Molecular Vision 2007; 13:1601-1607 <http://www.molvis.org/molvis/v13/a178/> Received 20 February 2007 | Accepted 5 September 2007 | Published 7 September 2007

Download Reprint

Neuroprotective effect of geranylgeranylacetone against ischemia-induced retinal injury

Chikako Harada,¹ Kazuaki Nakamura,¹ Xiaoli Guo,¹ Nobuyoshi Kitaichi,² Yoshinori Mitamura,³ Kazuhiko Yoshida,² Shigeaki Ohno,² Hiroshi Yoshida,⁴ Takayuki Harada^1,4
 
 

¹Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, ²Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo, ³Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba, ⁴Department of Neuro-ophthalmology, Tokyo Metropolitan Neurological Hospital, Fuchu, Tokyo, Japan

Correspondence to: Takayuki Harada, Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan; Phone: +81-42-325-3881; FAX: +81-42-321-8678; email: harada@tmin.ac.jp

Abstract

Purpose: This study was conducted to assess the effects of geranylgeranylacetone (GGA) on ischemia-induced retinal injury.

Methods: Adult C57BL/6J mice were given oral treatments of GGA at 200 mg/kg daily for seven days. Ischemic retinal injury was carried out, and the extent of retinal cell death was quantitatively examined after 7 days. Immunohistochemistry for single-stranded DNA, phosphorylated form of p38 mitogen-activated protein kinase (p38 MAPK), and cleaved caspase-3 were performed one day after ischemic injury.

Results: In GGA-treated mice, we found the number of surviving retinal neurons was significantly increased compared with vehicle-treated mice. Ischemia-induced phosphorylation of p38 MAPK, which mediates apoptosis of retinal ganglion cells, was suppressed by GGA treatment. In such retinas, cleaved caspase-3- and single-stranded DNA-positive cells were also decreased compared with vehicle-treated mice.

Conclusions: Oral GGA is a useful treatment for various retinal degenerative diseases that involve ischemic injury.

Introduction

Ischemic retinal injury is implicated in a number of pathological states, such as retinal artery occlusion, glaucoma, and diabetic retinopathy [1-5]. To find a treatment for this condition, it is necessary to understand the molecular regulation following ischemia. Ischemic injury is mainly associated with excessive concentrations of glutamate, which results in overactivation of glutamate receptors such as N-methyl-D-aspartate (NMDA) receptor, and initiates a cascade of events that leads to necrosis as well as apoptosis [6]. Several studies have suggested a possibility that retinal neurons can be protected by glutamate receptor antagonists [2,6,7], selective inhibition of N-acetylated-a-linked-acidic dipeptidase (NAALADase) [8], and activation of glutamate transporters [1,7]. In addition to typical glutamate neurotoxicity, reactive oxygen species, such as superoxide and hydrogen peroxide, are important mediators in damage caused by retinal ischemia [6,9,10]. Under stress conditions, several stress proteins, such as heat shock proteins (HSPs) are increased and function as molecular chaperones that protect native proteins from damage or refold defective polypeptides in an attempt to restore their native conformation [11]. For example, induction of HSPs by hyperthermic preconditioning contribute to retinal protection against NMDA-induced neurotoxicity [12] and light-induced photoreceptor degeneration [13].

Recent studies have shown that administration of geranylgeranylacetone (GGA), an acylic isoprenoid compound, upregulates HSP expression and exerts protective effects on a variety of organs, such as eye, brain, and heart [14-19]. In the retina, intraperitoneal injection of GGA increased HSP72 in the ganglion cell layer (GCL) and protected retinal ganglion cells (RGCs) in a rat model of glaucoma [14]. GGA also induced thioredoxin (Trx) in various tissues [20] that renders protection against ischemic injury [21] and glutamate neurotoxicity [22]. In mouse retina, GGA induced both HSP72 and Trx predominantly in the retinal pigment epithelium layer and protected photoreceptors from light damage [15]. GGA has been found to have an extremely low toxicity and has been clinically used for the treatment of gastric ulcers. In the present study, we investigated whether oral administration of GGA induces neuroprotective effect against ischemic retinal injury.

Methods

Animals

Experiments were performed using eight week-old C57BL/6J mice (CLEA Japan, Tokyo, Japan) in accordance with the ARVO statement for the Use of Animals in Vision Research. Light intensity inside the cages ranged from 100 to 200 lux under 12 light: 12 dark cycle. Food and tap water were given ad libitum.

