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Effect of geranylgeranylacetone on optic neuritis in experimental autoimmune encephalomyelitis
Neuroscience Letters 462 (2009) 281–285
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Effect of geranylgeranylacetone on optic neuritis in experimental autoimmune encephalomyelitis Xiaoli Guo a , Chikako Harada a , Kazuhiko Namekata a , Kenji Kikushima a , Yoshinori Mitamura b , Hiroshi Yoshida c , Yoh Matsumoto d , Takayuki Harada a,c,∗ a
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba, Japan Department of Neuro-ophthalmology, Tokyo Metropolitan Neurological Hospital, Fuchu, Tokyo, Japan d Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 28 May 2009 Received in revised form 3 July 2009 Accepted 13 July 2009 Keywords: Experimental autoimmune encephalomyelitis Multiple sclerosis Optic neuritis Neuroprotection
a b s t r a c t Optic neuritis is an acute inflammatory demyelinating syndrome of the central nervous system (CNS) that often occurs in multiple sclerosis (MS). Since it can cause irreversible visual loss, especially in the opticspinal form of MS or neuromyelitis optica (NMO), the present study was conducted to assess the effects of geranylgeranylacetone (GGA) on optic neuritis in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Myelin oligodendrocyte glycoprotein-induced EAE mice received oral administration of GGA at 500 mg/kg or vehicle once daily for 22 days. The effects of GGA on the severity of optic neuritis were examined by morphological analysis on day 22. Visual functions were measured by the multifocal electroretinograms (mfERG). In addition, the effects of GGA on severity of myelitis were monitored both on clinical signs and morphological aspects. The visual function, as assessed by the second-kernel of mfERG, was significantly improved in GGA-treated mice compared with vehicle-treated mice. GGA treatment decreased the number of degenerating axons in the optic nerve and prevented cell loss in the retinal ganglion cell layer. However, the severity of demyelination in the spinal cord remained unaffected with the treatment of GGA. These results suggest that oral GGA administration has beneficial effect on the treatment for optic neuritis in the EAE mouse model of MS. © 2009 Elsevier Ireland Ltd. All rights reserved.
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by localized areas of demyelination. Experimental autoimmune encephalomyelitis (EAE) serves as an animal model that recapitulates many features of MS. It can be induced by immunization of susceptible animals with a number of myelin antigen including myelin basic protein (MBP) [33], proteolipid protein (PLP) [6,7,30] and myelin oligodendrocyte glycoprotein (MOG) [8,22]. In EAE, the identity of the target autoantigen at least in part determines the disease phenotype and pattern of lesion distribution in the CNS. For example, immune response to MBP or PLP induces lesions located predominantly in the spinal cord whereas immunization with MOG generates lesions located predominantly in the optic nerve and spinal cord [1]. Optic neuritis is an acute inflammatory demyelinating syndrome of the CNS that often occurs in MS. Since it can cause irreversible visual loss, especially in the optic-spinal form of MS or neuromyelitis
∗ Corresponding author at: Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. Tel.: +81 42 325 3881; fax: +81 42 321 8678. E-mail address: [email protected] (T. Harada). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.07.028
optica (NMO) [11,18,20], and is incurable currently, it is important to find a treatment that will restore the visual function. Recent studies have shown that administration of geranylgeranylacetone (GGA), an acyclic isoprenoid compound, rapidly upregulates the expression of heat shock proteins in variety of tissues [4,17,24,28,29,32] and alleviates polyglutamine-mediated motor neuron disease [19]. In addition, we recently reported that GGA exerts anti-apoptotic effects against ischemic retinal injury in vivo [10]. GGA has been used clinically for the treatment of gastric ulcers with an extremely low toxicity. In the present study, we investigated whether oral administration of GGA exerts a protective effect against optic neuritis and clinical signs in MOG-induced EAE mice. Female C57BL/6J mice were maintained at the animal facilities of the Tokyo Metropolitan Institute for Neuroscience and were 6–8 weeks of age at the time of immunization. Animal treatments were performed in accordance with the Tokyo Metropolitan Institute for Neuroscience Guidelines for the Care and Use of Animals. EAE was induced in mice with rat MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) as previously reported [8]. Briefly, mice were subcutaneously injected with 100 g of MOG35–55 mixed with 500 g of heat-killed Mycobacterium tuberculosis H37RA (Difco,
