Biomedical subjects

W Y Shen

Publications and source records attributed to W Y Shen.

At least 19 recordsLinked to original sources

PubMed2002

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy.

Neovascularisation (NV) within the eye often results in visual loss. Vascular endothelial growth factor (VEGF) has been implicated in the development of ocular NV. Previous studies have shown that VEGF antagonists successfully suppressed retinal and choroidal NV in animal models. However, the systemic approach and transient nature of the delivery systems used in these studies hinder therapeutic application. To achieve stable and localised ocular anti-angiogenic therapy, we explored the use of recombinant adeno-associated virus (rAAV)-mediated secretion gene therapy (SGT). In this study, we generated a rAAV vector encoding soluble VEGF receptor 1, sFlt-1 (AAV-CMV.sflt) and determined its ability to inhibit cautery-induced corneal NV and laser-induced choroidal NV. Delivery of AAV-CMV.sflt into the anterior chamber resulted in transgene expression in the iris pigment epithelium and corneal endothelium, which reduced the development of corneal NV in the stroma of cauterised rats by 36% compared with cauterised control groups (P = 0.009). Subretinal delivery of AAV-CMV.sflt near the equator of the eye also suppressed choroidal NV at the laser lesions around the optic nerve by 19% (P = 0.002), indicating that there was diffusion of the secreted anti-angiogenic protein across the retina. Both results suggest that the long-term suppression of ocular NV is possible through the use of stable rAAV-mediated SGT.

Adenoviridae↗

PubMed2001

Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization.

Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.

Adenoviridae↗

PubMed2001

Combined effect of cyclosporine and sirolimus on improving the longevity of recombinant adenovirus-mediated transgene expression in the retina.

OBJECTIVES: To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants. METHODS: An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression was monitored in real time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in virus-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymphocytes, and CD68(+) macrophages, histologic findings, and dark-adapted electroretinography. Two synergistic immunosuppressants, cyclosporine and sirolimus, were used alone or in combination to prolong transgene expression by temporary immunosuppression. RESULTS: The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coincident with cell infiltration in virus-contacted area. Immunostaining showed that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocytes and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wave of dark-adapted electroretinogram were observed at 3 to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being the most efficient. The combined immunosuppression attenuated but did not retard the rAd-induced retinal damage. CONCLUSIONS: Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sirolimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer. CLINICAL RELEVANCE: The intraocular safety of rAd should be carefully considered before clinical trials are performed.

Adenoviridae↗

PubMed2001

Uptake dynamics and retinal tolerance of phosphorothioate oligonucleotide and its direct delivery into the site of choroidal neovascularization through subretinal administration in the rat.

This study aimed to investigate uptake dynamics and retinal tolerance of phosphorothioate oligonucleotides (PS-oligos) following subretinal injection. A fluorescent-labeled PS-oligo (FL-oligo) with random sequence was administered into the subretinal space of rat by transsclera-choroid-retinal pigment epithelium (RPE) injection at doses of 0.129, 1.29, and 12.9 microg in 2.0 microl solution. The uptake dynamics were evaluated by fundus fluorescent photography in real time and by fluorescence microscopy using flat mounts and cryosections. Immunophenotyping for CD4+, CD8+ cytotoxic lymphocytes, and CD68+ macrophages was performed to assess cellular infiltration in the retina. In addition, the FL-oligo was injected subretinally in a rat model of choroidal neovascularization (CNV) for direct delivery into the site of CNV. Subretinal administration of FL-oligo resulted in both dose-dependent and time-dependent distribution in the retina, where it accessed the RPE and all layers of the neuroretina. CD4+, CD8+ cytotoxic lymphocytes, and CD68+ macrophages were observed at the site of needle penetration. However, in areas far from the injection site where the FL-oligo appeared strongly, cellular infiltration was absent, and the retinal morphology was preserved very well. The FL-oligo was successfully delivered into the site of intense laser photocoagulation. It was predominantly localized to the RPE, macrophages, and some choroid cells and remained detectable for at least 56 days after injection. Our results demonstrate for the first time that subretinal injection efficiently introduced PS-oligo into the RPE and neuroretina with an acceptable level of safety. Subretinal administration of antiangiogenic oligonucleotides may hold great potential for the treatment of CNV.