Oral administration of geranylgeranylacetone

GGA was a gift from Eisai Co., Ltd. (Tokyo, Japan). GGA (200 mg/kg) was used as an emulsion with 5% gum arabic (Kozakai Seiyaku, Tokyo, Japan) and 0.2% tocopherol (Wako, Osaka, Japan). The vehicle contained these reagents except for GGA. All medicines were orally administered to animals dairy for seven days using feeding needles.

Ischemic retinal injury

Ischemia was achieved on the day seven, and the animals were treated essentially as previously described [3,23]. Briefly, we instilled sterile saline into the anterior chamber of the left eye at 110 cm H₂O pressure for 20 min while the right eye served as a non-ischemic control. Seven days after reperfusion, animals were deeply anesthetized with diethylether and perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer containing 0.5% picric acid at room temperature. The eyes were removed and postfixed overnight in the same fixative and then embedded in paraffin. The posterior part was sectioned sagittally at 7 μm thickness through the optic nerve. It was then mounted and stained with hematoxylin and eosin.

Quantification of retinal damage

Ischemic damage was quantified in three ways. First, the thickness of the inner retinal layer (IRL), located between the internal limiting membrane and the interface of the outer plexiform layer and the outer nuclear layer (ONL), were measured at about 0.5 about 1.0 mm from the optic nerve head. Second, in the same sections, the number of neurons in the GCL was counted from one ora serrata through the optic nerve to the other ora serrata. Third, RGCs were retrogradely labeled from the superior colliculus (SC) with the Fluoro-Gold (FG; fluorochrome, Englewood, CO) soon after ischemic injury [23]. Each midbrain was exposed. The skull was removed, using a micro drill, and 2 μl of 4% FG was injected into the SC to mark the RGCs by retrograde axonal transport. Seven days after FG application, the eyes were enucleated and the retinas were detached and prepared as flattened wholemounts in 4% PFA in 0.1 M PBS solution. GCL was examined in whole-mounted retinas with fluorescence microscopy to determine the RGC density. Four standard areas (0.04 mm²) of each retina at the point of 0.1 mm from the optic disc were randomly chosen. Labeled cells were counted by observers blinded to the identity of the mice, and the average number of RGCs/mm² was calculated. For statistical analysis, six animals were used for each vehicle- and GGA-treated group.

Immunohistochemistry

Frozen retinal sections were treated with 0.3% H₂O₂ for blocking endogenous peroxidase. After sections were washed in phosphate-buffered saline (PBS), they were blocked with PBS containing 10% normal goat serum and 0.8% Triton-X 100 for 1 h at room temperature. They were incubated overnight at 4 °C with a rabbit polyclonal antibody single-stranded DNA (ssDNA; 1:100; Dako Kyoto, Japan), phosphorylated form of p38 mitogen-activated protein kinase (p38 MAPK, 1:100; Promega, Madison, WI) or cleaved caspase-3 (R&D system, 1:10000; Minneapolis, MN). The sections were then incubated with goat anti-rabbit immunoglobulins conjugated to peroxidase labeled-dextran polymer (Dako EnVision, Dako) for 30 min and visualized with DAB substrate kit (Dako). Counting of ssDNA- and phosphorylated p38-positive cells was performed by two masked investigators and averaged.

Statistics

Data are presented as mean±SD except as noted. When statistical analysis was performed, Student's t-test was used to estimate the significance of the results. Statistical significance was accepted at p<0.05.