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Fig. 1. Effect of geranylgeranylacetone on visual response in mice of experimental autoimmune encephalomyelitis. (A) Averaged responses of second-order kernel from six independent animals are demonstrated by three-dimensional plots. The degree of retinal function is presented in the color bar. The higher score (red) indicates highly sensitive visual function and lower score (green) indicates retinal dysfunction. Values are given in nV per square degree (nV/deg2 ). (B) Quantitative analysis of the visual response amplitude in mice of experimental autoimmune encephalomyelitis. The sum of the response amplitudes for each stimulus element was divided by the total area of the visual stimulus. Data are means ± SEM of six independent animals in each group. Note the improved visual function in geranylgeranylacetone (GGA)-treated mice compared with vehicle-treated mice. Asterisk (*) indicates p < 0.001. Values are given in nV per square degree (nV/deg2 ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Detroit, MI) emulsified in complete Freund’s adjuvant. Each mouse also received intraperitoneal injections of 500 ng pertussis toxin (Seikagaku, Tokyo, Japan) immediately and 48 h after the immunization. To determine the effect of GGA, a kind gift from Eisai (Tokyo, Japan), MOG-immunized mice were administrated by oral gavage either GGA (500 mg/kg) or vehicle (5% gum Arabic and 0.2% tocopherol in distilled water) once daily throughout the whole experimental period. Clinical signs were scored daily as follows: 0, no clinical signs; 1, loss of tail tonicity; 2, flaccid tail; 3, impairment of righting reflex; 4, partial hind limb paralysis; 5, complete hind limb paralysis; 6, partial body paralysis; 7, partial forelimb paralysis; 8, complete forelimb paralysis or moribund; and 9, death. The second-order kernel of multifocal electroretinograms (mfERGs), which is a sensitive indicator of inner retinal dysfunction [13], was measured as previously reported [12]. Briefly, on day 22 after immunization, mice were anesthetized by an intraperitoneal injection of a mixture of xylazine (10 mg/kg) and ketamine (25 mg/kg). Pupils were dilated with 0.5% phenylephrine hydrochloride and 0.5% tropicamide. mfERGs were recorded using a VERIS 6.0 system (Electro-Diagnostic Imaging, Redwood City, CA, USA). On day 22 after immunization, mice were anesthetized with diethylether and perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer containing 0.5% picric acid. Eyes, optic nerves and lumbar spinal cords were removed, postfixed and processed as previously reported [8]. Briefly, eyes were embedded in paraffin wax and saggital sections through the optic nerve at the thickness of 7 m were collected and stained with hematoxylin and eosin (HE). To quantify the number of neurons in the ganglion cell layer (GCL) of the retina, cells were counted from one ora serrata through the optic nerve to the other ora serrata. On the other hand, optic nerves were embedded in paraffin wax or Epon812 resin, sectioned and stained with luxol fast blue (LFB) followed by HE. Transversal semithin sections (500 nm) of the
optic nerve were stained with toluidine blue [12]. Spinal cords were embedded in paraffin wax, sectioned at the thickness of 7 m, and stained with LFB followed by HE. Data are presented as mean ± SEM. Student’s t-test was used for statistical analyses and results were considered to be significant at p < 0.05. To assess the effect of GGA on the severity of optic neuritis in MOG-induced EAE mice, we first examined the visual function using mfERGs, an established non-invasive method [12]. Fig. 1A shows the averaged responses of second-order kernel in each group. The visual function in vehicle-treated EAE mice was impaired in all visual fields, which was clearly improved by GGA treatment (Fig. 1A). Furthermore, quantitative analysis revealed that the visual function was significantly improved in GGA-treated mice (7.7 ± 1.0 nV/deg2 ; n = 6) compared with vehicle-treated EAE mice (4.1 ± 0.4 nV/deg2 ; n = 6) (p = 0.0009, Fig. 1B). We next analyzed the histopathology of the optic nerve. In vehicle-treated EAE mice, inflammatory cells and demyelination appeared obviously in the optic nerve lesion, while this effect was clearly mild in GGA-treated mice (Fig. 2A–F). To further analyze morphological changes in the optic nerve, semithin transverse sections were collected and stained with toluidine blue. The degenerating axons in vehicle-treated EAE mice had abnormally dark axonal profiles, and the density of axons through the optic nerve was decreased compared with normal mice (Fig. 2H). In contrast, the number of degenerating axons was clearly reduced in GGAtreated EAE mice compared with the vehicle-treated EAE mice (Fig. 2I). Taken together, these data demonstrate that GGA treatment attenuates EAE-induced optic neuritis in both histological and functional aspects. Since GGA prevents retinal ganglion cell death during ischemic injury [8], GGA may also protect retinal neurons in EAE mice. To examine this hypothesis, we administered vehicle and GGA to EAE mice and performed histological evaluation. EAE induced a mild
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Fig. 2. Effect of geranylgeranylacetone on optic nerves in mice of experimental autoimmune encephalomyelitis. Mice were treated with vehicle or geranylgeranylacetone (GGA) for 22 days after immunization with myelin oligodendrocyte glycoprotein. (A–C) Luxol fast blue (LFB) and hematoxylin and eosin (HE) staining of optic nerves. (D–F) Higher magnifications of (A–C), respectively. (G–I) Toluidine blue staining of semithin transverse sections. Arrows in (H and I) point to the degenerating axons. Bar represents 110 m in A–C, 45 m in D–F and 20 m in G–I.
