Mónica S. Chianelli

Melatonin: a novel neuroprotectant for the treatment of glaucoma

Abstract:  Glaucoma is a leading cause of blindness. Although ocular hypertension is the most important risk factor, several concomitant factors such as elevation of glutamate and decrease in gamma‐aminobutyric acid (GABA) levels, disorganized NO metabolism, and oxidative damage could significantly contribute to the neurodegeneration. The aim of this report was to analyze the effect of melatonin on retinal glutamate clearance, GABA concentrations, NO synthesis, and retinal redox status, as well as on functional and histological alterations provoked by chronic ocular hypertension induced by intracameral injections of hyaluronic acid (HA) in the rat eye. In normal retinas, melatonin increased glutamate uptake, glutamine synthase activity, GABA turnover rate, glutamic acid decarboxylase activity, superoxide dismutase activity, and reduced glutathione (GSH) levels, whereas it decreased NOS activity, L‐arginine uptake, and lipid peroxidation. To assess the effect of melatonin on glaucomatous neuropathy, weekly injections of HA were performed in the eye anterior chamber. A pellet of melatonin was implanted subcutaneously 24 hr before the first injection or after six weekly injections of HA. Melatonin, which did not affect intraocular pressure (IOP), prevented and reversed the effect of ocular hypertension on retinal function (assessed by electroretinography) and diminished the vulnerability of retinal ganglion cells to the deleterious effects of ocular hypertension. These results indicate that melatonin could be a promissory resource in the management of glaucoma.

Therapeutic efficacy of melatonin in reducing retinal damage in an experimental model of early type 2 diabetes in rats

Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults, mostly affected by type 2 diabetes mellitus (T2DM). We have developed an experimental model of early T2DM in adult rats which mimics some features of human T2DM at its initial stages and provokes significant retinal alterations. The aim of this work was to analyze the effect of melatonin on retinal changes induced by the moderate metabolic derangement. For this purpose, adult male Wistar rats received a control diet or 30% sucrose in the drinking water. Three weeks after this treatment, animals were injected with vehicle or streptozotocin (STZ, 25 mg/kg). One day or 3 wk after vehicle or STZ injection, animals were subcutaneously implanted with a pellet of melatonin. Fasting and postprandial glycemia, and glucose, and insulin tolerance tests were analyzed. At 12 wk of treatment, animals which received a sucrose‐enriched diet and STZ showed significant differences in metabolic tests, as compared with control groups. Melatonin, which did not affect glucose metabolism in control or diabetic rats, prevented the decrease in the electroretinogram a‐wave, b‐wave, and oscillatory potential amplitude, and the increase in retinal lipid peroxidation, NOS activity, TNFα, Müller cells glial fibrillary acidic protein, and vascular endothelial growth factor levels. In addition, melatonin prevented the decrease in retinal catalase activity. These results indicate that melatonin protected the retina from the alterations observed in an experimental model of DR associated with type 2 diabetes.

Therapeutic Effect of Melatonin in Experimental Uveitis

Uveitis is a common ophthalmic disorder that can be induced in hamsters by a single intravitreal injection of bacterial lipopolysaccharide (LPS). To examine the therapeutic effects of melatonin on uveitis, a pellet of melatonin was implanted subcutaneously 2 hours before the intravitreal injection of either vehicle or LPS. Both 24 hours and 8 days after the injection, inflammatory responses were evaluated in terms of i) the integrity of the blood-ocular barrier, ii) clinical signs, iii) histopathological studies, and iv) retinal function. Melatonin reduced the leakage of proteins and cells in the anterior segment of LPS-injected eyes, decreased clinical signs such as dilation of the iris and conjunctival vessels, and flare in the anterior chamber, and protected the ultrastructure of the blood-ocular barrier. A remarkable disorganization of rod outer segment membranous disks was observed in animals injected with LPS, whereas no morphological changes in photoreceptor outer segments were observed in animals treated with melatonin. Furthermore, melatonin prevented a decrease in LPS-induced electroretinographic activity. In addition, melatonin significantly abrogated the LPS-induced increase in retinal nitric-oxide synthase activity, tumor necrosis factor-alpha, and nuclear factor kappaB p50 and p65 subunit levels. These results indicate that melatonin prevents the clinical, biochemical, histological, ultrastructural, and functional consequences of experimental uveitis, likely through a nuclear factor kappaB-dependent mechanism, and support the use of melatonin as a new therapeutic strategy for the treatment of uveitis.

