ISO-1 | Z-ietd-fmk

ISO-1, a macrophage migration inhibitory factor antagonist, prevents N-methyl-D-aspartate-induced retinal damage
Taeko Naruoka a, Tsutomu Nakahara a,n, Yo Tsuda a, Yuki Kurauchi a, Asami Mori a,
Kenji Sakamoto a, Jun Nishihira b, Kunio Ishii a
a Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
b Department of Medical Management and Informatics, Hokkaido Information University, 59-2, Nishi-Nopporo, Ebetsu City, Hokkaido 069-8585, Japan

A R T I C L E I N F O

Article history:
Received 10 June 2013 Received in revised form 26 July 2013
Accepted 26 August 2013
Available online 13 September 2013
Keywords:
Amacrine cells Excitotoxicity Glutamate
Macrophage migration inhibitory factor Neuronal cells

A B S T R A C T

Macrophage migration inhibitory factor (MIF) has been shown to play an important role in a variety of inﬂammatory and immune-mediated diseases. The inﬂammatory responses contribute to retinal neuronal degeneration. However, the role of MIF in the progression of retinal degeneration has not yet been elucidated. In this study, we determined whether pharmacological inhibition of MIF protects against the retinal damage induced by N-methyl-D-aspartate (NMDA) in rats. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer and reduction in the thickness of the inner plexiform layer, (2) an increase in apoptotic cells, (3) a decrease in parvalbumin-positive amacrine cells, (4) accumulation of leukocytes, and (5) microglia activation. Injection of (S,R)-3-(4-hydroxyphenyl)- 4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1, 100 nmol), a MIF antagonist, signiﬁcantly attenuated these NMDA-induced responses. These ﬁndings suggest that ISO-1 exerts protective effects against retinal injuries and that MIF may be a target for neuroprotective intervention in retinal diseases associated with glutamate-induced excitotoxicity.

1. Introduction

Glutamate is an excitatory neurotransmitter in the visual system, as well as in the central nervous system. Glutamate-induced excito- toxicity is implicated in certain retinal diseases, including diabetic retinopathy and glaucoma (Dreyer et al., 1996; Ambati et al., 1997; Pulido et al., 2007). In many cases, the neurotoxic effect of glutamate has been predominantly attributed to excessive stimulation of N-methyl-D-aspartate (NMDA) receptors (Lam et al., 1997; Solberg et al., 1997; Nucci et al., 2005). The mechanisms of NMDA-induced neuronal injury involve excessive Ca2þ inﬂux and subsequent activa- tion of Ca2þ-dependent responses, including formation of nitric oxide (NO) via neuronal NO synthase (Morizane et al., 1997; Vorwerk et al., 1997). In addition, upregulation of pro-inﬂammatory cytokines and inﬂammatory adhesion molecules and recruitment of leukocytes into the retina are involved (Nakazawa et al., 2007; Al-Gayyar et al., 2011). Thus, the excessive activation of NMDA receptors appears to affect retinal neuronal cell survival by indirect as well as direct mechanisms. Macrophage migration inhibitory factor (MIF) was originally discovered as a T lymphocyte-derived factor that inhibits macro- phage migration (David, 1966). It is now recognized that MIF is a multipotent cytokine involved in a variety of biological functions,

n Corresponding author. Tel./fax: þ 81 3 3444 6205.
E-mail address: [email protected] (T. Nakahara).

including pro-inﬂammatory actions and enhancement of immu- nological reactions (Nishihira, 2000; 2012). In the eye, MIF is constitutively expressed in the corneal epithelium and endothe- lium (Matsuda et al., 1996a), iris and ciliary epithelial cells (Matsuda et al., 1996b), and astrocytes, Müller cells, and pigment epithelial cells (Matsuda et al., 1997b). Furthermore, it was demonstrated that MIF is released from the corneal epithelial cells of an injured eye (Matsuda et al., 1997a). These ﬁndings suggest that MIF has physiological and pathological roles in the eye. However, to the best of our knowledge, the roles of MIF in retinal degeneration have not yet been determined.
To assess whether the pharmacological inhibition of MIF prevents the progression of neuronal cell death following intravi- treal injection of NMDA, we used (S,R)-3-(4-hydroxyphenyl)-4,5- dihydro-5-isoxazole acetic acid methyl ester (ISO-1). ISO-1 is a highly speciﬁc inhibitor to the catalytic site of MIF (Al-Abed et al., 2005) and has been shown to reduce the biological function of MIF (Dios et al., 2002; Lubetsky et al., 2002; Al-Abed et al., 2005).