cell loss in the ganglion cell layer (GCL) in vehicle-treated mice, but such degeneration was not detected in GGA-treated mice (Fig. 3A). Quantitative analysis revealed that the number of surviving cells in the GCL was significantly increased following the GGA treatment (from 363 ± 14 in vehicle-treated mice to 430 ± 10 in GGA-treated
mice; p = 0.004, Fig. 3B). These results suggest that GGA protects retinal ganglion cell death from optic neuritis. We examined the effect of GGA on EAE clinical signs during the course of GGA treatment from 0 to 22 days after the disease induction. All vehicle-treated (n = 12) and GGA-treated (n = 12) mice
Fig. 3. Effect of geranylgeranylacetone on retinal cell loss in mice of experimental autoimmune encephalomyelitis. (A) Histopathology analysis of the retina. (a–c) Hematoxylin and eosin staining of retinal sections in normal, vehicle-treated and geranylgeranylacetone (GGA)-treated mice. (d–f) Higher magnifications of the ganglion cell layers in (a–c), respectively. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Bar represents 100 m in a–c and 70 m in d–f. (B) Quantitative analysis of retinal cell number in mice of experimental autoimmune encephalomyelitis. The numbers of cells in the GCL in vehicle- and GGA-treated mice are presented. Results of five independent animals are presented as means ± SEM. Note the decreased retinal cell loss in GGA-treated mice compared with vehicle-treated mice. Asterisk (*) indicates p < 0.01.
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Fig. 4. Effect of geranylgeranylacetone on myelitis in mice of experimental autoimmune encephalomyelitis. (A) Effect of geranylgeranylacetone (GGA) on clinical scores of EAE mice. Mice were treated with vehicle or GGA for 22 days after immunization with myelin oligodendrocyte glycoprotein. Results of 12 independent animals are presented as means ± SEM. There was no significant difference between vehicle- and GGA-treated mice. (B) Effect of GGA on spinal cords in mice of experimental autoimmune encephalomyelitis. Lateral region of lumbar spinal cords was stained with luxol fast blue (LFB) and hematoxylin and eosin (HE) staining. Arrows indicate infiltrating cells in the white matter. Bar indicates 200 m.
developed EAE with an incidence of 100%, and showed a similar mean disease onset (10.4 ± 1.0 versus 11.0 ± 0.7 days; p = 0.64). In addition, the severity of EAE in GGA-treated mice was comparable with that in vehicle-treated mice (Fig. 4A). We also investigated the histopathology of spinal cord 22 days after EAE induction. The extents of demyelination were mild and numerous inflammatory cell infiltrates were observed in the white matter of both groups (arrows in Fig. 4B). Thus, our data suggest that GGA treatment improved optic neuritis while exerting no effect on EAE clinical signs. In the present study, we demonstrated that oral administration of GGA in EAE mice prevented neural cell death in the GCL and reduced the number of degenerating axons in the optic nerve without affecting the disease onset or clinical signs of EAE. In addition, the results from the second-order kernel of multifocal electroretinograms (mfERGs), which is a sensitive indicator of inner retinal function including retinal ganglion cells [12,13], were significantly improved in GGA-treated EAE mice in vivo. These results are consistent with former findings that GGA exerts protective effect on retinal ganglion cells [10,17]. We previously demonstrated that GGA suppresses both p38 mitogen-activated protein kinase (MAPK) and caspase-3 activation in the ischemic retina [10]. In addition, H2 O2 -induced cell death is attenuated in isolated retinal ganglion cells that lack apoptosis signal-regulating kinase 1 (ASK1) [9]. ASK1 is a MAPK kinase that is activated in response to various stimuli and relays its apoptotic signals to p38 MAPK [16,21]. Consistently, ischemia-induced phosphorylation of p38 MAPK and retinal ganglion cell death were suppressed in ASK1-deficient mice [9]. Together with previous findings that heat shock protein 72 is a negative regulator of ASK1 [25] and GGA induces this stress protein in various retinal cell types [17,29], ASK1-p38 MAPK pathway may be one of the targets of GGA. Since reactive oxygen species, such as superoxide and H2 O2 , are important mediators of MS [5,27], we are currently examining the severity of optic neuritis in ASK1-deficient mice. One point that needs to be addressed is that despite the longterm and high dose administration (500 mg/kg), GGA treatment
did not induce any beneficial effects on the demyelination of the spinal cord. A standard clinical dose of GGA for the treatment of gastric ulcers in human is 150 mg/day [15], which is in a dose range of 1–5 mg/kg for an adult. In rats, pretreatment with a single dose of 600–800 mg/kg of GGA leads to neuroprotection against cerebral infarction and spinal cord injury [3,23,31]. We previously reported that pre-treatment with GGA at 200 mg/kg daily for 7 days prevents retinal cell death from ischemic retinal injury in mice [10]. In the current study, we initially administered a dose of 200 mg/kg for the treatment of EAE mice, however, no beneficial effects on clinical signs were observed (data not shown). Moreover, our 30-day study (500 mg/kg daily) did not demonstrate any improvement of clinical signs in EAE mice (data not shown). Thus, our results suggest that the beneficial effect of GGA is selective for the optic nerve. We currently can not explain the lack of GGA effect on the spinal cord exactly. One possible explanation might be that the optic nerve, which is thinner and shorter than the spinal cord, is more sensitive to the treatment of GGA. Another possibility is that the extent of GGA-dependent upregulation of heat shock proteins is different between the optic nerve and spinal cord. In addition, GGA may induce distinct subtypes of heat shock proteins in different tissues, and some heat shock proteins could cross-react with myelin antigens, resulting in myelin destruction [2]. Currently, corticosteroid is the only treatment option available for acute demyelinating optic neuritis, but future therapies could include neuroprotective agents, strategies to induce remyelination, and optic nerve transplantation [14]. We recently reported that MW01-5-188WH, a novel drug that selectively inhibits glial activation in the central nervous system, is effective in ameliorating the severity of MOG-induced EAE [8,26]. Thus, interfering with the interactions between glial cells might be another potential therapeutic strategy for the treatment of MS/EAE. Although further studies are required to examine its long-term effect, considering the low toxicity, GGA may be suitable for the treatment of optic neuritis, in combination with new drugs including MW01-5-188WH and ASK1 inhibitors.
