Rat Optik Sinir Ezme Modelinde Topikal ve Sistemik Alfa Tokoferol Uygulaması Yoluyla Retina Gangliyon Hücre Korunması

1. 1Gazi University Medical Faculty, Department O Ophthalmology, Ankara, Turkey
2. 2Gazi University Medical Faculty, Department Of Pathology, Ankara, Turkey
3. 3Gazi University Faculty Of Pharmacy, Department Of Pharmaceutical Tecnology, Ankara, Turkey
4. 4Kahramanmaras State Hospital, Department Of Ophthalmology, Kahramanmaras, Turkey
5. 5Gazi University Faculty Of Pharmacy, Department Of Biochemistry, Ankara, Turkey
6. 6World Eye Hospital, Ankara, Turkey

Sorumlu Yazar: Vitamin E and its antioxidative properties have been known for years. Various studies including all tissues of the body demonstrated that vitamin E, especially αa-tocopherol is an efficient chain-breaking antioxidant during lipid peroxidation.8,9 It has also antioxidant properties in the eye. It has been reported that vitamin E deficiency might lead to retinal degeneration.10 Antioxidant effects of alpha-, gamma-, and succinate tocopherols were also found to have antioxidant effects in guinea pig retina during ischemia-reperfusion injury.                          Therapeutic effects of antioxidants and nutritional support in ocular diseases have been examined in various studies. Glaucoma is one of these ocular diseases that has been found to be treated by some natural agents to some extent. It is a progressive optic neuropathy presenting with optic nerve excavation and visual field damage clinically. Progressive visual field damage is known to result from progressive RGC apoptosis and death. Major preventable risk factor for the glaucoma progression is elevated IOP. Besides reducing the IOP, neuroprotection is other treatment of choice that aims to prevent progressive RGC death. In a large number of patients, glaucomatous neuropathy progresses in spite of the decreased IOP. Thus, neuroprotection and neuroprotective treatments have gained popularity over the last years. Up to date, various animal models have been used to investigate some neuroprotective treatment strategies. There are various natural compounds available, with neuroprotective properties.12 α-tocopherol is one of these natural compounds that shows neuroprotective effects in some experimental models. Besides its neuroprotective properties, αa-tocopherol was reported to have some antiproliferative effects and to inhibit human Tenon’s capsule fibroblast proliferation.13 It was found to have a positive effect on the success of filtrating surgery in rabbit.14 It was also reported to be protective against cataract formation in animals and also in humans.15,16 Glaucoma, as one of the leading causes of blindness worldwide, usually presented with characteristic appearance of the optic nerve secondary to underlying apoptotic death of RGCs. Although reducing IOP is one of the most important therapeutic targets for the prevention of disease progression, it may not be enough in some cases. Underlying mechanisms of disease progression other than increased IOP have been popular field of research over years. Thus, number of studies investigating therapeutic agents to prevent RGC death have been performed in current literature. To investigate the neuroprotective properties of some therapeutic agents, various animal models simulating slow RGC loss similar to that seen in glaucoma have been designed. Optic nerve crush model is one of these animal models that have been used to provoke slow RGC death. It has been shown that optic nerve crush induces optic nerve injury and causes retrograde degeneration of RGC of the rat.17 Optic nerve crush model is easy to perform and it causes slow RGC loss that is much more similar to the nature of glaucoma than that of optic nerve transection model. Optic nerve damage may be induced by crushing optic nerve with clamps, forceps or other mechanic devices.4 On the other hand, it is often difficult to compare the results of studies obtained by different investigators owing to different methods for measuring IOP and noncomparable pressure levels.4 In the current study, this problem was tried to be overcome to some extend by IOP measurements done by the same author (ZA). Increasing RGC survival is one of the important steps for neuroprotection. Neuroprotection implies prevention of neuronal death secondary to any injury or disease and is also of great importance in the treatment of glaucoma and some neurodegenerative diseases. In the present study, we aimed to investigate the neuroprotective effect of topical αa-tocopherol and compare its efficacy with that of systemic αa-tocopherol in optic nerve crush model. Once an agent said to be neuroprotective in eye, it must initially penetrate into vitreous, keep its concentration for a given period of time, and must have receptors on its target tissues that are expected to be