The effect of hyperglycaemia on permeability and the expression of junctional complex molecules in human retinal and choroidal endothelial cells.

Diabetic retinopathy is the leading cause of preventable blindness in the working population and its prevalence continues to increase as the worldwide prevalence of diabetes grows. Diabetic choroidopathy is less well studied and occurs in the late stages of diabetic eye disease. The main cause of visual loss in diabetic eye disease is diabetic macular oedema caused by an increase in microvascular endothelial permeability. Endothelial cell permeability is influenced by multiple factors which have not been fully elucidated, particularly in human models. In addition, the gene and protein expression between retinal and choroidal endothelial cells, even in humans, has been shown to be heterogeneous. The aim of this project was to determine, in vitro, the effect of high glucose (25 mM) on human paracellular permeability in retinal and choroidal endothelial cells. The expression of selected tight junction molecules (Occludin, Claudin-5, JAM-A and JAM-C) and adheren junction (VE-Cadherin) molecules was also compared between retinal and choroidal endothelial cells and with high glucose. High glucose conditions significantly increased the permeability in both retinal and choroidal endothelial cells monolayers although the increase was higher in retinal endothelial cells. Under normal glucose culture conditions microarray analysis determined that occludin and claudin-5 gene expression was higher in retinal endothelial cells than choroidal endothelial cells, and western blotting indicated that claudin-5 protein expression was also higher in retinal endothelial cells whilst JAM-A, and C and VE-Cadherin levels were similar. In retinal endothelial cells exposed to high glucose claudin-5, occludin and JAM-A was found to be reduced, whereas the expression of VE-Cadherin and JAM-C was unchanged when evaluated with western blotting, immunofluorescence and qPCR. None of the proteins were significantly decreased by high glucose in choroidal endothelial cells. The increase in retinal endothelial cell permeability is likely caused by a decrease in selective tight junction protein expression, leading to increased paracellular permeability. This may indicate differences in junctional molecule regulation of permeability in retinal compared to choroidal endothelial cells.

Ranibizumab for the treatment of choroidal neovascularisation secondary to pathological myopia: interim analysis of the REPAIR study.

AIMS: To evaluate the efficacy and safety of intravitreal ranibizumab in patients with choroidal neovascularisation secondary to pathological myopia (myopic CNV). Data are from a pre-planned, 6-month interim analysis. METHODS: Phase II, open-label, single arm, multicentre, 12-month study, recruiting patients (aged ≥18 years) with active primary or recurrent subfoveal or juxtafoveal myopic CNV, with a best-corrected visual acuity (BCVA) score of 24-78 Early Treatment Diabetic Retinopathy Study (ETDRS) letters in the study eye and a diagnosis of high myopia of at least -6 dioptres. Patients received 0.5 mg ranibizumab administered intravitreally to the study eye, followed by monthly injections given as needed (based on a predefined algorithm) for up to 11 months. RESULTS: At 6 months, mean BCVA improved from baseline by 12.2 letters, as did central macular thickness (in this interim analysis defined as a measure of either central subfield macular thickness or centre point macular thickness) from baseline by 108 µm in the 48 study eyes of 48 patients. Fewer patients had centre-involving intraretinal oedema (13.0% vs 91.5%), intraretinal cysts (10.9% vs 57.4%), or subretinal fluid (13.0% vs 66.0%) at 6 months than at baseline. Patients received a mean of 1.9 retreatments, were satisfied with ranibizumab treatment, and well being was maintained. No new safety signals were identified. CONCLUSIONS: Results from the planned interim analysis support the role of ranibizumab in the treatment of myopic CNV, with excellent efficacy achieved with a low number of injections and few serious adverse events.

Clinical presentation, treatment, and outcomes in presumed intraocular tuberculosis: experience from Newcastle upon Tyne, UK.