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Acknowledgements We thank A. Kimura, A. Okumura, K. Nakamura and K. Kohyama for technical assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (X.G., K.N., T.H.); Japan Society for the Promotion of Science for Young Scientists (C.H.); the Welfare and Health Funds from Tokyo Metropolitan Government (Y.M.), the Ministry of Health, Labour, and Welfare of Japan (Y.M., T.H.), the Uehara Memorial Foundation, the Naito Foundation, the Suzuken Memorial Foundation, the Daiwa Securities Health Foundation, the Takeda Science Foundation, and the Japan Medical Association (T.H.). References [1] T. Berger, S. Weerth, K. Kojima, C. Linington, H. Wekerle, H. Lassmann, Experimental autoimmune encephalomyelitis: the antigen specificity of T lymphocytes determines the topography of lesions in the central and peripheral nervous system, Lab. Invest. 76 (1997) 355–364. [2] G. Birnbaum, L. Kotilinek, Immunity to heat shock proteins and neurological disorders of women, Infect. Dis. Obstet. Gynecol. 7 (1999) 39–48. [3] M. Fujiki, Y. Furukawa, H. Kobayashi, T. Abe, K. Ishii, S. Uchida, T. Kamida, Geranylgeranylacetone limits secondary injury, neuronal death, and progressive necrosis and cavitation after spinal cord injury, Brain Res. 1053 (2005) 175–184. [4] M. Fujiki, T. Hikawa, T. Abe, S. Uchida, M. Morishige, K. Sugita, H. Kobayashi, Role of protein kinase C in neuroprotective effect of geranylgeranylacetone, a noninvasive inducing agent of heat shock protein, on delayed neuronal death caused by transient ischemia in rats, J. Neurotrauma 23 (2006) 1164–1178. [5] Y. Gilgun-Sherki, E. Melamed, D. Offen, The role of oxidative stress in the pathogenesis of multiple sclerosis: the need for effective antioxidant therapy, J. Neurol. 251 (2004) 261–268. [6] J.M. Greer, V.K. Kuchroo, R.A. Sobel, M.B. Lees, Identification and characterization of a second encephalitogenic determinant of myelin proteolipid protein (residues 178–191) for SJL mice, J. Immunol. 149 (1992) 783–788. [7] J.M. Greer, R.A. Sobel, A. Sette, S. Southwood, M.B. Lees, V.K. Kuchroo, Immunogenic and encephalitogenic epitope clusters of myelin proteolipid protein, J. Immunol. 156 (1996) 371–379. [8] X. Guo, K. Nakamura, K. Kohyama, C. Harada, H.A. Behanna, D.M. Watterson, Y. Matsumoto, T. Harada, Inhibition of glial cell activation ameliorates the severity of experimental autoimmune encephalomyelitis, Neurosci. Res. 59 (2007) 457–466. [9] C. Harada, K. Nakamura, K. Namekata, A. Okumura, Y. Mitamura, Y. Iizuka, K. Kashiwagi, K. Yoshida, S. Ohno, A. Matsuzawa, K. Tanaka, H. Ichijo, T. Harada, Role of apoptosis signal-regulating kinase 1 in stress-induced neural cell apoptosis in vivo, Am. J. Pathol. 168 (2006) 261–269. [10] C. Harada, K. Nakamura, X. Guo, N. Kitaichi, Y. Mitamura, K. Yoshida, S. Ohno, H. Yoshida, T. Harada, Neuroprotective effect of geranylgeranylacetone against ischemia-induced retinal injury, Mol. Vis. 13 (2007) 1601–1607. [11] T. Harada, T. Ohashi, T. Fukazawa, R. Miyagishi, F. Moriwaka, S. Chin, K. Yoshida, H. Matsuda, Visual function in patients with optic neuritis associated with acute transverse myelopathy in multiple sclerosis, Jpn. J. Ophthalmol. 39 (1995) 290–294. [12] T. Harada, C. Harada, K. Nakamura, H.A. Quah, A. Okumura, K. Namekata, T. Saeki, M. Aihara, H. Yoshida, A. Mitani, K. Tanaka, The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma, J. Clin. Invest. 117 (2007) 1763–1770. [13] S. Hasegawa, A. Ohshima, Y. Hayakawa, M. Takagi, H. Abe, Multifocal electroretinograms in patients with branch retinal artery occlusion, Invest. Ophthalmol. Vis. Sci. 42 (2001) 298–304. [14] S.J. Hickman, C.M. Dalton, D.H. Miller, G.T. Plant, Management of acute optic neuritis, Lancet 60 (2002) 1953–1962.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Effect of geranylgeranylacetone on optic neuritis in experimental autoimmune encephalomyelitis Xiaoli Guo a , Chikako Harada a , Kazuhiko Namekata a , Kenji Kikushima a , Yoshinori Mitamura b , Hiroshi Yoshida c , Yoh Matsumoto d , Takayuki Harada a,c,∗ a
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba, Japan Department of Neuro-ophthalmology, Tokyo Metropolitan Neurological Hospital, Fuchu, Tokyo, Japan d Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 28 May 2009 Received in revised form 3 July 2009 Accepted 13 July 2009 Keywords: Experimental autoimmune encephalomyelitis Multiple sclerosis Optic neuritis Neuroprotection
