Nanosecond laser therapy reverses pathologic and molecular changes in age-related macular degeneration without retinal damage.

Age-related macular degeneration (AMD) is a leading cause of vision loss, characterized by drusen deposits and thickened Bruch's membrane (BM). This study details the capacity of nanosecond laser treatment to reduce drusen and thin BM while maintaining retinal structure. Fifty patients with AMD had a single nanosecond laser treatment session and after 2 yr, change in drusen area was compared with an untreated cohort of patients. The retinal effect of the laser was determined in human and mouse eyes using immunohistochemistry and compared with untreated eyes. In a mouse with thickened BM (ApoEnull), the effect of laser treatment was quantified using electron microscopy and quantitative PCR. In patients with AMD, nanosecond laser treatment reduced drusen load at 2 yr. Retinal structure was not compromised in human and mouse retina after laser treatment, with only a discrete retinal pigment epithelium (RPE) injury, and limited mononuclear cell response observed. BM was thinned in the ApoEnull mouse 3 mo after treatment (ApoEnull treated 683 ± 38 nm, ApoEnull untreated 890 ± 60 nm, C57Bl6J 606 ± 43 nm), with the expression of matrix metalloproteinase-2 and -3 increased (>260%). Nanosecond laser resolved drusen independent of retinal damage and improved BM structure, suggesting this treatment has the potential to reduce AMD progression.

Expression of muscarinic receptor subtypes in tree shrew ocular tissues and their regulation during the development of myopia.

PURPOSE: Muscarinic receptors are known to regulate several important physiologic processes in the eye. Antagonists to these receptors such as atropine and pirenzepine are effective at stopping the excessive ocular growth that results in myopia. However, their site of action is unknown. This study details ocular muscarinic subtype expression within a well documented model of eye growth and investigates their expression during early stages of myopia induction. METHODS: Total RNA was isolated from tree shrew corneal, iris/ciliary body, retinal, choroidal, and scleral tissue samples and was reverse transcribed. Using tree shrew-specific primers to the five muscarinic acetylcholine receptor subtypes (CHRM1-CHRM5), products were amplified using polymerase chain reaction (PCR) and their identity confirmed using automated sequencing. The expression of the receptor proteins (M1-M5) were also explored in the retina, choroid, and sclera using immunohistochemistry. Myopia was induced in the tree shrew for one or five days using monocular deprivation of pattern vision, and the expression of the receptor subtypes was assessed in the retina, choroid, and sclera using real-time PCR. RESULTS: All five muscarinic receptor subtypes were expressed in the iris/ciliary body, retina, choroid, and sclera while gene products corresponding to CHRM1, CHRM3, CHRM4, and CHRM5 were present in the corneal samples. The gene expression data were confirmed by immunohistochemistry with the M1-M5 proteins detected in the retina, choroid, and sclera. After one or five days of myopia development, muscarinic receptor gene expression remained unaltered in the retinal, choroidal, and scleral tissue samples. CONCLUSIONS: This study provides a comprehensive profile of muscarinic receptor gene and protein expression in tree shrew ocular tissues with all receptor subtypes found in tissues implicated in the control of eye growth. Despite the efficacy of muscarinic antagonists at inhibiting myopia development, the genes of the muscarinic receptor subtypes are neither regulated early in myopia (before measurable axial elongation) nor after significant structural change.

Binding of dexamethasone by alpha-crystallin.

PURPOSE: Long-term steroid therapy is a known risk factor for the development of posterior subcapsular cataract. Previous work in this laboratory has found soluble lens proteins to bind dexamethasone, but this binding is not due to a glucocorticoid receptor. This study was undertaken to identify the soluble protein or proteins involved in lens glucocorticoid binding. METHODS: Bovine lens extract was incubated with 5.2 x 10(-)(8) M [(3)H]-dexamethasone for 3 hours, and the distribution of label assessed in the soluble and insoluble fractions after centrifugation. Soluble lens extract was fractionated using gel permeation chromatography to isolate and identify proteins involved in the binding. Total lens proteins, high-molecular-weight proteins, or alpha-crystallin were exposed to dexamethasone and the protein bound steroid measured after separation of free and bound ligand on a gel chromatography column. Scatchard analysis was used to determine dexamethasone-binding parameters. Sequence comparisons between bovine alphaA- and alphaB-crystallins and glucocorticoid-binding proteins were performed using a sequence-alignment program. RESULTS: Of the total dexamethasone bound in lens extract, soluble proteins were found to account for 52%. The majority of the soluble protein-bound dexamethasone coeluted with the high-molecular-weight proteins that consisted mainly of alpha-crystallin. Binding studies with isolated proteins showed that alpha-crystallin accounted for more than 98% of total soluble dexamethasone binding in the lens. Scatchard analysis of steroid binding showed it to be a nonspecific partitioning event. Sequence comparisons between alphaA- and alphaB-crystallins and various glucocorticoid-binding proteins showed the lens proteins to have three regions of sequence homology with yeast corticosteroid-binding protein. CONCLUSIONS: alpha-Crystallin is the principal soluble glucocorticoid binding protein in the lens. The steroid association is described by nonspecific partitioning and may be related to the unique structural characteristics of the protein. The nonspecific association with alpha-crystallin is not thought to be functional; however, it may aid in the increased covalent steroid modification observed for this protein.