2. Material and methods

2.1. Animals

Male Sprague-Dawley rats weighing 220–240 g were main- tained on standard rat chow and tap water ad libitum in a room

with constant temperature (2272 1C) and humidity (55% 7 5%) and a 12-h light/dark cycle. All animal procedures were performed in accordance with the Association for Research in Vision and
Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research and the Regulations for the Care and Use of Laboratory Animals in Kitasato University adopted by the Institu- tional Animal Care and Use Committee for Kitasato University.

2.2. Treatments

The animals were divided into 3 groups: NMDAþvehicle (n¼ 16), NMDAþ ISO-1 (20 nmol, Calbiochem, San Diego, CA, USA) (n¼ 7), and NMDA ISO-1 (100 nmol) (n 23). Under general anesthesia with 50 mg/kg pentobarbital sodium (Nacalai Tesque, Kyoto, Japan), ISO-1 (20 or 100 nmol) or vehicle (DMSO), mixed with 200 nmol of NMDA (Nacalai Tesque) in a total volume of 5 ml, was injected into the
vitreous cavity of 1 eye. The same volume of DMSO (5 ml) was injected
into the vitreous cavity of the other eye as a control. In a separate experiment (n 4), the effect of intravitreal injection of ISO-1 (100 nmol) alone on the retina was examined. The dose of ISO-1 was selected based on our preliminary studies showing that 100 nmol/eye was a maximum effective dose.

2.3. Assessment of morphological changes

The rats were anesthetized with pentobarbital sodium, and their eyes were enucleated at 7 days after the injection. The eyes were immersed in a ﬁxative mixture (37.5% ethanol, 9.3% formal- dehyde, 12.5% acetic acid, and 3% glutaraldehyde) for 12 h at room temperature as previously reported (Tsuda et al., 2012; Ueda et al., 2013). Fixed retinal tissues were embedded in parafﬁn, and 5-μm cross-sections were cut through the optic disc of the eye. The sections were stained with hematoxylin and eosin. The number of cells in the ganglion cell layer (GCL) was counted at a distance of 1000 to 1250 mm from the center of the optic nerve head on both
sides, and the thickness of the inner plexiform layer (IPL) was
measured in 5 areas approximately 1 mm adjacent to the optic nerve on both sides. The values were averaged for each eye. The data for the treated eye of each animal were normalized to those for the contralateral vehicle-treated control.

2.4. Vascular perfusion

For terminal deoxynucleotidyl transferase-mediated dUTP nick- end labeling (TUNEL) staining and immunostaining, systemic vascular perfusion was performed 6 or 24 h after injections. The rats were deeply anesthetized with pentobarbital sodium. The chest was opened rapidly and the vasculature was perfused for 5 min at a pressure of 120 mmHg with a ﬁxative (1% paraformal- dehyde in phosphate-buffered saline [PBS]; pH 7.4) from an 18- gauge cannula inserted into the aorta via an incision in the left ventricle. The right atrium was incised to create an exit route for the ﬁxative. After perfusion, the eyes were removed and were stored in the ﬁxative for 1 h at 4 1C. The eyes were rinsed several
times with PBS, inﬁltrated overnight with 30% sucrose in PBS at
4 1C, and frozen in an optimal-cutting-temperature (OCT) com- pound (Sakura Finetek, Torrance, CA, USA).