a b s t r a c t Optic neuritis is an acute inflammatory demyelinating syndrome of the central nervous system (CNS) that often occurs in multiple sclerosis (MS). Since it can cause irreversible visual loss, especially in the opticspinal form of MS or neuromyelitis optica (NMO), the present study was conducted to assess the effects of geranylgeranylacetone (GGA) on optic neuritis in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Myelin oligodendrocyte glycoprotein-induced EAE mice received oral administration of GGA at 500 mg/kg or vehicle once daily for 22 days. The effects of GGA on the severity of optic neuritis were examined by morphological analysis on day 22. Visual functions were measured by the multifocal electroretinograms (mfERG). In addition, the effects of GGA on severity of myelitis were monitored both on clinical signs and morphological aspects. The visual function, as assessed by the second-kernel of mfERG, was significantly improved in GGA-treated mice compared with vehicle-treated mice. GGA treatment decreased the number of degenerating axons in the optic nerve and prevented cell loss in the retinal ganglion cell layer. However, the severity of demyelination in the spinal cord remained unaffected with the treatment of GGA. These results suggest that oral GGA administration has beneficial effect on the treatment for optic neuritis in the EAE mouse model of MS. © 2009 Elsevier Ireland Ltd. All rights reserved.
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by localized areas of demyelination. Experimental autoimmune encephalomyelitis (EAE) serves as an animal model that recapitulates many features of MS. It can be induced by immunization of susceptible animals with a number of myelin antigen including myelin basic protein (MBP) [33], proteolipid protein (PLP) [6,7,30] and myelin oligodendrocyte glycoprotein (MOG) [8,22]. In EAE, the identity of the target autoantigen at least in part determines the disease phenotype and pattern of lesion distribution in the CNS. For example, immune response to MBP or PLP induces lesions located predominantly in the spinal cord whereas immunization with MOG generates lesions located predominantly in the optic nerve and spinal cord [1]. Optic neuritis is an acute inflammatory demyelinating syndrome of the CNS that often occurs in MS. Since it can cause irreversible visual loss, especially in the optic-spinal form of MS or neuromyelitis
∗ Corresponding author at: Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. Tel.: +81 42 325 3881; fax: +81 42 321 8678. E-mail address: [email protected] (T. Harada). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.07.028
optica (NMO) [11,18,20], and is incurable currently, it is important to find a treatment that will restore the visual function. Recent studies have shown that administration of geranylgeranylacetone (GGA), an acyclic isoprenoid compound, rapidly upregulates the expression of heat shock proteins in variety of tissues [4,17,24,28,29,32] and alleviates polyglutamine-mediated motor neuron disease [19]. In addition, we recently reported that GGA exerts anti-apoptotic effects against ischemic retinal injury in vivo [10]. GGA has been used clinically for the treatment of gastric ulcers with an extremely low toxicity. In the present study, we investigated whether oral administration of GGA exerts a protective effect against optic neuritis and clinical signs in MOG-induced EAE mice. Female C57BL/6J mice were maintained at the animal facilities of the Tokyo Metropolitan Institute for Neuroscience and were 6–8 weeks of age at the time of immunization. Animal treatments were performed in accordance with the Tokyo Metropolitan Institute for Neuroscience Guidelines for the Care and Use of Animals. EAE was induced in mice with rat MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) as previously reported [8]. Briefly, mice were subcutaneously injected with 100 g of MOG35–55 mixed with 500 g of heat-killed Mycobacterium tuberculosis H37RA (Difco,
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Fig. 1. Effect of geranylgeranylacetone on visual response in mice of experimental autoimmune encephalomyelitis. (A) Averaged responses of second-order kernel from six independent animals are demonstrated by three-dimensional plots. The degree of retinal function is presented in the color bar. The higher score (red) indicates highly sensitive visual function and lower score (green) indicates retinal dysfunction. Values are given in nV per square degree (nV/deg2 ). (B) Quantitative analysis of the visual response amplitude in mice of experimental autoimmune encephalomyelitis. The sum of the response amplitudes for each stimulus element was divided by the total area of the visual stimulus. Data are means ± SEM of six independent animals in each group. Note the improved visual function in geranylgeranylacetone (GGA)-treated mice compared with vehicle-treated mice. Asterisk (*) indicates p < 0.001. Values are given in nV per square degree (nV/deg2 ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Detroit, MI) emulsified