PURPOSE: To report the clinical manifestations and treatment outcomes of patients with presumed intraocular tuberculosis (TB) seen at the Newcastle Uveitis Service, Royal Victoria Infirmary, Newcastle upon Tyne, UK over a 10-year period. METHODS: Retrospective review of case notes. RESULTS: A total of 21 patients were identified. Occlusive retinal vasculitis was the commonest ophthalmological presentation (12 patients). Eight patients (38%) were found to have underlying active systemic TB (four with mediastinal lymphadenopathy, three with pulmonary TB, one with cutaneous TB). Constitutional or respiratory symptoms, elevated inflammatory markers, and an abnormal chest radiograph were poor indicators of active TB. Two patients had inactive intrathoracic TB. Eleven patients had latent TB. Eighteen patients received anti-tuberculous treatment. Final visual acuity was better than or equal to initial visual acuity in 14 out of 16 patients who completed at least 6 months of standard anti-tuberculous treatment. CONCLUSIONS: Most patients with presumed intraocular TB have latent TB, but a significant minority has hitherto undetected active TB. Our series suggests that either proven or presumed intraocular TB occurs frequently in the absence of constitutional or respiratory symptoms, elevated inflammatory markers, or an abnormal chest radiograph. A minimum of 6 months standard anti-tuberculous treatment provides good visual outcomes in the majority of patients.

Prevalence of biometry assessment experiences among trainee ophthalmologists in the United Kingdom.

PURPOSE: To determine the biometry assessment experiences of senior house officers (SHOs) during basic surgical training (BST). METHODS: This is a postal survey of SHOs in recognized UK surgical training posts from April 2005 to November 2005. Data collected included SHO proficiency in various types of biometry, practical biometry experience and opinions as to whether performing biometry is still a required skill. RESULTS: Of 460 SHOs who were sent questionnaires 279 (60.1%) responded. Only 13% of respondents received formal training in biometry while 43% received informal training in biometry from either senior colleagues or nurses. SHOs maintained they were proficient in different types of biometry including contact biometry (49%), non contact biometry (45%), immersion biometry (2.5%) and keratometry (81%). If their current unit, 84% of SHOs did not perform any biometry. Although the majority of SHOs reported familiarity with IOL power prediction formulae, there was no consistency among the SHOs working in the same unit. Despite the lack of exposure to biometry, most (88%) felt that biometry was still a required skill. CONCLUSION: This study highlights the lack of training and low prevalence of performing biometry among trainee ophthalmologists. As proficiency in biometry is part of BST and the majority of the trainees wished to learn the skill, it is hoped that this issue will be addressed in the new Ophthalmic Specialist Training curriculum.

The effects of growth factors on the proliferation and in vitro angiogenesis of human macular inner choroidal endothelial cells.

AIM: To investigate the effect of VEGF(165), FGF2, IGF-1, PDGF-AA, PDGF-BB and IL-1 beta on the proliferation and angiogenic tube formation of human macular inner choroidal endothelial cells (ICEC). METHODS: The proliferation of human macular ICECs after exposure to the aforementioned growth factors was determined by using both a WST-1 colorimetric assay and a cell-counting technique. The effect of growth factors on ICEC angiogenesis was assessed by sprout formation using a three-dimensional in vitro Matrigel duplex assay. RESULTS: Using both the WST-1 assay and a cell-counting technique, VEGF(165) and FGF2 both significantly increased human macular ICEC proliferation. The effect of equimolar concentrations of VEGF(165) and FGF2 was additive. There was no significant effect for IGF-1, PDGF-AA, PDGF-BB or IL-1 beta on proliferation up to a growth factor concentration of 1000 pmol/l. The angiogenesis assay found a significant effect on sprout formation for VEGF(165) and FGF-2. Again, the effect of equimolar concentrations of VEGF(165) and FGF2 was additive. There was no significant effect for IGF-1, PDGF-AA, PDGF-BB or IL-1 beta on sprout formation at 1000 pmol/l. CONCLUSIONS: Both VEGF(165) and FGF2 significantly increase human macular ICEC proliferation and sprout formation in an angiogenesis assay. When present together, their effect was additive. IGF-1, PDGF-AA, PDGF-BB and IL-1 beta did not have any significant effect on proliferation or sprout formation in vitro. These results suggest that targeting other growth factors such as FGF2, in addition to VEGF, may be beneficial in the treatment of neovascular age-related macular degeneration.