2.5. TUNEL staining

Previous studies have shown that excitotoxic cell death in the retina is mediated through an apoptotic pathway (Lam et al., 1999; Kwong and Lam, 2000; Manabe and Lipton, 2003). To determine whether ISO-1 protects retinal neurons from such cell-death processes, apoptotic cell death was determined using the TUNEL assay. Because TUNEL-positive cells were evident in the GCL

within 6 h of NMDA injection (Manabe and Lipton, 2003), the eyes obtained at 6 h after injections were assessed.
Tissue sections were cut with a cryostat at a thickness of 16 mm and were dried on glass slides. The TUNEL assay was performed according to the manufacturer’s instructions (In Situ Cell Death Detection Kit; Roche Diagnostics, Mannheim, Germany). For nuclear staining, the sections were mounted with VECTASHIELD with 4′,6-
diamidino-2-phenylindole (DAPI)(Vector Laboratories, Burlingame,
CA, USA). Images of the regions of the mid-peripheral retina were obtained from each retinal section by using the BZ-9000 ﬂuorescent microscope system (Keyence, Osaka, Japan). The TUNEL-positive cells were manually counted in the GCL at 1.0–2.0 mm (both sides) from the center of the optic disc. The average number of TUNEL-positive cells/eye was obtained from 3 sections of each retina.
2.6. Immunostaining

For immunohistochemical staining, the tissue sections were rinsed to remove the OCT compound and were subsequently incubated in blocking solution (5% normal hamster serum) in PBS containing 0.3% Triton X-100 for 0.5 to 1 h at room temperature. We used the following antibodies: rabbit polyclonal anti-parvalbumin antibody (1:200, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti- CD45 antibody (1:100, BD Biosciences, San Jose, CA, USA), and rabbit polyclonal anti-Iba1 antibody (1:500, Wako, Osaka, Japan). The tissue sections were incubated with primary antibodies overnight at 4 1C.
After several rinses with 0.3% Triton X-100, the sections were
incubated for 5 h at room temperature with 1 or 2 species-speciﬁc secondary antibodies (1:400; Jackson ImmunoResearch, West Grove, PA) that were diluted in 0.3% Triton X-100. The sections were rinsed in 0.3% Triton X-100 and were mounted with VECTASHIELD with DAPI (Vector Laboratories). The sections incubated in the absence of primary antibodies were used as controls; these sections were processed and evaluated for speciﬁcity or background levels of staining. As described above, images of the regions of the mid- peripheral retina were obtained from each retinal section and the parvalbumin-, CD45-, or Iba1-positive cells were counted. The average number of cells/eye was obtained from 3 sections of each retina.

2.7. Data analysis

The mean values were compared by using one-way analysis of variance followed by the Tukey’s multiple comparison test. P values less than 0.05 were considered statistically signiﬁcant. All values are presented as means 7S.E.M.

3. Results

Morphometric studies of transverse sections revealed that intravitreal injection of NMDA (200 nmol) decreased the cell number in the GCL and the thickness of the inner plexiform layer (IPL) compared to the vehicle-treated retina at 7 days after the injection (Fig. 1Aa and Ab). ISO-1 (100 nmol) signiﬁcantly rescued NMDA-induced cell loss in the GCL (Fig. 1Ad and B) and thinning in the IPL (Fig. 1Ad and C). Although the lower concentration (20 nmol) of ISO-1 showed a tendency to prevent retinal damage (Fig. 1Ac), it did not reach statistical signiﬁcance (Fig. 1B and C). The thicknesses of the inner nuclear layer (INL), the outer plexi- form layer, and the outer nuclear layer were unaffected by the treatments (Fig. 1A). ISO-1 (100 nmol) alone had no signiﬁcant effect on the retinas (cell number in the GCL: control, 7273 cells/ mm vs. ISO-1, 7172 cells/mm [n ¼ 4]; thickness of the IPL: control, 46.771.2 μm vs. ISO-1, 45.372.5 μm [n 4]).
The retinal ganglion cell apoptosis is thought to play an important
role in NMDA-induced retinal injury (Lam et al., 1999; Kwong and Lam, 2000; Manabe and Lipton, 2003). To determine whether ISO-1

Vehicle

NMDA

NMDA
+
ISO -1 (20)