Genetic screening for progressive retinal atrophy in the Australian population of Irish Setters.

OBJECTIVE: To determine whether the rcd-1 mutation causing progressive retinal atrophy (PRA) in Irish Setters is in the Australian breeding population. METHOD: DNA samples were tested for the mutation using the Polymerase Chain Reaction and specific primer nucleotides to amplify the phosphodiesterase gene followed by restriction enzyme cleavage and fragment size determination. RESULTS: No mutant alleles were found in 38 Irish Setters, representing over 80% of all major breeding stock in five Australian states. CONCLUSIONS: It is likely that the Australian population of Irish Setters is free of the rcd-1 form of PRA.

The frequency of the canine leukocyte adhesion deficiency (CLAD) allele within the Irish Setter population of Australia.

OBJECTIVE: To determine the frequency of the 107G-->C canine leukocyte adhesion deficiency (CLAD) mutation in Irish Setters from the Australian breeding population. METHOD: Genomic DNA was isolated from 87 Irish Setter blood samples and a region of the beta-2 integrin gene (ITGB2), encompassing the mutation, was amplified using real-time Polymerase Chain Reaction (PCR). Two fluorescently labelled probes were hybridised to the fragment, and fluorescence resonance energy transfer (FRET) was used to detect the 107G-->C mutation responsible for CLAD. RESULTS: Three new heterozygotes were identified among 87 healthy Irish Setters from Australia. All originated from a litter sired by a known heterozygote. A total of seven heterozygotes have now been identified in 92 dogs (7.6%), representing over 90% of all major breeding stock in five Australian states. Two of the heterozygotes were recently imported adult dogs and the others were their offspring. CONCLUSIONS: The frequency of the 107C allele in the Australian population of Irish Setters is lower than that in Europe. Selective breeding programs should be adopted to eliminate the mutant allele presently in two breeding lines.

Glutamate uptake in retinal glial cells during diabetes.

AIMS: Glutamate recycling is a major function of retinal Muller cells. The aim of this study was to evaluate the expression and function of glutamate transporters during diabetes. METHODS: Sprague-Dawley rats were rendered diabetic by a single dose of streptozotocin (50 mg/kg). Following 12 weeks of diabetes, immunolocalisation and mRNA expression of the two glial cell transporters, GLAST and EAAT4 were evaluated using indirect immunofluorescence and real-time PCR. The function of glutamate transport was investigated at 1, 4 and 12 weeks following induction of diabetes by measuring the level of uptake of the non-metabolisable glutamate analogue, D: -aspartate, into Muller cells. RESULTS: There was no difference in the localisation of either GLAST or EAAT4 during diabetes. Although there was a small apparent increase in expression of both GLAST and EAAT4 in diabetic retinae compared with controls this was not statistically significant. At 1, 4 and 12 weeks following diabetes, D: -aspartate immunoreactivity was significantly increased in Muller cells of diabetic rats compared to controls (p<0.001). The EC(50) was found to increase by 0.304 log units in diabetic Muller cells compared with controls, suggesting that glutamate uptake is twice as efficient. CONCLUSIONS: These data suggest that there are alterations in glutamate transport during diabetes. However, these changes are unlikely to play a significant role in glutamate-induced neuronal excitoxicity during diabetes. These results suggest that although Muller cells undergo gliosis at an early stage of diabetes, one of the most important functions for maintaining normal retinal function is preserved within the retina.

Is there a glucocorticoid receptor in the bovine lens?

Prolonged glucocorticoid therapy is a risk factor for cataract development. The mechanism remains unknown. If cataract results from the direct effect of steroids on lens function, a glucocorticoid receptor is required. In order to determine whether such a receptor was present in the bovine lens, metabolic and steroid binding experiments were undertaken. Cultured bovine lens epithelial cells were exposed to 10(- 4)and 10(-8) M dexamethasone or prednisolone and the uptake and incorporation of(14)C leucine,(14)C glucose and(3)H thymidine, examined. Neither glucocorticoid affected cell protein synthesis or glucose uptake. Both dexamethasone concentrations and the lower concentration of prednisolone had no effect on thymidine uptake or incorporation, however, the 10(-4) M prednisolone exposure reduced these by 15 +/- 5%. This regulation is thought to be due to membrane fluidity changes and not the action of the glucocorticoid receptor. As the glucocorticoid receptor is very heat labile in vitro, the effects of increasing temperature on dexamethasone binding by proteins from lens epithelium, lens nucleus and liver were examined. At 0 degree C, lens epithelial extract bound nine-fold more dexamethasone than liver extract. After exposure to 37 degrees C, liver binding decreased by 66% whereas that for lens epithelium increased by 18%. For both lens extracts, steroid binding increased with temperature up to 50 degrees C. Scatchard analysis of the steroid binding kinetics showed there to be no high affinity sites in lens epithelial extract, with the binding best described as a non-specific partitioning event. Western blotting with a specific glucocorticoid receptor antibody revealed protein bands of approximately 94 and 79 kDa in liver, which is known to contain significant levels of receptor. No immunoreactivity was observed for lens epithelial extract. Therefore, within the limits of detection, these results suggest the bovine lens does not contain a glucocorticoid receptor. This raises questions about the validity of receptor-mediated mechanisms proposed for cataract development.