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Background: Uveitis and optic neuritis are prevalent ocular inflammatory diseases, and highly damaging ocular conditions. Both diseases are currently treated with corticosteroids, but they do not have adequate efficacy and are often associated with severe side effects. Thus, uveitis and optic neuritis remain a challenging field to ophthalmologists and a significant public health concern. Objective: This review summarizes findings showing the benefits of a treatment with melatonin in experimental models of these inflammatory ocular diseases. Results: Oxidative and nitrosative damage, tumor necrosis factor, and prostaglandin production have been involved in the pathogeny of uveitis and optic neuritis. Melatonin is an efficient antioxidant and antinitridergic, and has the ability to reduce prostaglandin and tumor necrosis factor levels both in the retina and optic nerve. Moreover, melatonin not only prevents functional and structural consequences of experimental uveitis and optic neuritis, but it is also capable of suppressing the actively ongoing ocular inflammatory response. Conclusions: Since melatonin protects ocular tissues against inflammation, it could be a potentially useful anti-inflammatory therapy in ophthalmology.

Diabetic retinopathy is a leading cause of acquired blindness. Visual function disorders have been observed in diabetic patients with very early retinopathy or even before the onset of retinopathy. The aim of the present work was to analyze the visual pathway in an early stage of experimental diabetes. Diabetes was induced in Wistar rats by an i.p. injection of streptozotocin. A deficit in anterograde transport from the retina to the superior colliculus was observed 6 weeks after streptozotocin injection. At this time point, morphologic studies did not reveal retinal ganglion cell loss or substantial alterations in the superior colliculus. The optic nerve was morphometrically evaluated at intraorbital (unmyelinated and myelinated) and intracranial sections. In animals that had been diabetic for 6 weeks, a large increase in astrocyte reactivity occurred in the distal (but not the intraorbital) portion, which coincided with significant axon loss. Moreover, profound myelin alterations and altered morphologic features of oligodendrocyte lineage were observed at the distal (but not the proximal) optic nerve portion. The present results suggest that axoglial alterations at the distal portion of the optic nerve could be the first structural change in the diabetic visual pathway.

Diabetic retinopathy is a leading cause of acquired blindness. Available treatments are not very effective. We investigated the effect of a weekly application of retinal ischemia pulses (ischemic conditioning) on retinal damage induced by experimental diabetes. Diabetes was induced by an intraperitoneal injection of streptozotocin. Retinal ischemia was induced by increasing intraocular pressure to 120 mmHg for 5 minutes; this maneuver started 3 days after streptozotocin injection and was weekly repeated in one eye, whereas the contralateral eye was submitted to a sham procedure. Diabetic retinopathy was evaluated in terms of i) retinal function (electroretinogram and oscillatory potentials), ii) integrity of blood-retinal barrier (by albumin–Evans blue complex leakage and astrocyte glial fibrillary acidic protein IHC), iii) optical and electron microscopy histopathologic studies, and iv) vascular endothelial growth factor levels (using Western blot analysis and IHC). Brief ischemia pulses significantly preserved electroretinogram a- and b-wave and oscillatory potentials, avoided albumin–Evans blue leakage, prevented the decrease in astrocyte glial fibrillary acidic protein levels, reduced the appearance of retinal edemas, and prevented the increase in vascular endothelial growth factor levels induced by experimental diabetes. When the application of ischemia pulses started 6 weeks after diabetes onset, retinal function was significantly preserved. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies for diabetic retinopathy treatment.

The aim of the present study was to analyze the early events elicited by tumor necrosis factor α (TNF‐α) on monocyte‐derived dendritic cells (moDC) adhesion to fibronectin (FN) and the involvement of cAMP in the signal transduction mechanism. The intracellular concentration of cAMP and moDC adhesion to FN decreased after TNF‐α treatment. An inverted dose‐dependency for TNF‐α effect was observed for adhesion and cAMP levels. The presence of a phosphodiesterase (PDE) inhibitor (IBMX) and cAMP analogs (8Br‐cAMP, Db‐cAMP) reversed the observed TNF‐α effects. The role of cAMP was analyzed further by examining the cAMP levels in nonadhered and adhered, TNF‐α‐treated moDC. Nonadhered moDC showed lower cAMP levels compared with adhered moDC. Furthermore, nonadhered moDC showed higher IL‐12 content and allostimulatory ability compared with adhered moDC. The higher allostimulatory capacity was abolished in the presence of cAMP analogs and a PDE inhibitor. These results suggest that cAMP levels correlate with TNF‐α‐induced changes of moDC adhesion and allostimulatory capacity.

