Research ReportNeuroprotective effects of minocycline against in vitro and in vivo retinal ganglion cell damage
Introduction
Retinal ganglion cell (RGC) death—feature common to many ophthalmic disorders (e.g., glaucoma, and central retinal artery or vein occlusion)—may occur via a variety of mechanisms involving, for example, oxidative stress [6], excitatory amino acids [12], nitric oxide [31], apoptosis [27], or endoplasmic reticulum (ER) stress [32]. Importantly, RGC death leads to glaucomatous optic neuropathy and associated visual-field loss.
Glaucoma is the second leading cause of preventable blindness in the industrialized world. While an elevated intraocular pressure (IOP) is the most important risk factor for the development or progression of glaucomatous damage, it should be remembered that it only a risk factor, not the disease itself [34]. Nevertheless, current therapies for glaucoma aim at lowering IOP by reducing the production of aqueous humor in the eye and/or by increasing its outflow from the eye. However, it has been suggested that decreasing IOP is imperfect as a way of preventing the progressive glaucomatous optic nerve damage that occurs in many patients. Consequently, successful development of a product capable of directly protecting both the retina and the optic nerve from glaucomatous damage would represent a significant advance in the treatment of this sight-threatening disease. Indeed, it is now recognized that therapeutic aims other than a reduction in IOP (such as direct neuroprotection) may be particularly valuable in the treatment of glaucoma. Therefore, an assessment of retina-protecting effects is of clinical interest when evaluating the potential of any new anti-glaucoma drugs.
Minocycline, a highly lipophilic semi-synthetic derivative of tetracycline that is capable of crossing the blood–brain barrier, is able to exert anti-inflammatory actions that are distinct from its ability to inhibit bacterial protein synthesis [2], [20]. Thus minocycline is an exceptional tetracycline derivative in that it exerts biological effects that are separate and distinct from its antimicrobial action. It is clinically well tolerated and in clinical trials has been shown to be safe and efficacious against rheumatoid arthritis [15], [23]. Recently, minocycline has been found to exert broadly protective effects in neurologic disease models, including those for stroke, Huntington's disease, Parkinson's disease, and spinal cord injury featuring cell death [11], [38], [42], [48], [49]. It is currently being evaluated in clinical trials for potential use against amyotrophic lateral sclerosis (ALS) and Parkinson's disease.
However, to our knowledge, the effects of minocycline have not been examined in studies employing retinal ganglion cell cultures and an in vivo model of retinal ganglion cell damage. In the present study, we examined its effects against retinal damage using (a) in vitro serum deprivation-induced neurotoxicity among cultured retinal ganglion cells and (b) in vivo NMDA-induced retinal damage in mice. In addition, we investigated whether its mechanisms of action might include effects against oxidative stress and endoplasmic reticulum (ER) stress.
Section snippets
Drugs
Dulbeco's modified Eagles's medium (D-MEM) was purchased from Sigma-Aldrich (St. Louis, MO). The drugs used and their sources were as follows. Minocycline hydrochloride, trolox (water-soluble Vitamin E), and MK-801 (an NMDA antagonist) were obtained from Sigma-Aldrich. Isoflurane was from Nissan Kagaku (Tokyo, Japan), while fetal bovine serum (FBS) from VALEANT (Costa Mesa, CA).
Retinal ganglion cell line (RGC-5) culture
Cultures of RGC-5 were maintained in D-MEM containing 10% FBS, 100 U/ml penicillin (Meiji Seika Kaisha Ltd., Tokyo,
Effects of minocycline on serum deprivation-induced cell death in RGC-5
Representative fluorescence stainings of nuclei (with Hoechst 33342 and PI dyes) are shown in Fig. 1. Non-treated cells showed normal nuclear morphology and negative staining with PI dye (which stains late-stage apoptotic and necrotic cells) (Figs. 1A, E, and I). Serum deprivation led to shrinkage and condensation of nuclei and positive staining with PI dye (Figs. 1B, F, and J). Treatment with minocycline at 2 μM (Figs. 1C, G, and K) or with trolox at 100 μM (Figs. 1D, H, and L) reduced both
Discussion
In the present study, minocycline at 0.2–20 μM protected retinal ganglion cells (RGC-5; an established transformed rat retinal ganglion cell line) from serum deprivation-induced in vitro retinal damage (Fig. 1, Fig. 2). Trolox (a derivative of vitamin E) at 100 μM also offered such protection, but its effect was weaker than that of minocycline. Furthermore, minocycline at 90 mg/kg intraperitoneally administered 60 min before an NMDA intravitreal injection prevented the in vivo retinal damage
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