Results

Effect of geranylgeranylacetone on ischemia-induced retinal cell death

Since GGA may prevent neural cell apoptosis in adult retina, we examined the effect of vehicle and GGA during ischemic retinal injury. The morphology of vehicle- and GGA-treated mice was normal (Figure 1A,C). Histological evaluation demonstrated a significant decrease in ischemic damage in GGA-treated mice (Figure 1D) compared with vehicle-treated mice (Figure 1B). The thickness of the IRL after ischemia was decreased to 65±7 μm (n=6) in vehicle-treated mice, but 79±11 μm (n=6) in GGA-treated mice (Figure 2A). Second, the number of surviving cells in the GCL was also increased in GGA-treated mice (405±18/section; n=6) compared with vehicle-treated mice (339±13/section; n=6; Figure 2B). These results suggest that GGA protects inner retinal neurons in the ischemic retina. Since GCL contains RGCs, displaced amacrine cells, and other minor cell types [24], we next carried out retrograde labeling of RGCs with FG and tried to determine the effect of GGA on RGC survival. Figure 3A-H shows the representative results of RGC labeling in vehicle- and GGA-treated mice. Consistent with the results of GCL counting (Figure 2B), the extent of ischemia-induced RGC death seemed to be mild in GGA-treated mice (Figure 3D,H) compared with vehicle-treated mice (Figure 3B,F). Quantitative analysis revealed that FG-labeled (surviving) RGCs after ischemia was significantly increased in GGA-treated mice (83±3%; n=6) compared with vehicle-treated mice (66±2%; n=6, Figure 4). These results demonstrate that GGA has a protective effect on inner retinal neurons, including RGCs, after ischemic injury.

Effect of geranylgeranylacetone on cell death pathway in the ischemic retina

We next analyzed apoptotic cells in the retina by detection of ssDNA-immunopositive cells one day after ischemic injury [25]. Control retinas showed practically no signals in both vehicle- and GGA-treated mice (data not shown). In ischemic retinas, many ssDNA positive cells were observed in vehicle-treated mice (Figure 5A), but the number was significantly decreased (Figure 6) in GGA-treated mice (Figure 5B). Previous studies have suggested a possibility that activation of p38 MAPK pathway leads to ischemia-induced retinal cell apoptosis [23,26,27]. In control retinas, phosphorylated/activated p38-positive cells were absent (data not shown). In contrast, one day after ischemia, immunohistochemical analysis showed many phosphorylated p38-positive cells in the inner retina in vehicle-treated mice (Figure 5C), but the number was apparently decreased (Figure 6) in GGA-treated mice (Figure 5D). We previously demonstrated that ischemia-induced retinal cell apoptosis in the inner retina is executed mainly by caspase-3 [10,23]. We observed cleaved caspase-3-like immunoreactivity in the inner retina in vehicle-treated mice (Figure 5E), but noted they were sparse in GGA-treated mice (Figure 5F). These results suggest that GGA suppressed the activation of p38 MAPK and caspase-3, and prevented ischemia-induced retinal cell apoptosis.

Discussion

In the present study, we demonstrated that oral administration of GGA exerted anti-apoptotic effects against ischemic retinal injury in vivo. In addition, we found that GGA suppressed both p38 MAPK and caspase-3 activation in the ischemic retina. Our results are consistent with former studies describing that activation of p38 induces RGC apoptosis after axotomy of the optic nerve or mediated by glutamate neurotoxicity [26,27]. In addition to typical glutamate neurotoxicity, reactive oxygen species, such as superoxide and hydrogen peroxide, are important mediators of retinal damage caused by ischemia [6,9,10]. We previously demonstrated that hydrogen peroxide-induced cell death is attenuated in isolated RGCs that lack apoptosis signal-regulating kinase 1 (ASK1) [23]. ASK1 is a MAPK kinase kinase that is activated in response to various stimuli and relays its apoptotic signals to p38 [28,29]. Consistently, ischemia-induced phosphorylation of p38 and RGC death were suppressed in ASK1-deficient mice [23]. Together with previous findings that HSP72 is a negative regulator of ASK1 [30], and GGA has been found to induce this stress protein in various retinal cell types [14,15], ASK1-p38 pathway may be one of the targets of GGA. GGA also has been found to induce Trx, another possible factor that negatively regulates ASK1 activity [31], in the inner retina as well as the retinal pigment epithelial layer [15]. Recent studies have suggested the possibility that Trx is a kind of neurotrophic factor and may play a crucial role in maintaining retinal neurons from ischemic injury [21]. Since other trophic factors such as ciliary neurotrophic factor and glial cell line-derived neurotrophic factor supplied from Müller glial cells rescue neural cells [5,32-35], application of GGA as well as Trx, in combination with these trophic factors, may stimulate multiple cellular targets and activate separate mechanisms in order to rescue RGCs during ischemic injury. Further studies determining the effect of GGA on trophic factor productions in RGCs, Müller glial cells and RPE cells, will be needed.

