Pablo Sande

Effect of glaucoma on the retinal glutamate/glutamine cycle activity

Glutamate‐induced excitotoxicity has been proposed to mediate the death of retinal ganglion cells in glaucoma. The metabolic dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. Thus, the aim of the present work was to study the retinal glutamate/glutamine activity in eyes with hypertension induced by intracameral injections of hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 or 10 weeks of treatment, glutamate and glutamine uptake and release were assessed using [3H]‐glutamate and [3H]‐glutamine as radioligands, respectively. In addition, glutamine synthetase activity was assessed by a spectrophotometric assay, whereas glutaminase activity was measured through the conversion of [3H]‐glutamine to [3H]‐glutamate. At 3 weeks of treatment with HA, a significant decrease (P<0.01) in glutamate uptake and glutamine synthetase activity was observed. Glutamine uptake and release, as well as glutaminase activity, were significantly increased (P<0.01) in eyes injected with HA for 3 weeks compared with vehicle‐injected eyes, whereas [3H]‐glutamate release did not change in hypertensive eyes. Only the changes in glutamine synthetase activity persisted at 10 weeks of treatment with HA. These results indicate a significant alteration in the retinal glutamate/glutamine cycle activity in hypertensive eyes. Since these changes preceded both functional and histological alterations induced by ocular hypertension, these results support the involvement of glutamate in glaucomatous neuropathy.

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 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.

SIRT6 Is Required for Normal Retinal Function

The retina is one of the major energy consuming tissues within the body. In this context, synaptic transmission between light-excited rod and cone photoreceptors and downstream ON-bipolar neurons is a highly demanding energy consuming process. Sirtuin 6 (SIRT6), a NAD-dependent deacylase, plays a key role in regulating glucose metabolism. In this study, we demonstrate that SIRT6 is highly expressed in the retina, controlling levels of histone H3K9 and H3K56 acetylation. Notably, despite apparent normal histology, SIRT6 deficiency caused major retinal transmission defects concomitant to changes in expression of glycolytic genes and glutamate receptors, as well as elevated levels of apoptosis in inner retina cells. Our results identify SIRT6 as a critical modulator of retinal function, likely through its effects on chromatin.

Effect of chondroitin sulfate on intraocular pressure in rats.

PURPOSE To study the effect of intracameral injections of chondroitin sulfate (CS) on intraocular pressure (IOP), retinal function, and histology in rats. METHODS Acute or chronic injections of CS were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with vehicle. IOP was daily or weekly assessed by a tonometer. Retinal function was assessed by scotopic electroretinography (ERG) and the visual pathway by flash visual evoked potentials (VEPs), whereas the retinal and optic nerve head structure were examined by histologic analysis. RESULTS A single injection of 8 mg (but not 2 or 4 mg) CS induced a significant increase of IOP. The increase of IOP induced by a single injection of 8 mg CS lasted for 7 days, whereas chronic (weekly) administration during 10 weeks induced a significant and sustained increase in IOP compared with eyes injected with vehicle. A significant decrease of scotopic ERG a- and b- wave amplitude was observed after 6 and 10 weeks of CS administration. Moreover, a significant decrease in scotopic flash VEP N2-P2 component amplitude was observed in eyes treated with CS for 6 and 10 weeks. A significant loss of ganglion cell layer cells and optic nerve axons was observed in eyes receiving CS for 10 weeks. CONCLUSIONS These results suggest that exogenous CS simulates the accumulation of CS in primary open-angle glaucoma and that increased amounts of CS could play a key role in the IOP dysregulation characteristic of glaucoma.

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.

Effect of Experimental Diabetic Retinopathy on the Non-Image-Forming Visual System

Diabetic retinopathy is a leading cause of blindness. Intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin, are involved in non-image-forming visual responses such as photoentrainment of circadian rhythms and pupilary light reflex. Since several reports indicate that retinal ganglion cells are affected by diabetes, we investigated the non-image-forming visual system in an advanced stage of experimental diabetes in rats induced by streptozotocin. After 15 wks of diabetes induction, clear alterations in the visual function were observed and all animals developed mature cataracts. At this time point, concomitantly with a significant decrease in the number of Brn3a(+) retinal ganglion cells, no differences in the number of melanopsin-containing cells, melanopsin levels, and retinal projections to the suprachiasmatic nuclei and the olivary pretectal nucleus were observed. At high light intensity, afferent pupil light reflex appears to be conserved in diabetic animals. After 15 wks of diabetes induction, a significant decrease in light-induced c-Fos expression in the suprachiasmatic nuclei was found. In diabetic animals, the locomotor activity pattern was conserved, although a delay in the time needed for re-entrainment after a phase delay was observed. In diabetic animals, lensectomy reversed the alterations in c-Fos expression and in the locomotor activity rhythm. These results suggest that the neuronal substrate of the non-image-forming visual system remained largely unaffected at advanced stages of diabetes, and that lensectomy, a relatively easy and safe surgery, could partially restore circadian alterations induced by diabetes. (Author correspondence: ruthr@fmed.uba.ar)

Therapeutic benefit of melatonin in experimental feline uveitis

Abstract:  Uveitis is a frequent ophthalmic disorder which constitutes one of the main causes of blindness in domestic cats. The aim of this report was to analyze the effect of melatonin on experimentally induced uveitis in cats. Bacterial lipopolysaccharide (LPS) was injected intravitreally into one eye from intact cats, while the contralateral eye was injected with vehicle. Melatonin was orally administered every 24 hr to a group of ten cats, from 24 hr before until 45 days after intravitreal injections. Eyes were evaluated by means of clinical evaluation, intraocular pressure (IOP), blood–ocular barrier integrity (via measurement of protein concentration and cell content in samples of aqueous humor [AH]), electroretinogram (ERG), and histological examination of the retinas. In LPS‐treated eyes, several clinical signs were observed until day 45 postinjection. The treatment with melatonin significantly decreased clinical signs and prevented the reduction in IOP induced by LPS. In LPS‐injected eyes, melatonin significantly preserved the blood–ocular barrier integrity, as shown by a decrease in the number of infiltrating cells and protein concentration in the AH. Mean amplitudes of scotopic ERG a‐ and b‐waves were significantly reduced in eyes injected with LPS, whereas melatonin significantly prevented the effect of LPS. At 45 days after injection, LPS induced alterations in photoreceptors and at the middle portion of the retina, whereas melatonin preserved the retinal structure. These results indicate that melatonin prevented clinical, biochemical, functional, and histological alterations induced by LPS injection. Thus, melatonin might constitute a useful tool for the treatment of feline uveitis.