retina and the optic nerve. Since the αa-tocopherol is a liposoluble molecule, its penetrance to the vitreous in topical form might have been difficult. In our study, a-tocopherol was prepared as a nanosuspension with a mean size around 6 nm. Following the preparation of nanosuspension, it was administered topically twice a day. Its presence in the vitreous was demonstrated by HPLC method. High peaks were detected in vitreous samples that was supporting the presence of topically applied αa-tocopherol in vitreous. Microparticular structure of the formulation might have increased the penetration of the αa-tocopherol into the vitreous. Tanito et al18 also demonstrated increased concentration of αa-tocopherol and αa-tocopherol-3 in ocular tissues including neural retina after a 4-day application of 5µL pure αa-tocopherol and αa-tocopherol-3 (2.23±0.14 mg). However, topical a-tocopherol–3 was reported to reach much more drug concentration in ocular tissues than those detected with topical a-tocopherol. We used topical αa-tocopherol at a lower dose but for 45 days. Another study by Nagata et al19 demonstrated that a-tocopherol acetate concentration was increased in aqueous humor and lens after 3 weeks of topical application. In the current study, we demonstrated peaks in chromatogram after the topical administration of αa-tocopherol. Mean vitreous concentration of topically applied αa-tocopherol was 2.27±0.61 µg/mL. We also showed that αa-tocopherol protected RGCs at much more lower concentration than those reported in literature. In our study, α-tocopherol, given in both systemic (Group 1) and topical (Group 3) forms was found to increase RGC survival as determined by RGC count. Furthermore, RGC counts were found to be comparable in Groups 1 and 3 (p=0.216). a-tocopherol is a well-known antioxidant substance. Neuroprotective properties of some natural substances including a-tocopherol have been studied in various studies.12 It was found to be neuroprotective in ischemia-reperfusion models.11,19 On the other hand, in these studies, αa-tocopherol was usually administered intraperitoneally or subcutaneously. Engin et al6 investigated the neuroprotective effect of αa-tocopherol against glaucomatous damage. This was a clinical study in which orally administered αa-tocopherol was given to the patients. The authors reported that resistivity indexes and pulsatility indexes were significantly lower in patient groups in which αa-tocopherol supplementation was given. This study clinically demonstrated the therapeutic impact of αa-tocopherol in the treatment of glaucoma patients. On the other hand, there is no data showing the therapeutic effect of topically applied a-tocopherol in the current literature. Thus, in our experimental study, αa-tocopherol was given both systemically and topically, and the neuroprotective effects of αa-tocopherol in both forms were investigated and compared. We found that αa-tocopherol was neuroprotective in both systemic and topical forms. There is no experimental study demonstrating the neuroprotective effects of topically applied αa-tocopherol on RGCs. It is important to know that it’s also neuroprotective in topical form. There is also no other study investigating the neuroprotective effects of αa-tocopherol in optic nerve crush model of rat. Since it is easier to apply αa-tocopherol topically than systemically in ophthalmic practice, this might be advantageous for the patients who need RGC protection. To summarize, neuroprotective agents have been one of the most important components of the treatment of optic nerve diseases characterized by progressive loss of the RGCs such as glaucoma. There are various natural compounds including αa-tocopherol which were found to be promising in the treatment of glaucoma.12 To the best of our knowledge, this is the first study demonstrating the neuroprotective effects of topically applied αa-tocopherol in comparison with systemic αa-tocopherol. The results of our study clearly indicate that topically applied αa-tocopherol increases the survival of the RGCs as much as systemic αa-tocopherol application. As a strong antioxidant agent, a-tocopherol, may also be used topically for the treatment of glaucoma in the future. Further clinical and prospective studies are needed to demonstrate the neuroprotective effects of topically applied α-tocopherol. Acknowledgements This study was supported by Gazi University, Department of Scientific Investigations, Besevler, Ankara. We also would like to thank Prof. Berati Hasanreisoglu and Prof. Nevin Ozkan for their supports and advices. Address for Correspondence/Yazisma Adresi: Zeynep Aktas MD, Gazi University Medical Faculty, Department of Ophthalmology, Ankara, Turkey Phone: +90 312 202 63 15 E-mail: drzeynep2000@yahoo.com Received/Gelis Tarihi: 01.06.2012 Accepted/Kabul Tarihi: 11.01.2013