP in a map of 10 RFLP loci were obtained by multipoint linkage analysis. The XLRP locus was located telomeric to DXS7 in seven of the families and centromeric to DXS7 in two of the families. Under the hypothesis of two XLRP loci, a heterogeneity (admixture) test was performed, providing significant evidence of heterogeneity in XLRP (P less than .01). No correlation was detected between the clinical manifestations of XLRP and the two different disease loci.

Chromosome Mapping↗
PubMed1989

[Argon laser trabeculoplasty for open angle glaucoma--a four year follow-up].

79 patients (105 eyes) with uncontrolled open angle glaucoma underwent argon laser trabeculoplasty (ALT). The average pressure drop was 10.7 mmHg in 58 eyes at one year, 10.1 mmHg in 35 eyes at two years, 9.9 mm Hg in 48 eyes at three years and 10.2 mmHg in 24 eyes at four years. Teh mean success rate was 78%, failures occurred in the first six months in 21 of 23 eyes, the remaining eyes failed at three years, 18 eyes (17%) required filtering surgery. Average medication scores varied little from year 1 through year 4, 45 eyes (42.9%) were being treated with few medications after ALT. The results of ALT depend on the severity of glaucoma: the eyes with advanced disc damage (C/D greater than 0.9, visual field less than 20 degrees) had 59% rate of filtering surgery, while the eyes with C/D 0.6-0.8, visual field greater than 20 degrees at ALT had a filtering surgery rate of 3%.

Aged↗
PubMed1987

Biological and therapeutic aspects of proliferative vitreoretinopathy.

Conventional vitreoretinal surgery for proliferative vitreoretinopathy is unsatisfactory despite numerous innovations over the past 20 years. Silicone oil injections in this series resulted in 70% reattachment of these end-stage cases, but within twelve months over 90% had no useful visual result. The development of an in vitro model for 3-dimensional culture of cells has allowed evaluation of numerous potential drugs with anti-proliferative or anti-contractile activities. Amongst these, taxol has shown the most promise. There remain, however, doubts about possible long-term toxicity to the optic nerve and retina. It is concluded that a more physiological vitreous substitute and more specific anti-proliferative or anti-contractile pharmacological agents are needed.

Alkaloids↗
PubMed1982

Super viscous silicone liquid in retinal surgery.

Very high viscosity (12500 centistokes) liquid silicone was injected into the vitreous of 47 eyes of 46 patients with complicated retinal detachment over a two year period. Only eyes judged inoperable by scleral buckling and/or vitrectomy techniques were considered. Complete anatomical reattachment was achieved in 34 cases (72%) while partial reattachment was achieved in a further six cases (13%). All eyes in which complete or partial retinal reattachment was achieved showed improvement in vision but this was usually restricted to count fingers or 6/60 at 3 months to 2 years follow-up. Mounting evidence now suggests that previous clinical opinions claiming an unacceptable toxicity rate with liquid silicone may be misleading.

Child↗