J Mol Neurosci DOI 10.1007/s12031-014-0332-5

The Expression Changes of Myelin and Lymphocyte Protein (MAL) Following Optic Nerve Crush in Adult Rats Retinal Ganglion Cells Yongsheng Huang & Yue Xu & Qiaochu Cheng & Shanshan Yu & Yi Gao & Qinmeng Shu & Cheng Yang & Yuan Sun & Jiawei Wang & Fan Xu & Xiaoling Liang

Received: 1 April 2014 / Accepted: 13 May 2014 # Springer Science+Business Media New York 2014

Abstract Myelin and lymphocyte protein (MAL), a component of compact myelin, is highly expressed in oligodendrocytes and Schwann cells. It has been reported that MAL may play a vital role in the process of neuronal apoptosis following acute spinal cord injury. However, acquaintance regarding its distribution and possible function in the retina is limited. Therefore, in a rodent model of optic nerve crush (ONC), the dynamic changes of MAL in retina was detected. The expression of MAL was mainly located in the retinal ganglion cells (RGCs) and was increased strongly after ONC. The peak of MAL expression appeared on the third day. In addition, there was a concomitant upregulation of active-caspase-3, which also co-localized with MAL in RGCs. Moreover, colocalization of MAL with terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) was detected in RGCs after ONC. Collectively, all these results suggested that the upregulation of MAL might play an important role in the pathophysiology of RGCs after ONC.

Yongsheng Huang and Yue Xu contributed equally to this work Y. Huang : Y. Xu : Q. Cheng : S. Yu : Y. Gao : Y. Sun : J. Wang : F. Xu : X. Liang (*) State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong Province, People’s Republic of China e-mail: [email protected] Q. Shu Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People’s Republic of China C. Yang Department of Ophthalmology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong Province, People’s Republic of China

Keywords Optic nerve crush . MAL . Retinal ganglion cells . Apoptosis . Rats

Introduction Due to the inability of neurons to regenerate, the lesions in the neurons of the central nervous system (CNS) are usually irreparable. Mature retinal ganglion cells (RGCs) are typical CNS neurons and possess only weak intrinsic potential to regrow injured axons (Chen et al. 2000; GrandPre et al. 2000). After optic nerve trauma in adult rats, a rapid and massive reduction of the original population of RGCs is observed, and more than 90 % of the RGCs die within 2 weeks after the injury (Fischer et al. 2000; Nadal-Nicolas et al. 2009; Parrilla-Reverter et al. 2009). Previous studies have shown that optic nerve crush (ONC) can induce RGCs death, and ONC has been used as an animal model to investigate axonal degeneration of the CNS (Allcutt et al. 1984; Castano et al. 1996; Li et al. 1999). The reasons for the death of RGCs after ONC in adult animals are not known. Many studies have demonstrated that neurotrophic factor deprivation (Quigley et al. 2000), oxidative stress (Gupta et al. 2013; Tezel 2006), glutamate-induced excitotoxicity (Sucher et al. 1997), reactive gliosis (Neufeld and Liu 2003), and induction of caspase dependent apoptotic pathway (Joachim et al. 2014; Leung et al. 2008) have been hypothesized to underlie the processes of RGCs loss. The myelin and lymphocyte protein (MAL) was one of the first lipid-raft components that was isolated both in vitro from oligodendrocytes and epithelial cells (Kim et al. 1995; Zacchetti et al. 1995) and in vivo from CNS myelin and kidney membranes (Frank et al. 1998; Schaeren-Wiemers et al. 1995b). T lymphocyte maturation-associated protein is another name for MAL protein, which acts as a role of

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membrane trafficking and signaling in T lymphocytes (Alonso and Millan 2001). Recent studies have shown that MAL has been implicated in several malignancies including esophageal, ovarian, cervical, breast carcinoma, and epithelium cell carcinoma (Buffart et al. 2008; Cao et al. 2010; Horne et al. 2009; Overmeer et al. 2009) and can enhance apoptosis and block at the G1/S transition of cancer cells (Cao et al. 2010; Mimori et al. 2003). Furthermore, MAL is also a nonglycosylated integral membrane protein highly enriched in oligodendrocytes and Schwann cells myelin of CNS and peripheral nervous system (Schaeren-Wiemers et al. 1995a, b). A recent study has shown that MAL might play a vital role in the process of neuronal apoptosis following acute spinal cord injury (SCI) (Zhang et al. 2013). All of the above revealed that MAL might be related to neuronal apoptosis. However, its expression and function in the retina are still not well understood. In this study, significant upregulation of MAL in the ONC model was found. At its peak expression, MAL expressed mainly in RGCs. This pattern of MAL expression coincided well with the phases of the death of RGCs and physically coexisted with active-caspase-3 and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) during ONC. These data were conducted to gain greater insight into the functions of MAL and its roles in the cellular and molecular mechanisms underlying retina injury and repair.

Materials and Methods Animals and Optic Nerve Crush Male Sprague–Dawley rats (10 weeks; Laboratory of Zhongshan Ophthalmic Center, Guangzhou, China) with an average body weight of 250 g (220–275) were used in this study. All animals underwent ONC injury or sham operation in the left eye. ONC was performed as previously described with slight modification (Xu et al. 2013a, b). For ONC, the rats were deeply anesthetized with chloral hydrate (10 % solution) and surgery was performed under aseptic conditions. A conjunctival incision was made over the dorsal aspect of one eye, which was then gently rotated downward in the orbit. The superior and external rectus muscles were removed to expose 3–4 mm of the optic nerve. The epineurium was slit open along the long axis, and the nerve was crushed 2 mm behind the eye by squeezing the forceps for 10 s (Vigneswara et al. 2012). Before wound closure, the retinal perfusion was checked funduscopically. Animals with severe reduction of the perfusion were excluded. Sham operation was done with the same procedures but without crushing the optic nerve.

