1

P2.26 Neuroprotectıve effect of rıluzole and resveratrol admınıstratıon ın experımental glaucoma model Dilara Pirhan1, Nursen Yüksel2, Esra Emre2, Abdulkadir Cengiz3, Yusuf Çaglar2, Irem Özöver4, Demir Kürsat Yildiz4 1Department of Ophthalmology, State Hospital, Ministry of Health, Sanliurfa - Turkey, 2Department of Ophthalmology, Kocaeli University School of Medicine, Kocaeli - Turkey, 3Department of Technical Education, Kocaeli University

School of Medicine, Kocaeli - Turkey, 4Department of Pathology, Kocaeli University School of Medicine, Kocaeli - Turkey

Purpose: Glaucoma has the pathophysiological features of both chronic and neurodegenerative disease; consequently, there is a hope that riluzole and resveratrol hold promise for glaucoma management. To the best of our knowledge, the effect of riluzole and resveratrol in experimental glaucoma model has never been investigated. Based on these insights, we aimed to investigate whether riluzole and resveratrol, potent antiapoptotic drugs, have neuroprotective effect on the survival of retinal ganglion cells (RGCs) in experimental glaucoma. Methods: A total of 84 male Wistar albino rats were housed in temperature and light controlled rooms. All surgical manipulations were carried out under general anesthesia. A drop of topical anesthetics was instilled on eyes prior to IOP measurements. During recovery from anesthesia a topical ointment was applied to prevent corneal desiccation. Rats were anesthetized with ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (0.5 mg/kg) administered intraperitoneally. With a syringe and a 30-gauge needle, hyaluronic acid (Sigma catalog no. H1751; Sigma Chemical Co., St. Louis, MO) were injected into right eyes of anesthetized rats weekly (Image 1, A), while an equal volume of saline solution was injected into right eyes of control/saline group. In the chronic protocol, injections were applied at the corneoscleral limbus beginning at hour 12 and changing the site of the next injection hourly, by rotating the head to achieve better access to the limbus. Intraocular pressure (IOP) was measured under anesthesia before and after each glaucoma induction with a TonoPen XL applanation tonometer (Mentor, Norwell, MA, USA). Tonometry was performed within 3 minutes of

2

anesthesia onset and always between the hours of 9:00am and 11:00am. Almost all the animals had localized corneal edema for less than 24 hours, at the site of the injection. The rats were randomly grouped as follows: control group (C, n = 12), vehicle-treated glaucoma group (G, n = 12), riluzole-treated group in the early phase of glaucoma (ERL, n = 12), riluzole-treated group in the late phase of glaucoma (LRL, n = 12), resveratrol-treated group in the early phase of glaucoma (ERS, n = 12), resveratrol-treated group in the late phase of glaucoma (LRS, n = 12), and riluzole and resveratrol combined-treated group in the early phase of glaucoma (RR, n = 12). ERL group received a single daily dose of 8 mg/kg riluzole i.p. starting with the glaucoma induction for a period of six weeks. LRL group received the same dose of riluzole following the glaucoma induction for a period of three weeks. ERS group received a single daily dose of 10 mg/kg resveratrol i.p. starting with glaucoma induction for a period of six weeks. LRS group received the same dose of resveratrol following the glaucoma induction for a period of three weeks. RR group received same doses of riluzole and resveratrol together starting with the glaucoma induction for a period of six weeks. Riluzole (Rilutek®, S.A., Sanofi-Aventis, Istanbul, Turkey) was first dissolved with 0.05% ethanol in 0.9% saline. Resveratrol was dissolved with 2% ethanol in 0.9% saline. Control group received no medication, an equal volume of saline solution was injected into the right eyes, vehicle treated glaucoma group received i.p injection of physiological saline including 2% ethanol as a vehicle control for all drug groups complying with drug schedule for six weeks. The fluorescent tracer dextran tetramethylrhodamine (DTMR; 3000 MW; Molecular Probes, Inc. Eugene, OR, USA) was applied intraorbitally 2 days prior to sacrifice as previously described.1-4 For the DTMR injection, we followed the method described by Solomon et al.5 Then the animals were deeply anesthetized, perfused transcardially through the ascending aorta with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) as previously described.2, 3. Globes were marked with sutures for orientation from superior limbus. Enucleation was performed to the fixed eyes immediately after death of animals. The lens and vitreous were extracted by cutting the anterior chamber at the level of ora serrata. The optic nerves were carefully dissected and postfixed in formaldehyde. The eyecups were postfixed in the same fixative for 2 hour. Then, the retinas were removed, flat-mounted, with the RGC layer being uppermost using PBS/glycerol (1/1), and superior retina is marked for orientation. The retinas of rats were photographed shortly after mounting. Labeled RGCs per unit area were captured on the camera with