Angiogenesis Inhibitors↗

PubMed2000

Controlled production of active cathepsin D in retinal pigment epithelial cells following adenovirus-mediated gene delivery.

The transduction of a low cathepsin D-producing retinal pigment epithelial cell line with a recombinant adenovirus, Ad.proCatD, carrying a viral promoter and the precursor form of the lysosomal enzyme cathepsin D, procathepsin D, led to the upregulation of proCatD expression. However, the resultant aspartic protease activity did not exceed that observed in normal primary human retinal pigment epithelial cells. Following the injection of Ad. proCatD into rat eyes, immunohistochemistry and Western blot analysis localized the expression of procathepsin D to the retinal pigment epithelial cell layer and to the sclera/choroid/retinal epithelial cell layers, respectively. This upregulation of procathepsin D expression was accompanied by a limited increase in aspartic protease activity. The injected eyes did not demonstrate any of the retinal changes that have been associated with the overproduction and secretion of active cathepsin D. Immunoelectronmicroscopy of Ad.proCatD-transduced retinal pigment epithelial cells demonstrated the presence of cathepsin D not only in cytoplasmic vesicles and lysosomes but also in the nucleoli and, less strongly, elsewhere in euchromatic regions of some 10% of cells. In spite of the upregulated expression of procathepsin D, the production of active cathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells was strictly controlled. It is proposed that active cathepsin D production is controlled at the point of posttranslational modification by an intranuclear feedback mechanism initiated by the relative excess of procathepsin D in Ad. proCatD-transduced retinal pigment epithelial cells.

Adenoviridae↗

PubMed2000

Inhibition of diclofenac formulated in hyaluronan on angiogenesis in vitro and its intraocular tolerance in the rabbit eye.

PURPOSE: To investigate the effect of diclofenac, a potent nonsteroidal anti-inflammatory drug, formulated in hyaluronan (diclofenac/HA) on angiogenesis in vitro and its intraocular toxicity in vivo. METHODS: The effect of diclofenac/HA on angiogenesis was determined by choriocapillary endothelial cells on Matrigel stimulated by vascular endothelial growth factor (VEGF). The tube areas were quantified by image digital analysis. For toxicity study, diclofenac/HA was injected intravitreally with a dose range from 100 to 1080 microg in 26 rabbits following gas compression vitrectomy. Potential toxicity was assessed by indirect ophthalmoscopy and by histological studies (light and electron microscopy). Retinal function was monitored by electroretinography (ERG) in six rabbits that received 400 microg of diclofenac/HA. RESULTS: Diclofenac/HA, 180, 90 microg/ml, inhibited tube formation to 24% and 55% of the standard group (Media Ham's F12 plus 5% fetal calf serum and 50 ng/ml VEGF) respectively (P<0.01). Intravitreal injection of 540 microg or higher doses of diclofenac/HA resulted in ocular toxicity in the rabbit, demonstrated as cataract, vitreous haze and retinal damage observed by indirect ophthalmoscopy and light- and electron-microscopic examinations. No toxicity was observed in the eyes that received 400 microg or less diclofenac/HA, which was further supported by the normal ERG examined at 4 and 25 days post injection. CONCLUSIONS: Diclofenac/HA inhibits tube formation in vitro and is non-toxic to the rabbit retina at concentrations that are inhibitory to tube formation. Our results suggest diclofenac/HA may be an effective candidate to inhibit ocular neovascularisation related to granulomatous reaction in the eye.

Animals↗

PubMed2000

Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization.

Vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization found in age-related macular degeneration. Normally expressed in low levels, this study investigates whether the overexpression of VEGF in the retinal pigment epithelium is sufficient to cause choroidal neovascularization in the rat retina. A recombinant adenovirus vector expressing the rat VEGF(164) cDNA (AdCMV.VEGF) was constructed and injected into the subretinal space. The development of neovascularization was followed by fluorescein angiography, which indicates microvascular hyperpermeability of existing and/or newly forming blood vessels, and histology. VEGF mRNA was found to be overexpressed by retinal pigment epithelial cells and resulted in leaky blood vessels at 10 days postinjection, which was maintained for up to 31 days postinjection. By 80 days postinjection, new blood vessels had originated from the choriocapillaris, grown through the Bruch's membrane to the subretinal space, and disrupted the retinal pigment epithelium. This ultimately led to the formation of choroidal neovascular membranes and the death of overlying photoreceptor cells. By controlling the amount of virus delivered to the subretinal space, we were able to influence the severity and extent of the resulting choroidal neovascularization. These results show that even temporary overexpression of VEGF in retinal pigment epithelial cells is sufficient to induce choroidal neovascularization in the rat eye.

Adenoviridae↗

PubMed2000

Long-term real-time monitoring of adeno-associated virus-mediated gene expression in the rat retina.

PURPOSE: Previous studies have demonstrated that adeno-associated virus (AAV) efficiently transduced retinal pigmented epithelial (RPE) cells. The goal of this study was to further evaluate and characterize transgene expression within the RPE cells over time in vivo. METHODS: Adeno-associated virus-mediated gene transfer was monitored and quantified by retinal photography following subretinal injection of a recombinant AAV encoding the green fluorescent protein gene (rAAVCMV-gfp) into rat eyes. Retinal function of transduced rat eyes was measured by electroretinography. RESULTS: The maximum level of transgene expression was reached at 8 weeks postinjection followed by a gradual decrease throughout the experimental period. Interestingly, it was observed that while gfp expression was stable in some RPE cells, gfp fluorescence completely disappeared in other cells over the duration of the experiment. The expression of AAV-mediated gfp in RPE cells did not alter the retinal function for over 1 year CONCLUSIONS: These results confirm the importance of this direct visualization system to study vector transgene expression in vivo and support the use of AAV for diseases treatable by targeting RPE cells.

Animals↗

PubMed2000

The role of vascular endothelial growth factor (VEGF) in abnormal vascular changes in the adult rat eye.

The aim of this project was to determine if the subretinal delivery of a recombinant adenovirus encoding vascular endothelial growth factor (VEGF) was sufficient to induce changes resembling choroidal neovascularisation (CNV) in a rat model. A recombinant adenovirus was produced encoding vegf164 cDNA (Ad.RSV.VEGF). Transduction of cultured RPE cells confirmed VEGF expression and ensured the absence of Ad.RSV.VEGF-related toxicity. Following subretinal injection into rat eyes, fluorescein angiography indicated that the in vivo delivery of Ad.RSV.VEGF was associated with vascular leakage. Histological analysis demonstrated that changes resembling the early signs of CNV development were also present in the Ad.RSV.VEGF injected eyes. These results suggest that while a transient VEGF expression in the RPE layer is able to induce CNV-related changes, it may be insufficient for the development of a full neovascular membrane. This study demonstrates that virus-mediated gene delivery, in addition to its clinical applications, is a potentially efficient research tool for investigating gene expression-related physiological changes in vitro and in vivo.

Adenoviridae↗

PubMed2000

The use of adenovirus-mediated gene transfer to develop a rat model for photoreceptor degeneration.

