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.

Immunolocalization of the P2X4 receptor on neurons and glia in the mammalian retina.

Extracellular adenosine 5'-triphosphate (eATP) acts as a neurotransmitter within the retina and brain, activating a range of ionotropic P2X and metabotropic P2Y receptors. In this study, the specific localization of the P2X4 receptor (P2X4-R) subunit was evaluated in the retina using fluorescence immunohistochemistry and pre-embedding immuno-electron microscopy. Punctate P2X4-R labeling was largely localized to the inner and outer plexiform layers of mouse, rat and cat retinae. In the mouse outer retina, double-labeling of P2X4-R with the horizontal cell marker, calbindin, revealed P2X4-R immunoreactivity (P2X4-R-IR) on horizontal cell somata and processes. In the inner retina, P2X4-R expression was found closely associated with rod and cone bipolar cell terminals, and the punctate labeling was observed on calretinin-positive amacrine cells. Using immuno-electron microscopy, P2X4-Rs were observed on processes post-synaptic to photoreceptor and bipolar cell terminals, likely representing horizontal, amacrine and ganglion cells, respectively. Furthermore, P2X4-R expression was also observed on Müller cells, astrocytes and microglia. These data suggest a role for P2X4-Rs in the lateral inhibitory pathways of the retina, modulating neuronal function of photoreceptors and bipolar cells. The expression on macro- and microglial cells implicates a role for P2X4-Rs in glial signaling, tissue homeostasis and immunosurveillance within the mammalian retina.

Characterization of retinal function and glial cell response in a mouse model of oxygen-induced retinopathy.

Retinal neovascularization, such as that occurring in proliferative diabetic retinopathy and retinopathy of prematurity, can have serious effects on visual function. By using a mouse model of neovascularization, oxygen-induced retinopathy (OIR), the interplay among angiogenesis, neuronal function, and the macro- and micro-glial response was explored. OIR was induced by exposure of mice to 75% oxygen from postnatal day 7 (P7) to P11 and then room air until P18. Controls were reared in room air. Blood vessel development was assessed by using fluorescence histochemistry. Aberrant intravitreal neovascularization was present across all eccentricities of retina in mice with OIR, whereas the number of vessels present in the deep plexus was reduced in the central regions. Neuronal function of both the rod and cone pathways, assessed by using the electroretinogram, was found to be significantly reduced in OIR. This may in part be explained by an alteration in photoreceptor outer segment morphology and also a loss of neurons and their synapses in the inner nuclear and plexiform layers of the central retina. In addition, there was an increase in the number of gliotic Müller cells and microglia in mice with OIR and the increase in the number of these cells correlated with the absence of the deep plexus. This indicates that the activity of both macro- and microglia is altered in regions where the deep plexus blood supply is deficient. Treatments or genetic manipulations directed toward amelioration of proliferative retinopathy need to address not only the vascular changes but also the alterations in neuronal and macro- and microglial function.

Diisopropylfluorophosphate alters retinal neurotransmitter levels and reduces experimentally-induced myopia.

The excessive enlargement of the eye seen in myopia is known to be regulated, in part, by retinal neurotransmitters. One excitatory retinal neurotransmitter, acetylcholine, has been implicated in the signal cascade through the effectiveness of the muscarinic antagonist, atropine, in preventing myopia development. The present study therefore examined whether an indirect cholinomimetic, diisopropylfluorophosphate (DFP), could increase the level of experimental myopia, induced by the deprivation of pattern vision. Injections of either phosphate-buffered DFP or phosphate-buffered saline were made every 48 h into the vitreous chamber of one eye of chicks. The injected eye was then deprived of pattern vision by a translucent occluder. The fellow eye remained untreated and acted as a genetic control. At the end of the treatment period (8 days) axial ocular dimensions and cycloplegic refractive error were measured. To investigate the effects of DFP on retinal neurotransmitter levels, measurements of acetylcholine and dopamine contents were made on retinal tissue following either a single or multiple DFP injections, using reverse phase high performance liquid chromatography (HPLC). Rather than potentiating myopia and vitreous chamber elongation, a significant reduction in myopia (58%) was observed in DFP-injected deprived eyes, compared to saline controls. However, open eyes injected with DFP showed no difference in refraction or vitreous chamber depth compared to contralateral control eyes or saline controls. HPLC analysis revealed increased steady-state content of acetylcholine (+34 +/- 6 ng/mg protein, mean +/- SEM, P<0.01, equivalent to a 54% increase) and dopamine (+377 +/- 83 pg/mg protein, P<0.01, a 36% increase) in DFP-treated eyes compared to contralateral control eyes following a single DFP injection. No changes in either acetylcholine or dopamine content were found in saline-treated control animals. Injection of dopamine antagonists, in combination with DFP, indicated that the DFP-induced reduction in myopia is mediated, at least in part, through a D2 receptor mechanism. Findings argue against a direct cholinergic 'stop-go' pathway controlling ocular growth. Instead the reduction of induced myopia could be related to the action of DFP (directly or indirectly) on dopamine levels in the retina.