in complete Freund’s adjuvant. Each mouse also received intraperitoneal injections of 500 ng pertussis toxin (Seikagaku, Tokyo, Japan) immediately and 48 h after the immunization. To determine the effect of GGA, a kind gift from Eisai (Tokyo, Japan), MOG-immunized mice were administrated by oral gavage either GGA (500 mg/kg) or vehicle (5% gum Arabic and 0.2% tocopherol in distilled water) once daily throughout the whole experimental period. Clinical signs were scored daily as follows: 0, no clinical signs; 1, loss of tail tonicity; 2, flaccid tail; 3, impairment of righting reflex; 4, partial hind limb paralysis; 5, complete hind limb paralysis; 6, partial body paralysis; 7, partial forelimb paralysis; 8, complete forelimb paralysis or moribund; and 9, death. The second-order kernel of multifocal electroretinograms (mfERGs), which is a sensitive indicator of inner retinal dysfunction [13], was measured as previously reported [12]. Briefly, on day 22 after immunization, mice were anesthetized by an intraperitoneal injection of a mixture of xylazine (10 mg/kg) and ketamine (25 mg/kg). Pupils were dilated with 0.5% phenylephrine hydrochloride and 0.5% tropicamide. mfERGs were recorded using a VERIS 6.0 system (Electro-Diagnostic Imaging, Redwood City, CA, USA). On day 22 after immunization, mice were anesthetized with diethylether and perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer containing 0.5% picric acid. Eyes, optic nerves and lumbar spinal cords were removed, postfixed and processed as previously reported [8]. Briefly, eyes were embedded in paraffin wax and saggital sections through the optic nerve at the thickness of 7 m were collected and stained with hematoxylin and eosin (HE). To quantify the number of neurons in the ganglion cell layer (GCL) of the retina, cells were counted from one ora serrata through the optic nerve to the other ora serrata. On the other hand, optic nerves were embedded in paraffin wax or Epon812 resin, sectioned and stained with luxol fast blue (LFB) followed by HE. Transversal semithin sections (500 nm) of the
optic nerve were stained with toluidine blue [12]. Spinal cords were embedded in paraffin wax, sectioned at the thickness of 7 m, and stained with LFB followed by HE. Data are presented as mean ± SEM. Student’s t-test was used for statistical analyses and results were considered to be significant at p < 0.05. To assess the effect of GGA on the severity of optic neuritis in MOG-induced EAE mice, we first examined the visual function using mfERGs, an established non-invasive method [12]. Fig. 1A shows the averaged responses of second-order kernel in each group. The visual function in vehicle-treated EAE mice was impaired in all visual fields, which was clearly improved by GGA treatment (Fig. 1A). Furthermore, quantitative analysis revealed that the visual function was significantly improved in GGA-treated mice (7.7 ± 1.0 nV/deg2 ; n = 6) compared with vehicle-treated EAE mice (4.1 ± 0.4 nV/deg2 ; n = 6) (p = 0.0009, Fig. 1B). We next analyzed the histopathology of the optic nerve. In vehicle-treated EAE mice, inflammatory cells and demyelination appeared obviously in the optic nerve lesion, while this effect was clearly mild in GGA-treated mice (Fig. 2A–F). To further analyze morphological changes in the optic nerve, semithin transverse sections were collected and stained with toluidine blue. The degenerating axons in vehicle-treated EAE mice had abnormally dark axonal profiles, and the density of axons through the optic nerve was decreased compared with normal mice (Fig. 2H). In contrast, the number of degenerating axons was clearly reduced in GGAtreated EAE mice compared with the vehicle-treated EAE mice (Fig. 2I). Taken together, these data demonstrate that GGA treatment attenuates EAE-induced optic neuritis in both histological and functional aspects. Since GGA prevents retinal ganglion cell death during ischemic injury [8], GGA may also protect retinal neurons in EAE mice. To examine this hypothesis, we administered vehicle and GGA to EAE mice and performed histological evaluation. EAE induced a mild
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Fig. 2. Effect of geranylgeranylacetone on optic nerves in mice of experimental autoimmune encephalomyelitis. Mice were treated with vehicle or geranylgeranylacetone (GGA) for 22 days after immunization with myelin oligodendrocyte glycoprotein. (A–C) Luxol fast blue (LFB) and hematoxylin and eosin (HE) staining of optic nerves. (D–F) Higher magnifications of (A–C), respectively. (G–I) Toluidine blue staining of semithin transverse sections. Arrows in (H and I) point to the degenerating axons. Bar represents 110 m in A–C, 45 m in D–F and 20 m in G–I.