NMDA
+
ISO -1 (100)

GCL

IPL

INL OPL
ONL

NMDA
NMDA + ISO-1 (20) NMDA + ISO-1 (100)

100 100

75 75

50 50

25 25

0 0

Fig. 1. Effects of ISO-1 against retinal damage 7 days after intravitreal injection of N-methyl-D-aspartate (NMDA). A: Vehicle (a); NMDA (200 nmol) (b); NMDA (200 nmol) þ ISO-1 (20 nmol)(c); NMDA (200 nmol) þISO-1 (100 nmol)(d). The scale bar represents 30 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. B, C: Retinal damage was assessed quantitatively by counting the cell number in the GCL and measuring the thickness of the IPL. Data for the treated eye of each animal were normalized to those of the vehicle (saline)-treated control eye. Each column with a vertical bar represents the mean 7
S.E.M. from 7 to 12 animals. nP o 0.05.

(100 nmol) reduces apoptotic cell death resulting from NMDA excito- toxicity, we conducted TUNEL staining of the retina at 6 h after intravitreal injection of NMDA. Few TUNEL-positive cells were observed in the vehicle-treated retinas (Fig. 2Aa), whereas numerous TUNEL-positive cells were found in the GCL at 6 h after NMDA treatment (Fig. 2Ab). Simultaneous treatment with 100 nmol ISO-1 and NMDA signiﬁcantly reduced the number of TUNEL-positive cells in the GCL compared to NMDA treatment alone (Fig. 2Ac and B). These ﬁndings indicate that ISO-1 signiﬁcantly attenuates the NMDA- induced ganglion cell apoptosis.
In addition to ganglion cells, amacrine cells express NMDA re- ceptors in the rat retina (Ng et al., 2004) and excessive activation of NMDA receptors leads to amacrine cell death. Because parvalbumin is mainly expressed in AII amacrine cells of the INL (Wässle et al., 1993; Kim et al., 2010), we next examined the effects of ISO-1 (100 nmol) on NMDA-induced damage to parvalbumin-positive amacrine cells. In the control retina, parvalbumin-positive cells were observed in the inner part of the INL and their processes were found in the IPL (Fig. 3Aa). Some parvalbumin-positive cells were found in the GCL as previously reported (Wässle et al., 1993; Kim et al., 2010). The number of parvalbumin-positive amacrine cells decreased at 24 h after NMDA injection (Fig. 3Ab), but this phenomenon was prevented by simulta- neous injection of ISO-1 and NMDA (Fig. 3Ac and 3B). Thus, ISO-1 exerts a protective effect on amacrine cells.

Finally, the effects of ISO-1 (100 nmol) on the distribution and the number of cells positive for CD45 (a leukocyte marker) and Iba1 (a microglia marker) were determined because the contribution of leukocyte inﬁltration and microglial activation to the progression of retinal neuronal damage has been suggested (Zhang et al., 2005; Nakazawa et al., 2007). In the control retina, Iba1-positive microglia exhibited ramiﬁed morphology (Fig. 4Ad), whereas few CD45-positive cells were observed (Fig. 4Aa). However, at 24 h after NMDA injection, both CD45- and Iba1-positive cells increased abundantly (Fig. 4Ab and Ae) and Iba1-positive cells exhibited a transformation from the ramiﬁed form to an ameboid shape. Some of the CD45-positive cells were co-stained with Iba1 in the NMDA-treated retina (Fig. 4Ah). Simultaneous injection of ISO-1 with NMDA signiﬁcantly decreased the number of CD45-positive cells (Fig. 4Ac and B) and Iba1-positive cells (Fig. 4Af and C). These results indicate that ISO-1 prevents the leukocyte inﬁltration and microglial activation observed in the NMDA- treated retina.