Melatonin is being increasingly promoted as a therapeutic agent for the treatment of jet lag and insomnia, and is an efficient free radical scavenger. We have recently characterized a product for the reaction of melatonin with nitric oxide (NO), N‐nitrosomelatonin. In the present work, reaction pathways with N1, C2, C4, C6 and C7 as possible targets for its reaction with NO that yield the respective nitroso derivatives have been investigated using semiempirical AM1 computational tools, both in vacuo and aqueous solution. Specifically, two different pathways were studied: a radical mechanism involving the hydrogen atom abstraction to yield a neutral radical followed by NO addition, and an ionic mechanism involving addition of nitrosonium ion to the indolic moiety. Our results show that the indolic nitrogen is the most probable site for nitrosation by the radical mechanism, whereas different targets are probable considering the ionic pathway. These results are in good agreement with previous experimental findings and provide a coherent picture for the interaction of melatonin with NO.

ABSTRACT: By affecting the entrainment pathways of the biologic clock, melatonin has a major influence on the circadian and seasonal organization of vertebrates. In addition, a number of versatile functions that far transcend melatonin actions on photoperiodic time measurement and circadian entrainment have emerged. Melatonin is a free radical scavenger and antioxidant and it has a significant immunomodulatory activity, being presumably a major factor in an organism's defense toxic agents and invading organisms. Besides affecting specific receptors in cell membranes to exert its effects, the interaction of melatonin with nuclear receptor sites and with intracellular proteins, like calmodulin or tubulin‐associated proteins, as well as the direct antioxidant effects of melatonin, may explain many general functions of the pineal hormone.

DAILY variations in melatonin content and 2-[125I]melatonin specific binding in retinas of golden hamsters were studied. Both parameters showed significant variations throughout the 24 h period. Maximal specific binding was observed at 24.00 h, while melatonin content peaked at 04.00 h. Saturation studies performed at 12.00 and 24.00 h indicated that the maximal concentration of 2-[125I]melatonin binding sites (Bmax) was significantly higher at 24.00 h than at 12.00 h, whereas the dissociation constant (Kd) remained unchanged. As 2-[125I]melatonin specific binding decreased at times when retinal melatonin content was high, these findings suggest that daily variations in retinal melatonin levels may be implicated in the regulation of the density of melatonin binding sites, probably through mechanisms of up-and down-regulation induced by melatonin on its own binding sites.

Daily variations in melatonin content and 2-125Imelatonin specific binding in retinas of golden hamsters were studied. Both parameters showed significant variations throughout the 24 h period. Maximal specific binding was observed at 24.00 h, while melatonin content peaked at 04.00 h. Saturation studies performed at 12.00 and 24.00 h indicated that the maximal concentration of 2-125Imelatonin binding sites (Bmax) was significantly higher at 24.00 h than at 12.00 h, whereas the dissociation constant (Kd) remained unchanged. As 2-125Imelatonin specific binding decreased at times when retinal melatonin content was high, these findings suggest that daily variations in retinal melatonin levels may be implicated in the regulation of the density of melatonin binding sites, probably through mechanisms of up- and down-regulation induced by melatonin on its own binding sites.

Daily variations in cGMP, guanylate cyclase and phosphodiesterase activity in golden hamster retina were studied. Cyclic GMP content exhibited significant variations throughout the 24-hr cycle with maximal values during the dark phase. In order to establish the relative participation of nucleotide synthesis and breakdown during a 24-hr cycle, guanylate cyclase and phosphodiesterace activity were measured in hamsters killed at eight intervals. Guanylate cyclase activity increased at night, peaking at 22.00 hr. Phosphodiesterase activity did not change significantly throughout the light-lark cycle. Light exposure during the night inhibited the nocturnal increase in cGMP content and guanylate cyclase activity, while phosphodiesterase remained unchanged. From these results, it might be presumed that in response to continuous (in a range of hr) light or dark stimuli, the retina would process the photic signal in a different way from that in the short term (in a range of sec).