PURPOSE: To investigate the effects of recombinant adenovirus-mediated downregulation of cathepsin S (CatS) on the retinal pigment epithelium and/or neural retina in vivo. METHODS: The expression of green fluorescent protein (gfp) after subretinal injection of a recombinant adenovirus, Ad.gfp, into rat eyes was first established by in vivo fundus fluorescence photography and fluorescence microscopy. The autofluorescent debris accumulation in Ad.CatSAS (recombinant adenovirus carrying the antisense CatS gene)injected rat eyes was monitored by fluorescence microscopy, and the antisense CatS RNA expression was demonstrated by in situ hybridization. Changes in the retinal morphology were assessed by light microscopy. ResuLTS. The gfp expression was present in 30% to 90% of the injection area at 3 days and was absent 9 days after Ad.gfp injection. In Ad.CatSAS-injected eyes, the expression of antisense CatS RNA was demonstrated by in situ hybridization. Autofluorescent debris accumulation was significantly higher in Ad.CatSAS-injected eyes than in control eyes. The shortening of photoreceptor outer segments in Ad.CatSAS-injected eyes coincided with intense autofluorescent debris accumulation. The number of layers of photoreceptor cells decreased with time and were 11, 9, and 8 at 7, 14, and 28 days after Ad.CatSAS injection, respectively. In control eyes, the number of layers of photoreceptor cells (14) remained unchanged. CONCLUSIONS: These results demonstrate that recombinant adenovirus-mediated transient modulation of gene expression in retinal pigment epithelial (RPE) cells could induce changes in the retina, and, in spite of the low expression of endogenous CatS in RPE cells, this enzyme plays an important role in maintenance of normal retinal function.

Adenoviridae↗

PubMed2000

Matrix metalloproteinase (MMP) expression in experimental choroidal neovascularization.

PURPOSE: Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that degrade basement membrane and extracellular matrix proteins. To gain information on the possible role of MMPs in choroidal neovascularization (CNV), we have analyzed the mRNA expression of MMP-2 and MMP-9, two forms of MMPs implicated in ocular neovascularization, in a rat model. METHODS: Choroidal neovascularization was induced in pigmented rats by krypton laser photocoagulation of the fundus whereafter eyes were enucleated at 1, 3, 5, 7, 10 and 60 days. Antisense and sense riboprobes were generated using DNA complementary to MMP-2 and MMP-9, and mRNA expression was analyzed using in situ hybridization. RESULTS: In the untreated eyes MMP-2 mRNA expression was weakly detected in cells within the choroid. In laser-treated eyes MMP-2 mRNA expression was markedly increased and mainly localized to macrophage-like and retinal pigment epithelial (RPE)-like cells invading the choroid, subretinal space and inner retina. This increase in MMP-2 mRNA expression peaked at day 10 whereafter a decline was detected. MMP-9 mRNA expression was low in untreated eyes and did not increase following laser treatment. CONCLUSION: The results show that MMP-2 mRNA expression is increased in experimental CNV, and support of a role for MMP-2 in the development of CNV in age-related macular degeneration.

Animals↗

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↗

PubMed1999

Preferential adenovirus-mediated transduction of cells at the sites of laser photocoagulation in the rat eye.

PURPOSE: This study aimed to investigate the feasibility of recombinant adenovirus-mediated gene transfer into cells implicated in the development of choroidal neovascularization (CNV). METHODS: A rat model of CNV which used laser photocoagulation was developed. Gene delivery into the laser spots was investigated following subretinal injection of recombinant adenoviruses, AdRSVlacZ, AdCMVlacZ or AdCMVgfp. Immunohistochemical analysis was performed using a proliferating cell nuclear antigen antibody and a cytokeratin-specific antibody to identify the cell types transduced by the recombinant adenoviruses. RESULTS: At 7 days post-injection, lacZ expression was detected in 51.6 +/- 13.2% and 71.2 +/- 19.3% of laser spots in AdRSVlacZ- and AdCMVlacZ-injected eyes, respectively. By 28 days post-injection, lacZ expression was only present in AdCMVlacZ-injected eyes. In vivo fundus fluorescent photography of AdCMVgfp-injected eyes detected gfp expression in 79.9 +/- 12.7% and 35.6% +/- 19.7% of laser spots at 4 and 7 days post-injection, respectively. Although fundus fluorescent photography did not detect the gfp signal at 10 days post-injection, fluorescent microscopy revealed a gfp signal in 81.3 +/- 6.0% of laser spots. Immunohistochemical analysis detected retinal pigment epithelial (RPE) cells as the most predominant proliferating cell type in the laser spots, although several other proliferating cell types were also identified. X-gal staining showed that the majority of transduced cells were those present in the laser spots. CONCLUSIONS: It is proposed that following laser photocoagulation, proliferating RPE cells are susceptible to adenovirus-mediated gene delivery and that they may be suitable targets for the delivery of antiangiogenic factors by recombinant adenoviruses in order to inhibit developing CNV membranes.