cell loss in the ganglion cell layer (GCL) in vehicle-treated mice, but such degeneration was not detected in GGA-treated mice (Fig. 3A). Quantitative analysis revealed that the number of surviving cells in the GCL was significantly increased following the GGA treatment (from 363 ± 14 in vehicle-treated mice to 430 ± 10 in GGA-treated
mice; p = 0.004, Fig. 3B). These results suggest that GGA protects retinal ganglion cell death from optic neuritis. We examined the effect of GGA on EAE clinical signs during the course of GGA treatment from 0 to 22 days after the disease induction. All vehicle-treated (n = 12) and GGA-treated (n = 12) mice
Fig. 3. Effect of geranylgeranylacetone on retinal cell loss in mice of experimental autoimmune encephalomyelitis. (A) Histopathology analysis of the retina. (a–c) Hematoxylin and eosin staining of retinal sections in normal, vehicle-treated and geranylgeranylacetone (GGA)-treated mice. (d–f) Higher magnifications of the ganglion cell layers in (a–c), respectively. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Bar represents 100 m in a–c and 70 m in d–f. (B) Quantitative analysis of retinal cell number in mice of experimental autoimmune encephalomyelitis. The numbers of cells in the GCL in vehicle- and GGA-treated mice are presented. Results of five independent animals are presented as means ± SEM. Note the decreased retinal cell loss in GGA-treated mice compared with vehicle-treated mice. Asterisk (*) indicates p < 0.01.
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Fig. 4. Effect of geranylgeranylacetone on myelitis in mice of experimental autoimmune encephalomyelitis. (A) Effect of geranylgeranylacetone (GGA) on clinical scores of EAE mice. Mice were treated with vehicle or GGA for 22 days after immunization with myelin oligodendrocyte glycoprotein. Results of 12 independent animals are presented as means ± SEM. There was no significant difference between vehicle- and GGA-treated mice. (B) Effect of GGA on spinal cords in mice of experimental autoimmune encephalomyelitis. Lateral region of lumbar spinal cords was stained with luxol fast blue (LFB) and hematoxylin and eosin (HE) staining. Arrows indicate infiltrating cells in the white matter. Bar indicates 200 m.
developed EAE with an incidence of 100%, and showed a similar mean disease onset (10.4 ± 1.0 versus 11.0 ± 0.7 days; p = 0.64). In addition, the severity of EAE in GGA-treated mice was comparable with that in vehicle-treated mice (Fig. 4A). We also investigated the histopathology of spinal cord 22 days after EAE induction. The extents of demyelination were mild and numerous inflammatory cell infiltrates were observed in the white matter of both groups (arrows in Fig. 4B). Thus, our data suggest that GGA treatment improved optic neuritis while exerting no effect on EAE clinical signs. In the present study, we demonstrated that oral administration of GGA in EAE mice prevented neural cell death in the GCL and reduced the number of degenerating axons in the optic nerve without affecting the disease onset or clinical signs of EAE. In addition, the results from the second-order kernel of multifocal electroretinograms (mfERGs), which is a sensitive indicator of inner retinal function including retinal ganglion cells [12,13], were significantly improved in GGA-treated EAE mice in vivo. These results are consistent with former findings that GGA exerts protective effect on retinal ganglion cells [10,17]. We previously demonstrated that GGA suppresses both p38 mitogen-activated protein kinase (MAPK) and caspase-3 activation in the ischemic retina [10]. In addition, H2 O2 -induced cell death is attenuated in isolated retinal ganglion cells that lack apoptosis signal-regulating kinase 1 (ASK1) [9]. ASK1 is a MAPK kinase that is activated in response to various stimuli and relays its apoptotic signals to p38 MAPK [16,21]. Consistently, ischemia-induced phosphorylation of p38 MAPK and retinal ganglion cell death were suppressed in ASK1-deficient mice [9]. Together with previous findings that heat shock protein 72 is a negative regulator of ASK1 [25] and GGA induces this stress protein in various retinal cell types [17,29], ASK1-p38 MAPK pathway may be one of the targets of GGA. Since reactive oxygen species, such as superoxide and H2 O2 , are important mediators of MS [5,27], we are currently examining the severity of optic neuritis in ASK1-deficient mice. One point that needs to be addressed is that despite the longterm and high dose administration (500 mg/kg), GGA treatment
did not induce any beneficial effects on the demyelination of the spinal cord. A standard clinical dose of GGA for the treatment of gastric ulcers in human is 150 mg/day [15], which is in a dose range of 1–5 mg/kg for an adult. In rats, pretreatment with a single dose of 600–800 mg/kg of GGA leads to neuroprotection against cerebral infarction and spinal cord injury [3,23,31]. We previously reported that pre-treatment with GGA at 200 mg/kg daily for 7 days prevents retinal cell death from ischemic retinal injury in mice [10]. In the current study, we initially administered a dose of 200 mg/kg for the treatment of EAE mice, however, no beneficial effects on clinical signs were observed (data not shown). Moreover, our 30-day study (500 mg/kg daily) did not demonstrate any improvement of clinical signs in EAE mice (data not shown). Thus, our results suggest that the beneficial effect of GGA is selective for the optic nerve. We currently can not explain the lack of GGA effect on the spinal cord exactly. One possible explanation might be that the optic nerve, which is thinner and shorter than the spinal cord, is more sensitive to the treatment of GGA. Another possibility is that the extent of GGA-dependent upregulation of heat shock proteins is different between the optic nerve and spinal cord. In addition, GGA may induce distinct subtypes of heat shock proteins in different tissues, and some heat shock proteins could cross-react with myelin antigens, resulting in myelin destruction [2]. Currently, corticosteroid is the only treatment option available for acute demyelinating optic neuritis, but future therapies could include neuroprotective agents, strategies to induce remyelination, and optic nerve transplantation [14]. We recently reported that MW01-5-188WH, a novel drug that selectively inhibits glial activation in the central nervous system, is effective in ameliorating the severity of MOG-induced EAE [8,26]. Thus, interfering with the interactions between glial cells might be another potential therapeutic strategy for the treatment of MS/EAE. Although further studies are required to examine its long-term effect, considering the low toxicity, GGA may be suitable for the treatment of optic neuritis, in combination with new drugs including MW01-5-188WH and ASK1 inhibitors.