4. Discussion

The present study demonstrates that the MIF antagonist ISO-1 (100 nmol/eye) signiﬁcantly prevented the following changes induced by NMDA (200 nmol/eye):(1) cell loss in the GCL and

Vehicle NMDA

NMDA
+
ISO-1 (100)

GCL IPL
INL OPL
ONL

Vehicle NMDA
NMDA+ISO-1 (100)

Fig. 2. Effects of ISO-1 on the number of TUNEL-positive cells 6 h after intravitreal injection of N-methyl-D-aspartate (NMDA). A: Vehicle (a); NMDA (200 nmol)(b); NMDA (200 nmol) þ ISO-1 (100 nmol)(c). The scale bar represents 50 mm, blue nuclei: DAPI. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. B: Quantitative assessment of the number of TUNEL-positive cells per millimeter in the GCL in each retina. Each column with a vertical bar represents the mean 7 S.E.M. from 5 animals. nP o 0.05.

GCL IPL
INL

Vehicle NMDA

NMDA
+
ISO-1 (100)

75 * *

Vehicle NMDA
NMDA + ISO-1 (100)

0
Fig. 3. Effects of ISO-1 on the number of parvalbumin-expressing neurons 24 h after intravitreal injection of N-methyl-D-aspartate (NMDA). A: Vehicle (a); NMDA (200 nmol) (b); NMDA (200 nmol) þ ISO-1 (100 nmol)(c). The scale bar represents 50 mm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer. B: Quantitative assessment of the number of parvalbumin-expressing cells per millimeter in the INL in each retina. Each column with a vertical bar represents the mean 7 S.E.M. from 5 to 6 animals. nP o 0.05.

Vehicle NMDA

NMDA
+
ISO-1 (100)

GCL IPL INL

1000

750

Vehicle NMDA
NMDA + ISO-1 (100)

1000

750

Vehicle NMDA
NMDA + ISO-1 (100)

50 500

250 250

0 0
Fig. 4. Effects of ISO-1 on the number of CD45-positive cells and Iba1-positive cells 24 h after intravitreal injection of N-methyl-D-aspartate (NMDA). A: Vehicle (a, d and g); NMDA (200 nmol)(b, e and h); NMDA (200 nmol) þISO-1 (100 nmol)(c, f and i). The scale bar represents 50 mm, blue nuclei: DAPI. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer. B, C: Quantitative assessment of the number of CD45-positive cells and Iba1-positive cells present in the GCL and IPL of each retina. Each column with a vertical bar represents the mean 7 S.E.M. from 4 to 6 animals. nP o 0.05.

reduction in IPL thickness, (2) an increase in apoptotic cells, (3) a decrease in parvalbumin-positive amacrine cells, (4) accumulation of leukocytes, and (5) activation of microglia. These results suggest that ISO-1 exerts a protective effect against NMDA-induced retinal damage in rats. The increase in intracellular Ca2þ concentration and subsequent activation of Ca2þ-dependent mechanisms in neurons is thought to be important for the mechanisms of NMDA-induced retinal damage. In addition to such mechanisms, the present study provides evidence suggesting that MIF contri- butes to NMDA-induced retinal damage.
NMDA-induced retinal damage, which includes the loss of retinal ganglion cells and IPL thinning, is commonly observed in the inner retina because NMDA receptors are expressed by the ganglion and amacrine cells in the inner retina (Ng et al., 2004). It was suggested that apoptosis is involved in NMDA-induced retinal neuronal cell death (Lam et al., 1999; Manabe and Lipton, 2003) and TUNEL-positive cells were detected in the GCL and the INL at an early period, between 6 h and 24 h, after intravitreal injection of NMDA. At 6 h after injection, most of the TUNEL-positive cells were located in the GCL (Manabe and Lipton, 2003). Consistent with these results, we observed a signiﬁcant increase in the number of TUNEL-positive cells in the retina, especially the GCL,