Adenoviridae↗

PubMed1999

Dynamics of phosphorothioate oligonucleotides in normal and laser photocoagulated retina.

AIMS: To investigate the distribution, persistence, and stability of fluorescently labelled phosphorothioate oligonucleotides (PS-ODNs) in normal and laser photocoagulated retina following intravitreal injection in the rat. METHODS: Fluorescently labelled PS-ODNs were injected intravitreally into pigmented eyes at doses of 0.5-10.0 nmol in 2.0 microl solution. The dynamics of PS-ODNs was evaluated by fluorescent microscopy of cryosections and flat mounted retinal pigment epithelium (RPE)-choroid-sclera. Genescan analysis was used to assess the integrity of PS-ODNs in the retina after injection. The dynamics of PS-ODNs was also evaluated in the retina following krypton laser photocoagulation with a protocol producing choroidal neovascularisation (CNV). RESULTS: Following intravitreal injection the PS-ODNs demonstrated dose and time dependent distribution and persistence in the retina, where they accessed all neural layers. However, they preferentially accumulated in the RPE layer, demonstrated as bright granules in the cytoplasm of the cells. Injections of 5.0 and 7.5 nmol of PS-ODNs exhibited strong fluorescence in the retina for 6 weeks after injection. Genescan analysis demonstrated that the PS-ODNs remained almost completely intact for at least 12 weeks. Following laser treatment, the PS-ODNs were concentrated in the regions of laser photocoagulation and retained high intensity for at least 8 weeks after injection, particularly localised to macrophages, RPE, and the local choroidal tissue. CONCLUSIONS: These results indicate that PS-ODNs are stable and accessible to most neural layers of the retina, and they preferentially accumulate in the RPE layer following intravitreal injection. The successful delivery of PS-ODNs into normal and laser photocoagulated retina suggests that PS-ODNs may have potential in the development of therapy for attenuating retinal degenerations and CNV.

Animals↗

PubMed1999

[Biosynthesis of a single peptide chain containing human chorionic gonadotropin beta and C3D of complement].

In view of the strong immunity-enhancing function of HEL-C3d3 designed by Dr. Paul W. Dempsey, we made our efforts to produce a similar recombinant protein of hCG beta. With polymerase chain reaction, we introduced a Bam HI restriction site into the 3' terminal of hCG beta cDNA. The new cDNA and its terminal's correctness has been confirmed by sequencing. Then we have it covalently attached to the C3d3 cDNA at the pre-designed Bam HI/Bgl II site. Having the chimeric DNA correctly cloned into the protein nuclear polyhedrosis virus (AcNPV) expression vector pVL1393, we constructed the expression vector pVL1393-(hCG beta-C3d3). The insect cells were co-transfected with the expression vector and linearized nuclear polyhedrosis virus DNA, and recombinant viruses AcNPV-(hCG beta-C3d3) were screened out. Through anti-hCG beta immunoaffnity chromatography, the recombinant hCG beta-C3d3 chimera polypeptide was purified from culture supernatant of insect cells infected by the recombinant viruses. In RIA test, the expressed product competitively inhibits the binding of 125I-hCG beta to hCG beta-antibody. On SDS-PAGE and Western blot, the recombinant peptide hCG beta-C3d3 obviously appears to be with a molecular weight of 116KD. Therefore, we arrive at a conclusion that it has a normal immunogenic ability.

Animals↗

PubMed1998

Recombinant adenovirus-mediated gene delivery into the rat retinal pigment epithelium in vivo.

PURPOSE: The present paper describes changes following the subretinal injection of a recombinant replication-deficient adenovirus carrying the beta-galactosidase reporter gene construct into the rat retina. METHODS: Ad.RSV.betagal-mediated transduction of rat retinal pigment epithelium (RPE) cells in vitro and in vivo was examined following X-gal staining. RESULTS: There was a low level of beta-galactosidase expression in the RPE cells at 4 days postinjection. At 7 days postinjection, a strong transgene expression was present in RPE cells and the expression was maintained at 14 days postinjection. Except for the accumulation of cells at the site of the injection, the morphology of the rest of the retina remained normal. CONCLUSION: These results demonstrate that the RPE layer can be successfully targeted for gene delivery in the rat.