The pineal gland serves the function of a neuroendocrine transducer converting information about day length into the nocturnal release of melatonin. Melatonin acts on the brain, particularly on the hypothalamus, to affect several biological rhythms. By employing autoradiography and 2-[125I]melatonin as a radioligand, the hypothalamic suprachiasmatic nucleus (SCN) and the pars tuberalis of the adenohypophysis have been identified as sites for melatonin binding exhibiting dissociation constants (Kds) in the 10−10M range. These sites were also revealed in test-tube binding assays employing crude membrane fractions. Additionally, studies in either membrane or cytosol fractions using tritiated or radioiodine-labelled melatonin indicated location of another population of presumptive melatonin binding sites with Kds in the 10−8−10−9M range in several other brain areas, including the hippocampus, cerebral and cerebellar cortexes, as well as the pineal gland. Signal transduction processes for melatonin presumably involve interaction with G proteins to inhibit adenylate cyclase. Also, a decrease of Ca2+uptake, stimulation of guanylate cyclase and inhibition of cyclooxygenase occur at 10−8M melatonin concentrations. The time of administration of melatonin is critical for hormone action. In rodents and humans, a major late afternoon-early evening period of sensitivity is found for several central and peripheral effects of melatonin. Results in rats suggest that central synapses employing τ-aminobutyric acid (GABA) as an inhibitory transmitter are a target for pineal melatonin activity because: (a) pinealectomy (Px) disrupts circadian rhythmicity of brain GABA and benzodiazepine (BZP) binding; (b) low doses of melatonin counteract Px-induced modifications of BZP and GABA binding; (c) chronic melatonin treatment increases brain BZP and GABA binding; (d) melatonin administration accelerates brain GABA turnover rate; (e) melatonin increases glutamic acid decarboxylase activity and Cl−ion conductance in the medial basal hypothalamus-preoptic area, with maximal activity in the evening. As BZP, melatonin could affect circadian rhythmicity by modifying GABAergic mechanisms in the endogenous oscillator. Additionally, the epileptoid state described after Px and the mild sedation and torpor that follow administration of pharmacological amounts of melatonin can be explained by an effect on central GABAergic circuits.

Summary Two apparent affinities for Na+ -dependent,3H-GABA uptake were found in bovine pineal fragments in vitro i.e., a high affinity uptake (Km=37±5 µM) and a low affinity uptake (Km=435±50 uM). GABA or the neuronal and glial GABA uptake inhibitor nipecotic acid was significantly more effective than the inhibitor of the GABA glial uptake ß-alanine to decrease pineal3H-GABA uptake. High K+ concentration released3H-GABA in superfused bovine pineals, no differences in3H-GABA release among fragments taken from medial, proximal or distal pineal regions being apparent. Superfusion of pineal fragments in the absence of Ca2+ but in the presence of EGTA, Mg2+ or verapamil decreased significantly3H-GABA release induced by K+. In every case a Ca2+ -independent pineal GABA release was found. Preincubation with GABA or nipecotic acid, but not with ß-alanine, blunted subsequent3H-GABA release. GABA increased36Cr--influx in pineal homogenates, an effect blocked by picrotoxin. Incubation of pineal homogenates in the presence of aminooxyacetic acid decreased Vmax of glutamic acid decarboxylase, without modifying its Km. These results are compatible with a transmitter or modulator role of GABA in bovine pineal gland.

A characterization of nitric oxide synthase (NOS) in the golden hamster retina was performed. Enzymatic activity was partially Ca2and calmodulin-dependent, required NADPH, and was inhibited by L-arginine analogs. Retinal NOS activity was higher at midnight than at midday. When the hamster were placed under constant darkness for 24 or 48 h, and killed at equivalent time points, representing subjective day and subjective night respectively, the differences in NOS activity disappeared. One hour of darkness during the day increased, while the same period of light during the night decreased, retinal NOS activity. From these results, it might be presumed that hamster retinal NOS activity is regulated by the photic stimulus.

