Jose Garcia-Arumi

Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of proliferative diabetic retinopathy

Aims/hypothesisThe aim of this study was to compare the protein profile of vitreous fluid from diabetic patients with proliferative diabetic retinopathy (PDR) with that from non-diabetic patients with idiopathic macular holes (MH). The mRNA of proteins differentially produced was also assessed in the retinas from diabetic and non-diabetic donors.Materials and methodsVitreous humour from type 1 diabetic patients with PDR (n = 8) and from non-diabetic patients with MH (n = 10) closely matched in terms of age were studied. The comparative proteomic analysis was performed using fluorescence-based difference gel electrophoresis (DIGE). Differentially produced proteins (abundance ratio >1.4, p < 0.05) were identified by mass spectrometry. Expressions of mRNA were measured by real-time RT-PCR in retinas from ten human eyes obtained at post-mortem (five eyes from diabetic subjects and five eyes from non-diabetic subjects).ResultsEight proteins were highly produced in PDR patients in comparison with non-diabetic subjects: zinc-α2-glycoprotein (ZAG), apolipoprotein (apo) A1, apoH, fibrinogen A, and the complement factors C3, C4b, C9 and factor B). We found three proteins that were underproduced in PDR subjects: pigment epithelial derived factor (PEDF), interstitial retinol-binding protein (IRBP) and inter-α-trypsin inhibitor heavy chain (ITIH2). There was no overlap in the vitreous levels of the above-mentioned proteins between PDR patients and non-diabetic control subjects. The differential production of ZAG, C3, factor B, PEDF and IRBP was further confirmed by western blot, and was in agreement with mRNA levels detected in the retina.Conclusions/interpretationProteomic analysis by DIGE, which permits an accurate quantitative comparison, was useful in identifying new potential candidates involved in the pathogenesis of PDR.

Transpupillary thermotherapy for circumscribed choroidal hemangiomas

PURPOSE Various treatments have been described for circumscribed choroidal hemangiomas. We present our results with transpupillary thermotherapy (TTT) with infrared laser that indicate that the therapy is a new, effective treatment for this pathologic condition. DESIGN Noncomparative, prospective, interventional case series. PARTICIPANTS The treatment and follow-up of eight eyes of eight patients with circumscribed choroidal hemangiomas are presented. Each case was treated when the visual acuity (VA) decreased because of serous retinal detachment that affected the macula. METHODS Infrared diode laser was used at 810 nm and power between 800 and 1200 mW with a beam diameter of 2 or 3 mm depending on the diameter of the hemangioma, with 3 to 6 minutes of exposure time. MAIN OUTCOME MEASURES The final VA, the presence of serous subretinal fluid, funduscopic appearance of the tumor, and the degree of hyperfluorescence observed during fluorescein angiography were the main parameters. RESULTS In all eight cases, total reabsorption of the serous retinal detachment was achieved after one or repeated applications of TTT. Mild choroidal atrophy and minimal hyperpigmentation of the retinal pigment epithelium were observed in the treated eyes. CONCLUSIONS TTT can be considered an acceptable therapeutic option for choroidal hemangiomas and serous retinal detachment, and we believe that the role of this therapy will expand in the management of these patients.

Interphotoreceptor retinoid-binding protein (IRBP) is downregulated at early stages of diabetic retinopathy.

Aims/hypothesisInterphotoreceptor retinoid-binding protein (IRBP) plays a major role in the visual cycle and is essential to the maintenance of photoreceptors. The aim of this study was to determine whether a decrease in IRBP production exists in the early stages of diabetic retinopathy.MethodsVitreous samples from diabetic patients with proliferative and non-proliferative diabetic retinopathy (PDR, NPDR), and from non-diabetic patients with macular hole (control group) were selected for IRBP quantitative assessment by proteomic analysis (fluorescence-based difference gel electrophoresis) and western blot. Human post mortem eyes (n = 16) from diabetic donors without clinically detectable retinopathy and from non-diabetic donors (n = 16) were used to determine IRBP (also known as RBP3) mRNA levels (RT-PCR) and protein content (western blot and confocal microscopy). Retinal neurodegeneration was assessed by measuring glial fibrillar acidic protein (GFAP) and the apoptotic rate. Y79 human retinoblastoma cells were used to test the effects of glucose, TNF-α and IL-1β on IRBP expression and IRBP levels.ResultsIntravitreous IRBP concentration was significantly lower in PDR < NPDR < control in proteomic and western blot analysis. IRBP mRNA levels and IRBP protein content were significantly lower in the retinas from diabetic donors than in those from non-diabetic donors. Increased GFAP and a higher degree of apoptosis were observed in diabetic retinas compared with non-diabetic retinas. A dose-dependent downregulation of IRBP mRNA expression and IRBP content was detected with glucose, TNF-α and IL-1β in cultures of Y79 human retinoblastoma cells.Conclusions/interpretationUnderproduction of IRBP is an early event in the human diabetic retina and is associated with retinal neurodegeneration. The mechanisms leading to this deficit deserve further investigation.