Adenoviridae↗

PubMed1998

Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat.

AIMS: To investigate the longevity and reproducibility of choroidal neovascularisation (CNV) induced by krypton laser photocoagulation in the rat. The presence of cell adhesion molecules (CAMs) and vascular endothelial growth factor (VEGF) during the development of CNV was also studied. METHODS: 67 pigmented rats underwent retinal photocoagulation by krypton laser. The eyes were examined by either single or serial fluorescein angiography at 3 days, 1, 2-3, 4-5, 7-8, and 12 weeks post photocoagulation. The expression of CAMs (ICAM-1, E-selectin, and CD44) and VEGF post photocoagulation was studied by immunohistochemistry. RESULTS: CNV related fluorescein leakage appeared in 46.4% of 766 laser spots delivered to the 58 eyes that were tested at 2-3 weeks post treatment. The ratio of hyperfluorescent laser sites did not change significantly at 8 weeks post laser. The number of leaky spots was independent of the total number of lesions delivered to each eye (at 2-3 weeks post laser 10-15 spots/eye: 44% and 25-30 spots/eye: 49%; t = 0.7673; p = 0.3903). Nine eyes were followed by serial angiography between 2 and 12 weeks. The laser spots with fluorescein leakage at 2 weeks (51.5%) remained leaky at 12 weeks (51.5%). Histopathologically, macrophage accumulation peaked at 5 days and CNV was firstly observed at 1 week post photocoagulation. ICAM-1, E-selectin, CD44, and VEGF were maximally induced at 3-5 days post laser photocoagulation, and were localised to RPE, choroidal vascular endothelial, and inflammatory cells. VEGF was also detected in intravascular leucocytes at the sites of laser lesions. CONCLUSIONS: These studies demonstrated that krypton laser photocoagulation can be successfully used to produce lesions similar to those of human CNV. The response induced remained present for an extended period of time (12 weeks), thus offering a potential model to screen candidate CNV inhibitory agents. In addition, it is proposed that the expression of ICAM-1, E-selectin, CD44, and VEGF before new vessel formation might be linked to the initiation of CNV.

Animals↗

PubMed1997

[Closed reduction and percutaneous pinning for acute type III extension- Supracondylar fracture of distal humerus in children.].

GOAL OF SURGERY: Closed reduction of acute type III (according to Gartland) supracondylar extension fractures of the distal humerus and unicondylar pin fixation. INDICATIONS: Type III and unstable type II supracondylar fractures. CONTRAINDICATIONS: Swelling of the elbow. Compartment syndrome. PREOPERATIVE WORK UP: Radiographs of both elbows in the anterior-posterior and lateral projections. POSITIONING AND ANAESTHESIA: Supine with arm on arm board. General anaesthesia. SURGICAL TECHNIQUE: Closed reduction through manipulation under fluoroscopic control. Fixation with 2 Kirschner wires introduced percutaneously through the lateral condyle. Long arm cast for 4 to 5 weeks. POSTOPERATIVE MANAGEMENT: Radiographs on the first postoperative day, 1 week later and at time of cast and pin removal 4 to 5 weeks postoperatively. At that time active mobilisation of the elbow is started. Follow-up for 1 to 2 years is recommended. POSSIBLE COMPLICATIONS: Damage of ossific nucleus of physis. Damage to neurovascular structures. Wire migration. Pin tract infection. RESULTS: Out of 82 children (mean age 6.5 years) 73 had a follow-up of more than 6 months. 80.8% had good or excellent results. 15.1% had a decrease of the carrying angle of more than 10 degrees (4.1% more than 20 degrees ). There were 2 pin tract infections and 1 iatrogenic transient ulnar palsy.

English Abstract↗