Melatonin effect on retinal cyclic GMP accumulation, guanylate cyclase activity, cyclic GMP content and cyclic GMP phosphodiesterase activity was examined in the Syrian hamster retina. Melatonin increased significantly cyclic GMP accumulation at picomolar concentrations and in a time-dependent manner. The kinetic analysis of guanylate cyclase activity revealed a significant increase of both apparentVmax andKm, induced by 10 nM melatonin. The effect of melatonin was higher in the absence, than in the presence of the phoshodiesterase inhibitor (IBMX), suggesting an effect on cyclic GMP catabolism. Phosphodiesterase activity was significantly decreased by melatonin. The results show a dual effect of melatonin on cyclic GMP levels, i.e. by increasing the synthesis and inhibiting the degradation, both resulting in an increase of cyclic GMP levels. Taking into account the key role of cyclic GMP in visual mechanisms, the results would suggest the participation of melatonin in retinal physiology.

Summary 1.3H-γ-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pinealsin vitro.2.The release of3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+.3.In a Ca2+-free medium3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted.4.Norepinephrine (NE; 10−6–10−4M) stimulated3H-GABA release from rat pineal explants in a dose-dependent manner.5.The activity of 10−5M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (α1- andα1/α2-adrenoceptor blockers, respectively) and was unaffected by propranolol (β-adrenoceptor blocker).6.Theα1-adrenoceptor agonist phenylephrine (10−7-10−5M) and theβ-adrenoceptor agonist isoproterenol (10−5M) mimicked the GABA releasing activity of NE, while 10−7M isoproterenol failed to affect it; theα2-adrenoceptor agonist clonidine (10−7–10−5M) did not modify3H-GABA release.7.The addition of 10−4M GABA or of the GABA transminase inhibitorγ-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures.8.GABA at concentrations of 10−5M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants.9.The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release.10.The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels.11.These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2+ dependent; (b) NE releases rat pineal GABA via interaction withα1-adrenoceptors; (c) GABA inhibits melatonin productionin vitro via interaction with GABA type A receptor sites; and (d) GABA's effect on NE-induced melatonin release does not correlate with the lack of effect on the NE-induced decrease in pineal 5-HT content.

Non-exudative age-related macular degeneration, a prevalent cause of blindness, is a progressive and degenerative disease characterized by alterations in Bruch's membrane, retinal pigment epithelium, and photoreceptors exclusively localized in the macula. Although experimental murine models exist, the vast majority take a long time to develop retinal alterations and, in general, these alterations are ubiquitous, with many resulting from non-eye-specific genetic manipulations; additionally, most do not always reproduce the hallmarks of human age-related macular degeneration. Choroid vessels receive sympathetic innervation from the superior cervical ganglion, which, together with the parasympathetic system, regulates blood flow into the choroid. Choroid blood flow changes have been involved in age-related macular degeneration development and progression. At present, no experimental models take this factor into account. The aim of this work was to analyze the effect of superior cervical gangliectomy (also known as ganglionectomy) on the choroid, Bruch's membrane, retinal pigment epithelium and retina. Adult male C57BL/6J mice underwent unilateral superior cervical gangliectomy and a contralateral sham procedure. Although superior cervical gangliectomy induced ubiquitous choroid and choriocapillaris changes, it induced Bruch's membrane thickening, loss of retinal pigment epithelium melanin content and retinoid isomerohydrolase, the appearance of drusen-like deposits, and retinal pigment epithelium and photoreceptor atrophy, exclusively localized in the temporal side. Moreover, superior cervical gangliectomy provoked a localized increase in retinal pigment epithelium and photoreceptor apoptosis, and a decline in photoreceptor electroretinographic function. Therefore, superior cervical gangliectomy recapitulated the main features of human non-exudative age-related macular degeneration, and could become a new experimental model of dry age-related macular degeneration, and a useful platform for developing new therapies.

Daily variations in melatonin content of retinas of pinealectomized and sham-operated golden hamsters were studied. Melatonin content showed significant daily variations with maximal values at night (i.e. early in the night in pinealectomized hamsters and late at night in sham-operated animals). Moreover, mean retinal melatonin levels augmented significantly after pinealectomy. In vitrothe augmented melatonin levels found in retinas incubated in darkness for 8 h was suppressed by exposure to light, indicating the ability of hamster retina to regulate melatonin synthesis in isolated conditions. Taken together, the in vivoand in vitroresults support daily variations of melatonin content of exclusive retinal origin.