at 6 h after NMDA treatment. NMDA-induced apoptosis was signiﬁcantly prevented when ISO-1 was co-administered intravi- treally. Thus, the neuroprotective effect of ISO-1 could be partly attributed to inhibition of apoptosis.
The effects of NMDA on amacrine cells could be evaluated by parvalbumin immunostaining because parvalbumin is mainly expressed in the AII amacrine cells of the INL in the rat retina (Wässle et al., 1993; Kim et al., 2010). The number of parvalbumin immunoreactive cells was reduced in the injured retina after retinal ischemia-reperfusion (Kim et al., 2010) and intravitreal injection of NMDA (Oikawa et al., 2012). Indeed, we found that the number of parvalbumin-positive cells in the INL decreased at 24 h after NMDA treatment, and this reduction was prevented by simultaneous treatment with ISO-1. This ﬁnding suggests that NMDA damages both amacrine cells and ganglion cells and that ISO-1 exerts protective effects in both cell types.
Inﬁltration of leukocytes and activation of microglia are com- monly observed in the injured retina (Szabo et al., 1991; Tsujikawa et al., 1999; Zhang et al., 2005; Chang et al., 2006). Inﬁltrated leukocytes release oxidants and proteases, which induce inﬂam- matory reactions that further increase damage to the injured retina (Szabo et al., 1991; Hangai et al., 1995). Leukocyte adherence

to the vascular endothelium and subsequent inﬁltration into retinal tissues are proposed to be indirect mechanisms that are involved in NMDA-induced retinal damage (Nakazawa et al., 2007). Our present study demonstrated an increase in the CD45- positive cell number at 24 h after NMDA injection, indicating inﬁltration of leukocytes in the retina. The response was attenu- ated by co-injection of ISO-1 with NMDA. Therefore, the reduction of recruited leukocytes in the injured retina by ISO-1 may contribute to the prevention of additional retinal damage.
The microglial cell has been recognized as a “sensor” for pathological events in the central nervous system (Kreutzberg, 1996). In the injured retina, microglial activation characterized by morphologic transformation from the ramiﬁed form to an ame- boid shape has been demonstrated (Zhang et al., 2005). At 24 h after NMDA injection, both the morphologic transformation from the ramiﬁed form to an ameboid shape and an increased number of Iba1-positive cells were observed. Because some of the Iba1- positive cells were co-stained with CD45 in the NMDA-treated retina, the increase in Iba1-positive cells may be partly because of the differentiation of recruited leukocytes to microglia. Microglial cells have been shown to play dual roles in the progression of neurodegenerative disorders (Walter and Neumann, 2009). Although the exact role of microglia in the NMDA-treated retina is presently unclear, ISO-1 can normalize changes in morphology and the number of microglia induced by NMDA.
In the rat retina, MIF expression is localized to astrocytes, Müller cells, and pigment epithelial cells (Matsuda et al., 1997b). It is well known that retinal glial cells and pigment epithelial cells play an active role in inﬂammatory and immunological responses in the retina. Therefore, the constitutive expression of MIF in these cells suggests that this protein contributes to regulation of retinal tissue inﬂammation as well as local immunity. In preliminary studies, we examined the distribution and expression of MIF in rat retinas by using immunohistochemical techniques, and consistent with the study by Matsuda et al. (1997b), found that MIF is expressed in astrocytes, Müller cells, and pigment epithelial cells. The MIF levels in the retina were slightly decreased at 2 h after NMDA treatment, but returned close to the basal level by 24 h. Thus, an almost similar MIF expression pattern was observed in the NMDA-treated retina, indicating that neuronal damage does not alter the pattern of MIF expression in the retina.
In conclusion, we found that the MIF antagonist ISO-1 protects against NMDA-induced excitotoxic retinal injuries in rats. These results suggest that pharmacological interventions that block the signaling pathway involving MIF promote retinal ganglion cell survival under excitotoxic conditions. Glutamate levels are elevated in the eyes of patients with diabetic retinopathy (Ambati et al., 1997; Pulido et al., 2007) or certain types of glaucoma (Dreyer et al., 1996; Dkhissi et al., 1999). The blockade of MIF may be a candidate pharmacological intervention for preventing the development of retinal diseases associated with glutamate-induced excitotoxicity, including glaucoma and diabetic retinopathy.

Acknowledgments

The authors thank Atsuko Ichikawa and Ikumi Hayashi for technical assistance. This study was supported in part by a Grant-in Aid for Scientiﬁc Research (C) (No. 23590112, T.N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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