Ischemic injury is now implicated in a number of retinal diseases including glaucoma [1-3,6,7]. There is evidence that HSPs are involved in the glaucomatous eyes of humans [36] and in experimental models of primary open angle glaucoma (POAG) [37]. Ishii et al. demonstrated that intraperitoneal injection of GGA protected RGCs in a rat model of POAG [14].

In addition, we showed that oral administration of GGA also protected RGCs in response to acute ischemic injury. Although further studies are needed to examine its long-term effect, GGA may be available for the treatment of neurodegenerative diseases including glaucoma.

Acknowledgements

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Tokyo Biochemical Research Foundation, Naito Foundation, and Uehara Memorial Foundation.

References

1. Harada T, Harada C, Watanabe M, Inoue Y, Sakagawa T, Nakayama N, Sasaki S, Okuyama S, Watase K, Wada K, Tanaka K. Functions of the two glutamate transporters GLAST and GLT-1 in the retina. Proc Natl Acad Sci U S A 1998; 95:4663-6.

2. Lipton SA. Retinal ganglion cells, glaucoma and neuroprotection. Prog Brain Res 2001; 131:712-8.

3. Harada C, Harada T, Quah HM, Namekata K, Yoshida K, Ohno S, Tanaka K, Parada LF. Role of neurotrophin-4/5 in neural cell death during retinal development and ischemic retinal injury in vivo. Invest Ophthalmol Vis Sci 2005; 46:669-73.

4. Mitamura Y, Harada C, Harada T. Role of cytokines and trophic factors in the pathogenesis of diabetic retinopathy. Current Diabetes Review 2005; 1:73-81.

5. Harada C, Mitamura Y, Harada T. The role of cytokines and trophic factors in epiretinal membranes: involvement of signal transduction in glial cells. Prog Retin Eye Res 2006; 25:149-64.

6. Osborne NN, Casson RJ, Wood JP, Chidlow G, Graham M, Melena J. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog Retin Eye Res 2004; 23:91-147.

7. Harada T, Harada C, Nakamura K, Quah HM, Okumura A, Namekata K, Saeki T, Aihara M, Yoshida H, Mitani A, Tanaka K. The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma. J Clin Invest 2007; 117:1763-70.

8. Harada C, Harada T, Slusher BS, Yoshida K, Matsuda H, Wada K. N-acetylated-alpha-linked-acidic dipeptidase inhibitor has a neuroprotective effect on mouse retinal ganglion cells after pressure-induced ischemia. Neurosci Lett 2000; 292:134-6.

9. Bonne C, Muller A, Villain M. Free radicals in retinal ischemia. Gen Pharmacol 1998; 30:275-80.

10. Harada T, Harada C, Wang YL, Osaka H, Amanai K, Tanaka K, Takizawa S, Setsuie R, Sakurai M, Sato Y, Noda M, Wada K. Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo. Am J Pathol 2004; 164:59-64.

11. Hirsch C, Gauss R, Sommer T. Coping with stress: cellular relaxation techniques. Trends Cell Biol 2006; 16:657-63.

12. Kwong JM, Lam TT, Caprioli J. Hyperthermic pre-conditioning protects retinal neurons from N-methyl-D-aspartate (NMDA)-induced apoptosis in rat. Brain Res 2003; 970:119-30.

13. Barbe MF, Tytell M, Gower DJ, Welch WJ. Hyperthermia protects against light damage in the rat retina. Science 1988; 241:1817-20.

14. Ishii Y, Kwong JM, Caprioli J. Retinal ganglion cell protection with geranylgeranylacetone, a heat shock protein inducer, in a rat glaucoma model. Invest Ophthalmol Vis Sci 2003; 44:1982-92.

15. Tanito M, Kwon YW, Kondo N, Bai J, Masutani H, Nakamura H, Fujii J, Ohira A, Yodoi J. Cytoprotective effects of geranylgeranylacetone against retinal photooxidative damage. J Neurosci 2005; 25:2396-404.

16. Fujiki M, Hikawa T, Abe T, Uchida S, Morishige M, Sugita K, Kobayashi H. 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 2006; 23:1164-78.

17. Yasuda H, Shichinohe H, Kuroda S, Ishikawa T, Iwasaki Y. Neuroprotective effect of a heat shock protein inducer, geranylgeranylacetone in permanent focal cerebral ischemia. Brain Res 2005; 1032:176-82.