Isolation, culture, and characterisation of human macular inner choroidal microvascular endothelial cells.

AIM: To develop a method for the reliable isolation of adult human macular inner choroidal endothelial cells (ICECs) and to subsequently characterise them for their expression of a range of endothelial cell associated surface markers. METHOD: Human ICECs were isolated after manual dissection of maculas from fresh human posterior segments. Following enzyme digestion to form a single cell suspension, the ICECs were isolated using anti-CD31 coated Dynabeads. The isolated cells were grown in culture and examined for typical endothelial cell morphology, surface expression of vWf, CD 31, CD 105, VEGF receptors 1 and 2, and expression of E-selectin after stimulation with TNF-alpha. The cells were also examined for their ability to form fenestrations and capillary-like tubes in Matrigel. RESULTS: The method enabled the rapid isolation of viable cells that demonstrated typical endothelial cobblestone morphology in culture. The cells stained positive for CD31, vWf, CD105, VEGF receptors 1 and 2, and E-selectin (after stimulation with TNF-alpha). The cells stained negative for alpha smooth muscle actin and fibroblast surface protein. The cells also developed fenestrations when cultured on fibronectin coated plates and formed capillary-like tubes structures when cultured on Matrigel. CONCLUSIONS: This technique isolates cells from the human macular inner choroid that display features consistent with vascular endothelial cells. These cells could subsequently be used to further the understanding of the pathophysiological mechanisms of diseases of the inner choroid, such as choroidal neovascularisation.

A randomised, double-blind trial of topical ketorolac vs artificial tears for the treatment of episcleritis.

PURPOSE: To determine whether topical ketorolac (Acular) is more effective than artificial tears in treating the signs and symptoms of idiopathic episcleritis. METHODS: In this prospective, randomised, double-blind study, 38 eyes of 37 patients presenting with idiopathic episcleritis were allocated to receive either topical ketorolac (0.5%) or artificial tears three times a day for 3 weeks. The severity of patients' signs (episcleral injection and the number of clock hours affected) were recorded at weekly intervals. Patients' symptoms (perceived redness and pain scores) were recorded using a daily diary. RESULTS: There was no significant difference in the ophthalmic signs between the two groups at each assessment, including intensity of episcleral injection and the number of clock hours affected. No significant difference was found in the time to halve the baseline redness intensity scores (4.4 vs 6.1 days, P=0.2) or pain scores (3.6 vs 4.3 days, P=0.55). Significantly more patients on ketorolac reported stinging at the first follow-up visit (P<0.001). CONCLUSION: Topical ketorolac is not significantly better than artificial tears in treating the signs or symptoms of idiopathic episcleritis.

The effect of corneal thickness on intraocular pressure measurement in patients with corneal pathology.

BACKGROUND/AIM: To compare intraocular pressure (IOP) measurements taken by the Goldmann applanation tonometer, the Tono-Pen and the ocular blood flow pneumotonometer in eyes with varying central corneal thickness (CCT) due to penetrating keratoplasty (PK), keratoconus (KC), and Fuchs' endothelial dystrophy (FED). METHODS: IOP was measured with the Goldmann applanation tonometer, Tono-Pen XL, and OBF pneumotonometer in 127 eyes with the following corneal abnormalities. There were 56 eyes that had undergone PK, 37 eyes with KC, and 34 eyes with FED. CCT was measured using an ultrasound pachymeter after IOP determinations had been made. RESULTS: Mean IOP measurements in all three patient groups were significantly higher when measured by OBF pneumotonometer. Linear regression analysis showed that patients with FED had a significant increase in IOP with increasing CCT of 0.18 mm Hg/10 microm using the Goldmann tonometer, 0.15 mm Hg/10 microm with the Tono-Pen, and 0.26 mm Hg/10 microm with the OBF pneumotonometer. In patients with KC and after PK, linear regression analysis did not show a significant effect of CCT on IOP. A multivariate linear regression model controlling for age, sex, graft size, and patient group, showed that the effect of CCT on IOP for Tono-Pen (0.13 mm Hg/10 microm CCT) and Goldmann (0.14 mm Hg/10 microm CCT) were significantly lower than for the OBF pneumotonometer (0.26 mm Hg/10 microm CCT). CONCLUSIONS: This study found that mean IOP measurements using the OBF pneumotonometer were significantly higher than those made using the Goldmann applanation tonometer or Tono-Pen in eyes with a variety of cornel pathologies. The OBF pneumotonometer was found to be most affected by variation in CCT. For all three instruments, the relation between IOP and CCT depended on the corneal pathology and was greatest for FED.