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Acknowledgements We thank A. Kimura, A. Okumura, K. Nakamura and K. Kohyama for technical assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (X.G., K.N., T.H.); Japan Society for the Promotion of Science for Young Scientists (C.H.); the Welfare and Health Funds from Tokyo Metropolitan Government (Y.M.), the Ministry of Health, Labour, and Welfare of Japan (Y.M., T.H.), the Uehara Memorial Foundation, the Naito Foundation, the Suzuken Memorial Foundation, the Daiwa Securities Health Foundation, the Takeda Science Foundation, and the Japan Medical Association (T.H.). References [1] T. Berger, S. Weerth, K. Kojima, C. Linington, H. Wekerle, H. Lassmann, Experimental autoimmune encephalomyelitis: the antigen specificity of T lymphocytes determines the topography of lesions in the central and peripheral nervous system, Lab. Invest. 76 (1997) 355–364. [2] G. Birnbaum, L. Kotilinek, Immunity to heat shock proteins and neurological disorders of women, Infect. Dis. Obstet. Gynecol. 7 (1999) 39–48. [3] M. Fujiki, Y. Furukawa, H. Kobayashi, T. Abe, K. Ishii, S. Uchida, T. Kamida, Geranylgeranylacetone limits secondary injury, neuronal death, and progressive necrosis and cavitation after spinal cord injury, Brain Res. 1053 (2005) 175–184. [4] M. Fujiki, T. Hikawa, T. Abe, S. Uchida, M. Morishige, K. Sugita, H. Kobayashi, Role of protein kinase C in neuroprotective effect of geranylgeranylacetone, a noninvasive inducing agent of heat shock protein, on delayed neuronal death caused by transient ischemia in rats, J. Neurotrauma 23 (2006) 1164–1178. [5] Y. Gilgun-Sherki, E. Melamed, D. Offen, The role of oxidative stress in the pathogenesis of multiple sclerosis: the need for effective antioxidant therapy, J. Neurol. 251 (2004) 261–268. [6] J.M. Greer, V.K. Kuchroo, R.A. Sobel, M.B. Lees, Identification and characterization of a second encephalitogenic determinant of myelin proteolipid protein (residues 178–191) for SJL mice, J. Immunol. 149 (1992) 783–788. [7] J.M. Greer, R.A. Sobel, A. Sette, S. Southwood, M.B. Lees, V.K. Kuchroo, Immunogenic and encephalitogenic epitope clusters of myelin proteolipid protein, J. Immunol. 156 (1996) 371–379. [8] X. Guo, K. Nakamura, K. Kohyama, C. Harada, H.A. Behanna, D.M. Watterson, Y. Matsumoto, T. Harada, Inhibition of glial cell activation ameliorates the severity of experimental autoimmune encephalomyelitis, Neurosci. Res. 59 (2007) 457–466. [9] C. Harada, K. Nakamura, K. Namekata, A. Okumura, Y. Mitamura, Y. Iizuka, K. Kashiwagi, K. Yoshida, S. Ohno, A. Matsuzawa, K. Tanaka, H. Ichijo, T. Harada, Role of apoptosis signal-regulating kinase 1 in stress-induced neural cell apoptosis in vivo, Am. J. Pathol. 168 (2006) 261–269. [10] C. Harada, K. Nakamura, X. Guo, N. Kitaichi, Y. Mitamura, K. Yoshida, S. Ohno, H. Yoshida, T. Harada, Neuroprotective effect of geranylgeranylacetone against ischemia-induced retinal injury, Mol. Vis. 13 (2007) 1601–1607. [11] T. Harada, T. Ohashi, T. Fukazawa, R. Miyagishi, F. Moriwaka, S. Chin, K. Yoshida, H. Matsuda, Visual function in patients with optic neuritis associated with acute transverse myelopathy in multiple sclerosis, Jpn. J. Ophthalmol. 39 (1995) 290–294. [12] T. Harada, C. Harada, K. Nakamura, H.A. Quah, A. Okumura, K. Namekata, T. Saeki, M. Aihara, H. Yoshida, A. Mitani, K. Tanaka, The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma, J. Clin. Invest. 117 (2007) 1763–1770. [13] S. Hasegawa, A. Ohshima, Y. Hayakawa, M. Takagi, H. Abe, Multifocal electroretinograms in patients with branch retinal artery occlusion, Invest. Ophthalmol. Vis. Sci. 42 (2001) 298–304. [14] S.J. Hickman, C.M. Dalton, D.H. Miller, G.T. Plant, Management of acute optic neuritis, Lancet 60 (2002) 1953–1962.
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