Effect of melatonin on the retinal glutamate/glutamine cycle in the golden hamster retina

Glutamate is the main excitatory neurotransmitter in the retina, but it is neurotoxic when present in excessive amounts. The metabolic dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. Since melatonin has been shown to be neuroprotective in several systems, in the present report, its effect on the glutamate/glutamine cycle activity was examined in the golden hamster retina. Melatonin (0.1–10 nM) significantly increased retinal glutamine synthetase activity but it did not affect L‐glutamine release. A characterization of the hamster retinal L‐ glutamine uptake mechanism was performed. This mechanism was partly Na+‐dependent, and it was significantly inhibited by 2‐aminobicyclo (2, 2, 1) heptane 2‐carboxylic acid (BCH, a selective antagonists for the L‐type system) and by α‐(methylamino)‐isobutyric acid (MeAIB, substrate characteristic for the A ‐type transporter) suggesting the coexistence of these transport systems in the hamster retina. Melatonin (0.1–10 nM) significantly increased total glutamine uptake as well as the BCH and the MeAIB‐insensitive transporters activity. On the other hand, melatonin significantly decreased retinal glutaminase activity. On the basis of these results, it might be presumed that hamster retinal glutamate/glutamine cycle activity is regulated by physiological concentrations of melatonin. Furthermore, these findings suggest that a treatment with melatonin could be considered as a new approach to handling glutamate‐mediated neuronal degeneration.

Involvement of glutamate in retinal protection against ischemia/reperfusion damage induced by post-conditioning

Retinal ischemia could provoke blindness and there is no effective treatment against retinal ischemic damage. Brief intermittent ischemia applied during the onset of reperfusion (i.e., post‐conditioning) protects the retina from ischemia/reperfusion injury. Multiple evidences support that glutamate is implicated in retinal ischemic damage. We investigated the involvement of glutamate clearance in post‐conditioning‐induced protection. For this purpose, ischemia was induced by increasing intra‐ocular pressure for 40 min, and 5 min after reperfusion, animals underwent seven cycles of 1 min/1 min ischemia/reperfusion. One, three, or seven days after ischemia, animals were subjected to electroretinography and histological analysis. The functional and histological protection induced by post‐conditioning was evident at 7 (but not 1 or 3) days post‐ischemia. An increase in Müller cell glial fibrillary acidic protein (GFAP) levels was observed at 1, 3, and 7 days after ischemia, whereas post‐conditioning reduced GFAP levels of Müller cells at 3 and 7 days post‐ischemia. Three days after ischemia, a significant decrease in glutamate uptake and glutamine synthetase activity was observed, whereas post‐conditioning reversed the effect of ischemia. The intravitreal injection of supraphysiological levels of glutamate mimicked electroretinographic and histological alterations provoked by ischemia, which were abrogated by post‐conditioning. These results support the involvement of glutamate in retinal protection against ischemia/reperfusion damage induced by post‐conditioning.

Post-ischemic environmental enrichment protects the retina from ischemic damage in adult rats

The aim of this study was to elucidate whether post-ischemic enriched environment (EE) housing protects the retina from ischemic damage in adult rats, and the involvement of glutamate in retinal protection induced by EE housing. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. After ischemia, animals were housed in a standard environment (SE) or EE and subjected to electroretinography and histological analysis. EE housing afforded significant functional protection in eyes exposed to ischemia/reperfusion injury. A marked reduction in retinal thickness and ganglion cell number, and an increase in Müller cell glial fibrillary acidic protein (GFAP) levels were observed in ischemic retinas from SE-housed animals, which were reversed by EE housing. A deficit in anterograde transport from the retina to the superior colliculus was observed in SE- but not in EE-housed animals. In SE-housed animals, ischemia induced a significant decrease in retinal glutamate uptake and glutamine synthetase activity, whereas EE housing reversed the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate partially reproduced retinal alterations induced by ischemia/reperfusion, which were abrogated by EE housing. These results indicate that EE housing significantly protected retinal function and histology from ischemia/reperfusion injury in adult rats, likely through a glutamate-dependent mechanism.

Effect of bacterial lipopolysaccharide on ischemic damage in the rat retina.