Visual outcome of malignant hypertension in young people.

A retrospective review was carried out of patients under 16 years old with malignant hypertension, who had been referred to a teaching hospital ophthalmology department because of reduced visual acuity. Four patients (three girls, one boy) were seen between 1994 and 2000 with a mean age at presentation of 11.5 years (range 9-15). In the short term, visual acuity improved after control of blood pressure in all four patients. However, in the long term, two patients were registered blind one to two years after presentation, one because of a choroidal neovascular membrane developing at the macula, and the other because of progressive optic neuropathy. Both of these patients had a longer duration of symptoms before diagnosis, worse visual acuity, and higher blood pressure at presentation when compared with the patients who made a good visual recovery. These observations suggest that early diagnosis of malignant hypertension in children is essential in reducing the likelihood of permanent severe visual damage.

p38 mitogen-activated protein kinase activates peroxisome proliferator-activated receptor alpha: a potential role in the cardiac metabolic stress response.

The expression of enzymes involved in fatty acid beta-oxidation (FAO), the principal source of energy production in the adult mammalian heart, is controlled at the transcriptional level via the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Evidence has emerged that PPARalpha activity is activated as a component of an energy metabolic stress response. The p38 mitogen-activated protein kinase (MAPK) pathway is activated by cellular stressors in the heart, including ischemia, hypoxia, and hypertrophic growth stimuli. We show here that PPARalpha is phosphorylated in response to stress stimuli in rat neonatal cardiac myocytes; in vitro kinase assays demonstrated that p38 MAPK phosphorylates serine residues located within the NH(2)-terminal A/B domain of the protein. Transient transfection studies in cardiac myocytes and in CV-1 cells utilizing homologous and heterologous PPARalpha target element reporters and mammalian one-hybrid transcription assays revealed that p38 MAPK phosphorylation of PPARalpha significantly enhanced ligand-dependent transactivation. Cotransfection studies performed with several known coactivators of PPARalpha demonstrated that p38 MAPK markedly increased coactivation specifically by PGC-1, a transcriptional coactivator implicated in myocyte energy metabolic gene regulation and mitochondrial biogenesis. These results identify PPARalpha as a downstream effector of p38 kinase-dependent stress-activated signaling in the heart, linking extracellular stressors to alterations in energy metabolic gene expression.

The effect of sub-Tenon's anaesthesia on intraocular pressure.

PURPOSE: To assess the effect of sub-Tenon's anaesthesia on intraocular pressure (IOP) prior to cataract surgery. METHODS: Fifty consecutive patients undergoing phacoemulsification of cataract were recruited. Routine sub-Tenon's anaesthesia was administered with 5 ml unpreserved 2% lignocaine. IOPs were measured immediately prior to and at 1, 3, 5 and 10 min after injection. Efficacy was assessed subjectively by the operating surgeon. No ocular compression was used. Pre- and post-injection IOPs were compared using the Wilcoxon signed rank test, whereas all other results were compared with baseline using Student's two-tailed paired t-tests. RESULTS: All patients achieved good analgesia and akinesis. There was no significant difference between the IOP prior to and 1 min after injection. At all time intervals after 3 min there was a significant reduction in IOP compared with the pre-injection measurement. At 5 min, the mean IOP reduction was 2.72 mmHg and at 10 min IOP was lowered by 2.92 mmHg. Both reductions were statistically significant compared with baseline. CONCLUSIONS: Sub-Tenon's anaesthesia does not cause any significant rise in IOP, thereby possibly making it the anaesthetic technique of choice when an increase in IOP is undesirable. There is no indication for the use of an ocular pressure-reducing device when sub-Tenon's anaesthesia is employed.