3

same magnification (200x) and exposure on the same day.(Image 1, C) Counts were taken from comparable areas of four quadrants of each retina along 2 radii in 4 directions (i.e., superior, temporal, inferior and nasal) centered on the position of optic nerve head ,in a distance of 2 mm from the optic disc. Six fields 1 mm away from each other were counted along each radius, yielding a total of 24 fields of per retina (Image 1, B). RGCs counting process was carried out by an experienced observer who was masked to the procedure that had been carried out and to the treatments that was given. Images were counted by image analysis software Image Pro Plus (IPP 5.1 for Windows®; Media Cybernetics, Silver Spring, MD, USA). Optic nerve transection was obtained at approximately 2 mm posterior to the nerve’s emanation from the globe (Image 2). Sections were counterstained with GFAP Ab-7 (ASTRO5+ASTRO6) mouse monoclonal antibody (Lab Vision Corporation, CA, USA), and viewed under microscope (model Olympus BX51; Olympus Optical Co, Tokyo, Japan). For each optic nerve cross section, photographs were taken at low and high magnification (x20, x100 respectively) and compared to each other qualitatively (Image 3). The statistical analysis was performed by using a commercially available statistical software package (SPSS for Windows, version 13.0; SPSS, Chicago, Illinois, USA). The mean cell density of surviving RGCs in mm2 per retina was calculated. The neuroprotective effects of riluzole and were evaluated by comparing the number of surviving RGCs in the treated groups with the number of surviving RGCs in the non-treated groups using analysis of variance (ANOVA) with Bonferroni post hoc test with significance set at p = 0.05 (Image 1, D). Results: All eyes that inducted to glaucoma (n = 72), developed elevated IOP within the 1 day of treatment. In all groups, adequate IOP values were reached during the 6 weeks. The mean IOP in the glaucomatous eyes was significantly higher than in the controls for each treatment group (p < 0.01). The mean IOP of the glaucoma group and the 5 different treatment groups was similar without any significant difference, suggesting that any difference in RGC survival could be attributed to the treatment. The mean density of DTMR labeled the RGC density (mean cell number/mm2 ± standard deviation) was 1207 ± 56 in control group, 404 ± 65 in G group, 965 ± 56 in ERL group, 714 ± 25 in LRL group, 735 ± 29 in ERS group, 667 ± 20 in LRS group, 1071 ± 49 in RR group, respectively (Image 3). Six weeks after induction of glaucoma, retinal ganglion cell loss in the average when compared with the control group was 66% ± 5 glaucoma group, % 19 ± 5 in

4

ERL, 40% ± 3 in LRL, % 38 ± 3 in ERS, 44 ± 5% in LRS, 11 ± 3% in RR group, respectively. Optic nerve head sections 2 mm behind the optic nerve were evaluated after staining with GFAP (glial fibrillary acidic protein). Especially in tissue sections taken from G group, glial tissue increase were common, and astrocyte morphological changes were found to be due to an increase in the astrocyte bulk (Image 4). Reticulated and skein like architectural organization of astrocytes demonstrated a disorganized architecture with round and ovoid nodular appearance. Hypertrophy and hyperplasia of astrocytic extensions were observed. The delicate and fine staining pattern seen in control group was replaced by an amorphous appearance in morphology. Qualitative changes in the glaucoma group, as seen in the treatment groups, was showed to be less than. Conclusions: The results of this study showed that systemic treatment with riluzole and/or resveratrol, potent antiapoptotic drugs, is neuroprotective in experimental model of glaucoma. Both were found to have a significant neuroprotective effect on RGC survival. The combined use of these drugs showed a statistically significantly higher survival rate of the RGCs than other single treatment groups (Image 5). Riluzole has been shown to prevent or decrease pressure induced apoptosis and enhance ERG wave recovery, highlighting the benefits of targeting multiple receptors in excitotoxic cell death6. The animals that were given riluzole and resveratrol showed a statistically significantly higher survival rate of the RGCs than animals that underwent the same standardized glaucoma induction procedure and that only received vehicle injections, but the combination therapy that was given at the start of the damage, protection provided was almost close to the control group. In the literature although there is any publications conducted on glaucoma that have been reported riluzole and resveratrol are neuroprotective effects on RGCs, although neuroprotective effects of these drugs have been reported in many publications. To the best of our knowledge, this study is the first one demonstrating the neuroprotective effect of riluzole and resveratrol, based on RGC count in experimental glaucoma. One of the studies in the literature showing the neuroprotective effects of riluzole in retinal ischemia model made by Ettaiche et al.7 In this study before the ischemia provided by sudden IOP rise, 8 mg/kg riluzole was given. The neuroprotective effect was researched by electroretinography, and evaluation of apoptosis and histopathologic examination. They reported for the first time the potent neuroprotective effect against retinal ischemia by reducing the reported changes in the retinal cytoskeleton, necrotic cell