Comparison of different techniques for purification of triamcinolone acetonide suspension for intravitreal use

Background: Intravitreal triamcinolone has increasingly been used for the treatment of oedematous and neovascular diseases and purification of triamcinolone suspension may be important in order to avoid the potential toxic effects of the vehicle. The aim was to evaluate different techniques used to reduce the solvent agent benzyl alcohol (9.9 mg/ml) from a commercially prepared triamcinolone acetonide suspension. Methods: Different techniques were used to reduce the solvent agent benzyl alcohol: filter techniques using 0.22 μm or 5 μm pore size, and non-filter techniques using sedimentation or centrifugation. Quantification of triamcinolone acetonide and benzyl alcohol was performed by high pressure liquid chromatography (HPLC). Results: Benzyl alcohol concentration was decreased significantly in all the techniques used compared with the original commercial suspension (p<0.05), with no significant differences among them. The reduction was approximately one tenth of its original concentration. However, triamcinolone acetonide concentration differed significantly depending on the method used. Centrifugation method showed no differences versus the original commercial solution; sedimentation technique reduced the expected dose only 25%; the filter technique using a 5 μm pore size membrane reduced the expected dose to one fourth, while the filter technique using a 0.22 μm pore size membrane reduced the expected dose to 45%. Conclusions: All the different techniques employed effectively reduced the concentration of benzyl alcohol. However, the final concentration of triamcinolone was much lower than expected using the filter techniques. The pore size membrane inversely influenced the final concentration, with part of the triamcinolone crystals probably being entrapped in the filter. Centrifugation is recommended as the best way of administering the drug.

The db/db mouse: a useful model for the study of diabetic retinal neurodegeneration.

Background To characterize the sequential events that are taking place in retinal neurodegeneration in a murine model of spontaneous type 2 diabetes (db/db mouse). Methods C57BLKsJ-db/db mice were used as spontaneous type 2 diabetic animal model, and C57BLKsJ-db/+ mice served as the control group. To assess the chronological sequence of the abnormalities the analysis was performed at different ages (8, 16 and 24 weeks). The retinas were evaluated in terms of morphological and functional abnormalities [electroretinography (ERG)]. Histological markers of neurodegeneration (glial activation and apoptosis) were evaluated by immunohistochemistry. In addition glutamate levels and glutamate/aspartate transporter (GLAST) expression were assessed. Furthermore, to define gene expression changes associated with early diabetic retinopathy a transcriptome analyses was performed at 8 week. Furthermore, an additional interventional study to lower blood glucose levels was performed. Results Glial activation was higher in diabetic than in non diabetic mice in all the stages (p<0.01). In addition, a progressive loss of ganglion cells and a significant reduction of neuroretinal thickness were also observed in diabetic mice. All these histological hallmarks of neurodegeneration were less pronounced at week 8 than at week 16 and 24. Significant ERG abnormalities were present in diabetic mice at weeks 16 and 24 but not at week 8. Moreover, we observed a progressive accumulation of glutamate in diabetic mice associated with an early downregulation of GLAST. Morphological and ERG abnormalities were abrogated by lowering blood glucose levels. Finally, a dysregulation of several genes related to neurotransmission and oxidative stress such as UCP2 were found at week 8. Conclusions Our results suggest that db/db mouse reproduce the features of the neurodegenerative process that occurs in the human diabetic eye. Therefore, it seems an appropriate model for investigating the underlying mechanisms of diabetes-induced retinal neurodegeneration and for testing neuroprotective drugs.

Therapeutic options for capillary papillary hemangiomas

PURPOSE To describe the results and therapeutic complications of treatment of papillary capillary hemangiomas over the last 13 years. DESIGN Retrospective, noncomparative, interventional case series. PARTICIPANTS Five cases of unilateral capillary papillary hemangiomas were reviewed. Three patients had von Hippel-Lindau disease, and two had no associated familial or systemic disease. METHODS Two patients were treated with argon green laser, one patient received argon green and diode photocoagulation, and two patients underwent argon green photocoagulation and diode transpupillary thermotherapy (TTT); two eyes also underwent vitreoretinal surgery. Follow-up ranged from 1 to 13 years. MAIN OUTCOME MEASURES The presence of exudative retinal detachment associated with the activity of the hemangioma and the visual acuity were the two main parameters. RESULTS Prompt argon green laser treatment was the most effective therapy; -II was ineffective. Vitreoretinal surgery, transscleral drainage, and argon endolaser photocoagulation in one case resolved bullous retinal detachment secondary to tumor exudation. Pretreatment visual acuity (VA) levels ranged from 20/25 to counting fingers; posttreatment VA levels ranged from 20/25 to light perception. CONCLUSIONS If left untreated, papillary hemangiomas may evolve to exudative retinal detachment and marked VA decreases. Although we have not established an ideal therapy, we recommend appropriate treatment on diagnosis. Close follow-up and multiple treatments with argon laser are likely the best therapeutic course.

Management of macular edema in branch retinal vein occlusion with sheathotomy and recombinant tissue plasminogen activator.

