Neuroprotective effects of minocycline against in vitro and in vivo retinal ganglion cell damage

doi:10.1016/j.brainres.2005.06.053

Brain Research

Volume 1053, Issues 1–2, 16 August 2005, Pages 185-194

https://doi.org/10.1016/j.brainres.2005.06.053 Get rights and content

Abstract

The purpose of this study was to determine whether minocycline, a semi-synthetic tetracycline derivative, reduces (a) the in vitro neuronal damage occurring after serum deprivation in cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed using E1A virus) and/or (b) the in vivo retinal damage induced by N-methyl-d-aspartate (NMDA) intravitreal injection in mice. In addition, we examined minocycline's putative mechanisms of action against oxidative stress and endoplasmic reticulum (ER) stress. In vitro, retinal damage was induced by 24-h serum deprivation, and cell viability was measured by Hoechst 33342 staining or resazurin reduction assay. In cultures of RGC-5 cells maintained in serum-free medium for up to 24 h, the number of cells undergoing cell death was reduced by minocycline (0.2–20 μM). Serum deprivation resulted in increased oxidative stress, as revealed by an increase in the fluorescence intensity for 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), a reactive oxygen species (ROS) indicator. Minocycline at 2 and 20 μM inhibited this ROS production. However, even at 20 μM minocycline did not inhibit the retinal damage induced by tunicamycin (an ER stress inducer). Furthermore, in mice in vivo minocycline at 90 mg/kg intraperitoneally administered 60 min before an NMDA intravitreal injection reduced the NMDA-induced retinal damage. These findings indicate that minocycline has neuroprotective effects against in vitro and in vivo retinal damage, and that an inhibitory effect on ROS production may contribute to the underlying mechanisms.

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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Findings from this study alone cannot determine whether microglial phagocytosis is beneficial or harmful during neurodegeneration because microglia have no effects on the severity of RGC death in the ONI model (49, 50), which is an acute model. It has been reported that minocycline suppresses RGC death in animal models of glaucoma (66–68), strongly supporting the role of microglial activation in this disease, and microglia–glia and microglia–neuron interactions may play important roles in the survival or death of retinal neurons (69–71). Future studies will include how microglial DOCK8 affects retinal degeneration in a chronic model of glaucoma (72) and photoreceptor degeneration (69, 70) using microglia-specific DOCK8−/− mice.
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Research ReportNeuroprotective effects of minocycline against in vitro and in vivo retinal ganglion cell damage

Abstract

Introduction

Section snippets

Drugs

Retinal ganglion cell line (RGC-5) culture

Effects of minocycline on serum deprivation-induced cell death in RGC-5

Discussion

Exp. Eye Res.

Gen. Pharmacol.

J. Chromatogr. B.

Am. J. Ophthalmol.

Prog. Neurobiol.

Cell

Med. Clin. North Am.

Mol. Brain Res.

Life Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Invest. Dermatol.

Brain Res.

Surv. Ophthalmol.

Biochem. Pharmacol.

FEBS Lett.

Brain Res.

A novel mechanism of action of tetracyclines: effects on nitric oxide synthases

Proc. Natl. Acad. Sci. U. S. A.

Pharmaco-therapeutics of the newer tetracyclines

J. Am. Vet. Med. Assoc.

An investigation of the neuroprotective effects of tetracycline derivatives in experimental models of retinal cell death

Mol. Pharmacol.

Relation between lipophilicity and pharmacological behavior of minocycline, doxycycline, tetracycline, and oxytetracycline in dogs

Antimicrob. Agents Chemother.

Effects of melatonin, vitamin E and octreotide on lipid peroxidation during ischemia-reperfusion in the guinea pig retina

Eur. J. Ophthalmol.

Brain injury, edema, and rescular permeability changes induced by oxygen-derived free radicals

Neurology

Doxycycline

Ther. Drug Monit.

Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease

Proc. Natl. Acad. Sci. U. S. A.

Research Report
Neuroprotective effects of minocycline against in vitro and in vivo retinal ganglion cell damage