5

damage, and DNA fragmentation. The mechanism by which riluzole and resveratrol protects the RGCs from glaucomatous damage is not completely understood and, the remainder of this discussion is therefore rather speculative, may open up a recent novel, and may seek after potential therapeutic approaches for glaucoma management. RGCs and optic nerve, integral parts of the central nervous system, points out that glaucoma demonstrates the similar mechanisms of cell death observed in Alzheimer disease and is a neurodegenerative disease8-10. Riluzole, which has been used for the treatment of ALS, is an antiglutamate agent. It has inhibitory effect on blockade of Postsynaptic NMDA and universe type glutamate receptors, as well as voltage dependent Na + channels. Riluzole has anti-ischemic, sedative and anti-epileptic effects11 and it has shown to slow down the progression of ALS. Although the exact mechanism of neuroprotective effect of riluzole has not been clarified yet, Na+ channel inhibition is thought to be at least partially trusted,12 because inhibition of Na+ channel is associated with post ischemic neuronal protection. The protective effect of resveratrol has been shown in many tissues of cells. However the effects on retinal tissues are still not well known yet. Luna et al.16 investigated the effect of resveratrol in trabecular cell culture by creating a chronic oxidative stress, and demonstrated that oxidative stress dependent markers inhibit the production and suggested the idea of resveratrol could prevent or halt the damage in trabecular meshwork in patients with primary open angle glaucoma. In our study, the experimental model of glaucoma was created and RGCs were counted. This study is the first study showing the neuroprotective effect of resveratrol based on the count of the RGCs.

In several studies, the effects of resveratrol are defined.17-19 Recent studies indicate that neuroprotective effect of resveratrol is due to the mechanism of anti-apoptotic regardless of anti-oxidant properties.20 Resveratrol plays a role in agonist of Sirtulins (SIRT protein, silent information regular two) from Histone deacetylase family.21, 22 It has been reported that Sirtülin-1 activators (like resveratrol) have revealed neuroprotective activity in models of rat optic neuritis, and multiple sclerosis23. Ganglion cell death is multi-factorial and drugs with different mechanism of action are possible to demonstrate additive effects. Chen et al.24 investigated the effect of memantine and tea polyphenols in order to protect mouse brain cells from excitotoxicity and found that combined use is more effective. RGCs die at different times is an idea to explain increasing vision and visual field loss in glaucoma.25 This may show some RGCs are more sensitive than others. It is yet unclear to express it as “Some RGCs begin to die before some others” or