PURPOSE The purpose of this study was to investigate whether bacterial lipopolysaccharide (LPS) induces ischemic preconditioning in the rat retina, and, if so, whether nitric oxide (NO) is involved in this process. METHODS Rats were intravitreously injected with different doses of LPS (0.1, 1, or 5 microg) in one eye and vehicle in the contralateral eye 24 hours before retinal ischemia induced by increasing intraocular pressure to 120 mm Hg for 40 or 60 minutes. Subsequently, 7 or 14 days after ischemia, the rats were subjected to electroretinography and histologic analysis. One group of animals received intraperitoneal injections of NOS inhibitors, N-nitro-L-arginine methyl ester (L-NAME) aminoguanidine or N-(3-(aminomethyl)benzyl)acetamidine (W1400) before the injection of LPS or vehicle. Retinal nitric oxide synthase (NOS) activity was assessed through the conversion of (3)H-L-arginine to (3)H-L-citrulline. RESULTS One microgram (but not 0.1 or 5 microg) LPS afforded significant morphologic and functional protection in eyes exposed to ischemia-reperfusion injury. The beneficial effect of LPS was reversed by treatment with L-NAME, aminoguanidine, or W1400. LPS (1 and 5 microg, but not 0.1 microg) significantly increased retinal NOS activity. CONCLUSIONS These results indicate that LPS provides retinal protection against ischemia-reperfusion injury in a dose-dependent manner, probably through an inducible NOS-dependent mechanism.

Induction of ischemic tolerance protects the retina from diabetic retinopathy.

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.

Neuroprotective effect of melatonin in experimental optic neuritis in rats

Optic neuritis (ON) is an inflammatory, demyelinating, and neurodegenerative condition of the optic nerve, which might induce permanent vision loss. Currently, there are no effective therapies for this disorder. We have developed an experimental model of primary ON in rats through a single microinjection of 4.5 μg of bacterial lipopolysaccharide (LPS) into the optic nerve. Since melatonin acts as a pleiotropic therapeutic agent in various neurodegenerative diseases, we analyzed the effect of melatonin on LPS‐induced ON. For this purpose, LPS or vehicle were injected into the optic nerve from adult male Wistar rats. One group of animals received a subcutaneous pellet of 20 mg melatonin at 24 hr before vehicle or LPS injection, and another group was submitted to a sham procedure. Melatonin completely prevented the decrease in visual evoked potentials (VEPs), and pupil light reflex (PLR), and preserved anterograde transport of cholera toxin β‐subunit from the retina to the superior colliculus. Moreover, melatonin prevented microglial reactivity (ED1‐immunoreactivity, P < 0.01), astrocytosis (glial fibrillary acid protein‐immunostaining, P < 0.05), demyelination (luxol fast blue staining, P < 0.01), and axon (toluidine blue staining, P < 0.01) and retinal ganglion cell (Brn3a‐immunoreactivity, P < 0.01) loss, induced by LPS. Melatonin completely prevented the increase in nitric oxide synthase 2, cyclooxygenase‐2 levels (Western blot) and TNFα levels, and partly prevented lipid peroxidation induced by experimental ON. When the pellet of melatonin was implanted at 4 days postinjection of LPS, it completely reversed the decrease in VEPs and PLR. These data suggest that melatonin could be a promising candidate for ON treatment.

Retinal changes in an experimental model of early type 2 diabetes in rats characterized by non-fasting hyperglycemia

Diabetic retinopathy is a leading cause of acquired blindness in young, but also in elder adults, mostly affected by type 2 diabetes mellitus (T2DM). The aim of this work was to develop an experimental model of early human T2DM in adult rats, and to analyze retinal functional, morphological, and biochemical changes arising during the early stages of the moderate metabolic derangement. For this purpose, animals were divided in four groups: adult male Wistar rats receiving: tap water and citrate buffer i.p. (group 1), tap water with 30% sucrose and citrate buffer i.p. (group 2), tap water and 25mg/kg i.p streptozotocin (STZ, group 3), or 30% sucrose and STZ (group 4). Fasting and postprandial glycemia, fructosamine and serum insulin levels were assessed. In addition, i.p. glucose and insulin tolerance tests were performed. Retinal function (electroretinogram, ERG) and morphology (optical microscopy), retinal nitric oxide synthase (NOS) activity (using (3)H-arginine), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), and TNFα levels (ELISA) were evaluated. At 6 and 12 weeks of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in most metabolic tests, as compared with the other groups. At 12 weeks of treatment, a significant decrease in the ERG a- and b- wave and oscillatory potential amplitudes, and a significant increase in retinal NOS activity, TBARS, TNFα, glial fibrillary acidic protein in Müller cells, and vascular endothelial growth factor levels were observed. These results indicate that the combination of diet-induced insulin resistance and a slight secretory impairment resulting from a low-dose STZ treatment mimics some features of human T2DM at its initial stages, and provokes significant retinal alterations.