Experimental Design Ninety-nine SD rats were used in this study. All animals were killed at different survival times after injury. In the ONC groups, no animals were lost before these determined time points. For Western blotting analysis, they were divided into seven groups (n=9 at each group): the control (without ONC), the first day, the second day, the third day, the fifth day, and the seventh day after ONC and the sham-operated group (the third day after sham operation). For TUNEL staining, they were divided into two groups (n=9 at each group): the control (without ONC) and the third day after ONC. For immunofluorescence studies, they were divided into two groups (n=9 at each group): the control (without ONC) and the third day after ONC. All experiments involving animals were carried out in accordance with the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals published by the US National Academy of Sciences and approved by the Administration Committee of Experimental Animals, Guangdong Province, China. Western Blotting Analysis For western blot analysis, eyes were enucleated. Then, the cornea, lens, and vitreous were removed; retina was extruded gently from the eye cup. Total protein was obtained by lysing in a buffer (containing 1 M TrisHCl at pH 7.5, 1 % Triton X-100, 1 % Nonidet p-40, 10 % SDS, 0.5 % sodium deoxycholate, 0.5 M EDTA, 10 lg/ml leupeptin, 10 lg/ml aprotinin, and 1 mM PMSF) and then centrifuged at 10,000 × g for 30 min to collect the supernatant. Protein was separated with sodium dodecyl sulfate-PAGE and transferred to polyvinylidine difluoride filter membranes (Millipore, Bedford, MA). The membranes were incubated overnight with MAL (anti-Rabbit, 1:500; Abcam), or active caspase-3 (anti-mouse, 1:500; Cell Signaling), and βactin (anti-mouse, 1:1,000; Sigma) at 4 °C. At last, the membrane was incubated with second antibody goat-anti-rabbit or goat-anti-mouse conjugated horseradish peroxidase (1:2,000; Southern-Biotech) for 2 h and visualized using an enhanced chemiluminescence system (ECL; Pierce Company, USA). Immunofluorescence After defined survival times, rats were terminally anesthetized and perfused through the ascending aorta with saline, followed by 4 % paraformaldehyde. After perfusion, the sham and injured retina were removed and postfixed in the same fixative for 6 h and then replaced with 20 % sucrose for 1 day, following 30 % sucrose for 2– 3 days. The whole eyeball was embedded in OCT (Sakura

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Finetek, Inc., CA, USA), fast frozen in liquid nitrogen and 7-μm-thick sections of the tissues were cut. The sections were blocked with 1 % BSA (BSA) to avoid unspecific staining. Then, sections were incubated overnight at 4 °C with rabbit primary antibodies for anti-MAL (1:200; Abcam), mouse monoclonal primary antibodies antiNeuN (1:500; Millipore), and goat polyclonal antibody for anti-active caspase-3 (1:500; Cell Signaling). After washing in PBS, a mixture of fluorescein isothiocyanateand Cy3-conjugated secondary antibodies was added. The stained sections were examined with Leica confocal microscope (Germany).

Results

TUNEL Staining

To determine the cellular localization of MAL in retina after ONC at different survival times, we then performed immunohistochemistry experiments on transverse cryosections of the retinal tissues. In normal retina, weakly positive signals of MAL protein could be detected in the GCL and inner nuclear layer (INL) (Fig. 2a). At the third day after ONC, a significant increase in MAL protein was detected in the GCL only (Fig. 2b). To further investigate the cell types expressing MAL after ONC, we used double fluorescence labeling confocal microscope technique with a RGCs-specific marker: NeuN to identify the cell type. We found that positive MAL was both in cytoplasm and nuclei in RGCs, which was consistent with previous studies (Zhang et al. 2013) describing the cellular expression of MAL in neurons (Fig. 2c–e).

TUNEL staining was performed using the In Situ Cell Death Detection Kit, Fluorescence (Millipore, Billerica, MA, USA). Frozen tissue sections were rinsed with PBS and treated with 1 % Triton-100 in PBS for 2 min on ice. Slides were rinsed in PBS and incubated for 60 min at 37 °C with 50 μl of TUNEL reaction mixture. After washing with PBS, the slides were analyzed by Leica confocal microscope (Germany). Quantitative Analysis Cell quantitation in ganglion cell layer (GCL) was performed in an unbiased manner as our previous described (Shu et al. 2014; Xu et al. 2014). To avoid counting the same cell in more than one section, we counted every fifth section (50 μm apart). The number of MALpositive cells in the GCL was counted at ×400 magnification. For each section, three separate GCL regions were examined. The cell counts in the three or four sections were then used to determine the total number of MAL-positive cells per square millimeter. The number of cells double-labeled for MAL and TUNEL used in the experiment was quantified. To identify the proportion of TUNEL-positive cells expressing MAL, a minimum of 200 TUNEL-positive cells were counted in each section. Then, double-labeled cells for MAL and TUNEL were recorded. Statistical Analysis All data were analyzed with Stata 7.0 statistical software. The OD of the immunoreactivity is represented as mean ± SEM one-way ANOVA followed by the Tukey’s post hoc multiple comparison tests was used for statistical analysis. P values