6

“All RGCs begin to die at the same time, but their functionality remains at different rates”. In fact, as fast as these considerations that are important in terms of progression of glaucoma get clear, and as much as knowledge on RGCs death we have, it will be easier to develop a neuroprotectant agent. Because, a good neuroprotectant should slow down this progression. How is this going to happen? Neuroprotectant agent should block retinal neurons’ death directly or indirectly. This indirect effect can be in the form of producing chemicals which disable the damage of other retinal neurons (neuron or glia) to ganglion cells. If the RGCs starts to die at different speeds and at times, and the dead cells damage the other intact cells with secondary damage, neuroprotective agent should aim at preventing this secondary injury. If all RGCs happen to die at the same time, but in reality they die at different speeds, this process of death should be slowed down by neuroprotective substance. It is mentioned that indirect effect can be shown by editing the environmental factors. Normally, they provide transmitters of astrocytes and Muller cells such as the extracellular ion, glucose and other metabolites, water, pH, glutamate and GABA.26 Current evidences make us think of astrocytes and microglia become activated as a result of optic nerve damage and ischemia, and excretes toxic substances (glutamate, D-serine, NO, TNFα and β) into extracellular space. These factors can cause ganglion cell damage27. In our study, small numbers of cells were preserved in late riluzole and late-resveratrol group compared to the early riluzole and early resveratrol group. This shows that the initiation of treatment in the beginning of neuronal damage is important in reducing the progression of the damage. In the experimental optic nerve compression model of Vorwerk et al, memantine, riluzole and nimodipine was started and basal glutamate levels were measured in the vitreous. An inverse relationship was observed between the number of retinal ganglion cells and levels of glutamate. The measurements support that memantine and riluzole block the increase of intravitreal glutamate which is induced by optic nerve damage and have the neuroprotective effect. Lower levels of glutamate in vitreous was found in the group drug was treated a week before, than the damaged and the untreated group. Early drug treatment was supported to have effective protection of neurons as supported in our study28. Late riluzole group can be explained as less effective if the protective effect of these drugs to apoptotic cell death of ganglion cells in chronic glaucoma model will not be accepted. It can be considered that riluzole and resveratrol treat in the early period of secondary neuronal damage started can play significant role in preventing apoptosis process and in the late period the treat can protect the intact cells from

7

apoptotic process and slow down or halt the progression of the disease. The interaction of riluzole with other neurotransmitters, secondary messenger systems and ion channels plays a role as well as it inhibits the release of glutamate in the formation of neuroprotective effect29. Currently, although the etiopathogenesis of glaucoma is still not fully clarified, it is well known that RGCs and their axons are affected particularly in glaucomatous damage. In our study, GFAP was used as astrocytes marker in optic nerve sections for eyes with glaucoma. The changes in morphology of GFAP-positive astrocytes in these regions in the treatment given groups were noticeably less than that in the glaucoma group and this makes us think of riluzole and resveratrol reduce reactive astrocytes and are effective in preventing the RGCs from the secondary damage. In treated groups, especially RR and ERL groups than in the other groups, more RGCs to be preserved, the death of neurons to be restricted and astrocytes functions to show protective behavior, riluzole and resveratrol can be shown the ideal neuroprotectants for slowing down the progression of glaucomatous optic neuropathy. It can be considered that riluzole and resveratrol treat in the early period of secondary neuronal damage started can play significant role in preventing excitotoxity process and in the late period the treat can protect the intact cells from apoptotic process and slow down or halt the progression of the disease. Our data shows that neuroprotective efficacy of riluzole and resveratrol is significantly higher at the beginning of the glaucoma process. It is obvious that no neuroprotective treatment will have an effect on heavy damage received, or a dead ganglion cell. In our study, compared to the other treatment groups, a small number of cells were preserved in LRL and LRS groups. At this point, the usage of the neuroprotective agents at the beginning of the neuronal damage is seen to be important in reducing the progression and retaining functionality in cells. Because in the early period, the treatment protects the cells maintained at different levels of resistance to the factors that cause damage, and nevertheless, presence of the cells adapted to disturbed conditions, in a smaller number but strong, shows that the treatment applied in the advanced stage can halt the cell death. All of these results seem to support more and more accepted idea of glutamate based excitotoxicity to be effective on neuronal damage in glaucoma. In summary, we have found that riluzole and resveratrol treatment may exert their neuroprotective effects through distinct mechanisms such as reduction of glutamate toxicity, inhibition of the apoptotic cascade, antioxidant properties, and/or enhancing the expression of BDNF. We found a synergistic effect on

8

neuroprotection when both treatments were applied simultaneously. These multimodal drugs have various pharmacologic features that act on several molecules and actually have a potent effect for the treatment of glaucoma as other neurodegenerative diseases. Further experiments are required in order to study in complexity of the direct mechanisms of riluzole and resveratrol on RGCs survival and glia. As in our study, clinical availability of riluzole and resveratrol, the experimental efficacy of which has been shown in glaucoma, will be proven as a result of today’s ongoing large-scale, randomized clinical studies.

9

10