18. Ooie T, Takahashi N, Saikawa T, Nawata T, Arikawa M, Yamanaka K, Hara M, Shimada T, Sakata T. Single oral dose of geranylgeranylacetone induces heat-shock protein 72 and renders protection against ischemia/reperfusion injury in rat heart. Circulation 2001; 104:1837-43.

19. Suzuki S, Maruyama S, Sato W, Morita Y, Sato F, Miki Y, Kato S, Katsuno M, Sobue G, Yuzawa Y, Matsuo S. Geranylgeranylacetone ameliorates ischemic acute renal failure via induction of Hsp70. Kidney Int 2005; 67:2210-20.

20. Hirota K, Nakamura H, Masutani H, Yodoi J. Thioredoxin superfamily and thioredoxin-inducing agents. Ann N Y Acad Sci 2002; 957:189-99.

21. Yamamoto M, Ohira A, Honda O, Sato N, Furuke K, Yodoi J, Honda Y. Analysis of localization of adult T-cell leukemia-derived factor in the transient ischemic rat retina after treatment with OP-1206 alpha-CD, a prostaglandin E1 analogue. J Histochem Cytochem 1997; 45:63-70.

22. Inomata Y, Nakamura H, Tanito M, Teratani A, Kawaji T, Kondo N, Yodoi J, Tanihara H. Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina. J Neurochem 2006; 98:372-85.

23. Harada C, Nakamura K, Namekata K, Okumura A, Mitamura Y, Iizuka Y, Kashiwagi K, Yoshida K, Ohno S, Matsuzawa A, Tanaka K, Ichijo H, Harada T. Role of apoptosis signal-regulating kinase 1 in stress-induced neural cell apoptosis in vivo. Am J Pathol 2006; 168:261-9.

24. Perry VH. Evidence for an amacrine cell system in the ganglion cell layer of the rat retina. Neuroscience 1981; 6:931-44.

25. Maeda M, Sugiyama T, Akai F, Jikihara I, Hayashi Y, Takagi H. Single stranded DNA as an immunocytochemical marker for apoptotic change of ischemia in the gerbil hippocampus. Neurosci Lett 1998; 240:69-72.

26. Kikuchi M, Tenneti L, Lipton SA. Role of p38 mitogen-activated protein kinase in axotomy-induced apoptosis of rat retinal ganglion cells. J Neurosci 2000; 20:5037-44.

27. Manabe S, Lipton SA. Divergent NMDA signals leading to proapoptotic and antiapoptotic pathways in the rat retina. Invest Ophthalmol Vis Sci 2003; 44:385-92.

28. Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi M, Matsumoto K, Miyazono K, Gotoh Y. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 1997; 275:90-4.

29. Matsuzawa A, Ichijo H. Stress-responsive protein kinases in redox-regulated apoptosis signaling. Antioxid Redox Signal 2005; 7:472-81.

30. Adams JC. A veterinary-controlled meat and poultry inspection centre in Canada. Vet Rec 1975; 97:473-4.

31. Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, Kawabata M, Miyazono K, Ichijo H. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J 1998; 17:2596-606.

32. Harada T, Harada C, Nakayama N, Okuyama S, Yoshida K, Kohsaka S, Matsuda H, Wada K. Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration. Neuron 2000; 26:533-41.

33. Harada T, Harada C, Kohsaka S, Wada E, Yoshida K, Ohno S, Mamada H, Tanaka K, Parada LF, Wada K. Microglia-Muller glia cell interactions control neurotrophic factor production during light-induced retinal degeneration. J Neurosci 2002; 22:9228-36.

34. Harada C, Harada T, Quah HM, Maekawa F, Yoshida K, Ohno S, Wada K, Parada LF, Tanaka K. Potential role of glial cell line-derived neurotrophic factor receptors in Muller glial cells during light-induced retinal degeneration. Neuroscience 2003; 122:229-35.

35. Harada T, Harada C, Parada LF. Molecular regulation of visual system development: more than meets the eye. Genes Dev 2007; 21:367-78.

36. Tezel G, Hernandez R, Wax MB. Immunostaining of heat shock proteins in the retina and optic nerve head of normal and glaucomatous eyes. Arch Ophthalmol 2000; 118:511-8.

37. Park KH, Cozier F, Ong OC, Caprioli J. Induction of heat shock protein 72 protects retinal ganglion cells in a rat glaucoma model. Invest Ophthalmol Vis Sci 2001; 42:1522-30.