Purpose: The common adventitial sheath that surrounds the retinal venule and arteriole at the crossing site plays a crucial role in branch retinal vein occlusion (BRVO). The purpose of this study was to report the surgical recanalization of the occluded vein using a bimanual technique and recombinant tissue plasminogen activator (tPA) and its effect on final visual acuity. Methods: Arteriovenous sheathotomy was performed, using a bimanual technique, followed by fluid-air exchange and injection of 25 mg of recombinant tPA over the area of the occluded vein. Results: Intraoperative sectioning of the common arteriovenous sheath was achieved in all 40 patients. Thrombus release was observed in 11 cases (27.5%) and was correlated with early surgery (P < 0.001) and better final visual recovery (P < 0.06). Optical coherence tomography showed macular thickness that decreased by greater than 40% in 31 patients (77.5%) compared with preoperatively, and correlated to postoperative visual acuity (P < 0.001). The mean visual acuity increased from 20/100 to 20/40, with 70% of patients gaining three or more lines of visual acuity (Pearson 0.378, P = 0.016). Conclusion: Surgical venous decompression and injection of recombinant tPA may effectively manage macular edema secondary to BRVO, thus improving anatomic and visual outcome. Early surgical intervention may obtain maximum final visual recovery.

Vitreoretinal surgery and endoresection in high posterior choroidal melanomas.

Purpose Eyes with posterior choroidal melanomas more than 9 mm in thickness frequently are enucleated because of the potential complications of radiotherapy. The aim of this study was to evaluate the safety and efficacy of internal resection of these tumors. Methods Twenty-five consecutive patients with high posterior choroidal melanomas with a diameter less than 15 mm and a thickness greater than 9 mm were treated. If the retina was not invaded by the tumor, a vitrectomy was performed, followed by posterior hyaloid dissection, 120° anterior retinotomy, endophotocoagulation 2 mm past the tumor margin, melanoma removal with the vitrectomy probe, retinal reattachment with liquid perfluorocarbon and air, and silicone oil exchange. If the tumor invaded the retina, the laser was applied through the retina, and the retina and tumor were removed together. Results The mean patient age was 46.6 years. The tumor thicknesses ranged from 9.1 to 12.8 mm, and the tumor diameter ranged from 8.9 to 14.8 mm. The mean preoperative visual acuity was 20/60. In 11 patients, the tumor had invaded the retina. We removed the entire tumor from all 25 eyes. The main postoperative complications were cataract (40%), retinal detachment (16%), macular traction (16%), and epiretinal macular proliferation (8%). The mean postoperative visual acuity was 20/100. No tumors recurred, and there was no evidence of metastasis. The follow-up ranged from 12 to 72 months. Conclusions These data suggest that internal resection of high posterior melanomas may conserve ocular and functional vision and does not seem to increase the risk of metastatic disease. Longer follow-up is necessary to establish the safety of the procedure.

Surgical Embolus Removal in Retinal Artery Occlusion

Aims: To evaluate the anatomical outcomes, safety and functional effectiveness of surgical embolus removal in retinal artery occlusion (RAO). Methods: Prospective study of seven patients with RAO of <36 h duration. All eyes underwent pars plana vitrectomy and a longitudinal incision of the anterior wall of the occluded arteriole in an attempt to remove the embolus. Outcome measures included visual acuity and arteriolar reperfusion, as evaluated with fluorescein angiography. Results: Surgical removal of the embolus was achieved in six of the seven (87.5%) patients, visual acuity improved from a median of 20/400 (range: hand movements 20/25) to 20/40 (range: hand movements 20/25), and reperfusion of the occluded vessel was angiographically confirmed in four of the six patients in whom the embolus was successfully removed. Conclusion: Surgical removal of retinal arterial emboli seems to be an effective and safe treatment for RAO, but a randomised and controlled clinical trial will be necessary to establish an evidence base for the role, if any, of this intervention.

Vitreoretinal surgery for cystoid macular edema associated with retinitis pigmentosa

PURPOSE To evaluate the anatomic and functional outcome of vitreoretinal surgery in eyes with retinitis pigmentosa (RP) and macular edema. DESIGN Prospective noncomparative case series. PARTICIPANTS Twelve consecutive eyes of eight patients with RP and a documented decrease in visual acuity (VA) to 20/60 or worse caused by macular edema refractory to medical therapy. METHODS Pars plana vitrectomy was performed in the 12 eyes, followed by posterior hyaloid dissection, removal of the posterior inner limiting membrane after staining with indocyanine green, and gas tamponade. Preoperative best-corrected VAs ranged from 20/60 to 20/400 (mean, 20/115). MAIN OUTCOME MEASURES Changes in VA and foveal thickness as determined by optical coherence tomography (OCT). RESULTS The mean preoperative retinal thickness at the fovea was 477 micro m. Optical coherence tomography showed a decrease in macular thickness of >40% in 10 eyes (83.3%), with a mean postoperative foveal thickness of 260 micro m. The mean VA increased from 20/115 to 20/45, with an average of three lines of improvement. CONCLUSIONS Our results suggest that vitreoretinal surgery may effectively manage macular edema in RP.