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Melanopsin-expressing retinal ganglion cells are relatively resistant to excitotoxicity induced by N-methyl-d -aspartate
Neuroscience Letters 662 (2018) 368–373
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Research article
Melanopsin-expressing retinal ganglion cells are relatively resistant to excitotoxicity induced by N-methyl-D-aspartate Songtao Wanga, Dandan Gua, Peng Zhanga, Jing Chena, Yan Lia, Honglei Xiaoa, Guomin Zhoua,b, a b
T ⁎
Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, PR China Key Laboratory of Medical Imaging Computing and Computer Assisted Intervention of Shanghai, Shanghai 200032, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Melanopsin Retinal ganglion cell Intrinsically photosensitive retinal ganglion cell N-methyl-D-aspartate Excitotoxicity
Excitotoxicity plays an important role in neuronal loss in glaucoma. Previous studies indicate melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are more injury-resistant. However, whether melanopsin-expressing ipRGCs are resistant to N-methyl-D-aspartate (NMDA) induced excitotoxicity is not well understood. In the present study, we investigated the effects of NMDA-induced excitotoxicity in conventional retinal ganglion cells (RGCs) and melanopsin-expressing ipRGCs in adult mice. The loss of RGCs and the reduction of the thickness of inner plexiform layer (IPL) were studied by histology, immunofluorescence, TUNEL assay and optical coherence tomography (OCT). The remaining conventional RGCs and ipRGCs were quantified on the 1st, 3rd, 7th, and 21 st day after NMDA injection using immunofluorescence. NMDA mediated acute and severe damage of conventional RGCs damage in a time-dependent manner, and approximately 85% of the conventional RGCs were lost on the 21 st days. Furthermore, a significant reduction of the IPL thickness was observed. Moreover, compared to the PBS-injected eyes, the density of total melanopsin-positive RGCs decreased by 25% on the 1 st day after NMDA injection, and then the density was constant at other time points. Our results suggest that melanopsin-expressing ipRGCs are relatively resistant to excitotoxicity induced by NMDA.
1. Introduction Excitotoxicity, caused by excessive release of excitatory neurotransmitters such as glutamate and N-methyl-D-aspartate (NMDA), is known to induce neuronal cell death. It is reportedly involved in various diseases of the central nervous system (CNS) including Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's disease, and Alzheimer’s disease (AD) [21,28]. Excitotoxicity is known to play an important role in retinal ischemia/reperfusion injury [17,22] and neuronal loss in glaucoma [9]. NMDA-induced excitotoxicity is thought to be underlie in the pathogenesis of ischemic retinal damage characterized with the death of retinal ganglion cells (RGCs) [5]. Death of RGCs is a common feature of many ophthalmic disorders, such as glaucoma, diabetic retinopathy, and central retinal artery or vein occlusions. Therefore, the animal model of NMDA-induced excitotoxic damage is widely used to study the molecular mechanisms of RGC apoptosis and/or its prevention using
neuroprotective agents. Although different methods are applied to label RGCs including Nissl-staining [31], retrograde tracing technique [29,33] and immunodetection [14,32], each method has its own disadvantages. Retrograde tracing technique is one of the most common methods to label RGCs, and many retrograde tracers have been used such as DiI and Fluoro-Gold (FG). However a critical pitfall of this method is the underestimation of RGC number, and incomplete uptake the tracer resulting in incomplete labeling [30]. Another method is Nissl-staining; however it is difficult to distinguish two main types of retinal neurons in the ganglion cell layer (GCL), the RGCs and displaced amacrine cells. The third method for identifying RGCs is the immunodetection of proteins specifically expressed in RGCs. However, many RGC-specific antigens (Thy-1, γ-synuclein, and Bex1/2) are not suitable for quantification analysis because of the expression sites. Recently, transgenic approaches have been used successfully to identify RGCs. However, this technology is not widely applied in mice [25,26], since there are about
Abbreviations: PD, Parkinson’s disease; MS, multiple sclerosis; ALS, amyotrophic lateral sclerosis; CNS, central nervous system; NMDA, N-methyl-D-aspartate; TUNEL, terminal-deoxynucleoitidyl transferase mediated nick end labeling; ChAT, choline acetyltransferase; RT, room temperature; DAPI, 4 6-diamidino-2-phenylindole; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; TEM, transmission electron microscope; RGC, retinal ganglion cell; OCT, optical coherence tomography; PBS, phosphate buffered saline; MNU, N-methyl-N-nitrosourea; RGCs, retinal ganglion cells; FG, fluoro-gold; ipRGCs, intrinsically photosensitive retinal ganglion cells ⁎ Corresponding author at: Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Shanghai Medical college, Fudan University Shanghai 200032, PR China. E-mail addresses: [email protected], [email protected] (G. Zhou). http://dx.doi.org/10.1016/j.neulet.2017.10.055 Received 9 September 2016; Received in revised form 18 March 2017; Accepted 27 October 2017 Available online 02 November 2017 0304-3940/ © 2017 Elsevier B.V. All rights reserved.
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paraformaldehyde. Following that, the retinas were isolated, and four radial cuts were performed. Subsequently, the retinas were pasted on the nitrocellulose membrane with the GCL upturned and washed 6 times with 0.1 M PBS to remove the paraformaldehyde. Further, the retinas were blocked for 1 h at room temperature (RT; 20–25 °C) and incubated with the appropriate antibodies diluted in blocking buffer (PBS, 5% normal donkey serum, 1% Triton X-100) for 3 days at 4 °C. After 3 washes (10 min, RT), the tissues were incubated for 2 h at RT in fluorophore-conjugated secondary antibodies (Alexa Fluor 594 Donkey anti goat, A11058, Invitrogen; Alexa Fluor 488 Donkey anti rabbit, A21206, Invitrogen) diluted at 1:1000 in the same blocking buffer. Finally, the retinas were thoroughly washed with PBS, mounted onto gelatin-subbed slides, covered with anti-fading solution (Southern biotech). Photographic documentations were accomplished using a confocal laser-scanning microscope.
17–22 morphological distinct ganglion cell populations [34]. Thus, to date, the degree of NMDA-mediated RGCs damage is not well-understood. Intrinsically photosensitive retinal ganglion cells (ipRGCs), a new subtype of RGCs that have been reported to express melanopsin and are intrinsically photosensitive, were discovered more than a decade ago [3]. Studies have shown that ipRGCs are more injury-resistant following optic nerve injury [23], in both several experimental animal models of glaucoma [10,19] and even patients with mitochondrial optic neuropathies, Leber’s hereditary optic neuropathy and dominant optic atrophy [6,15]. A recently published report further pointed out that these cells are resistant to NMDA-induced excitotoxicity [7]. Although the expression levels of Melanopsin (Opn4) mRNA were unchanged after NMDA injection, it still remains unclear whether the cell density of ipRGCs is constant and whether they are selectively spared for a long duration after NMDA injection. To assess the degree of NMDA-mediated RGC damage and the longterm resistance of ipRGCs to excitotoxicity more systematically, we investigated the effect of NMDA injection in mice on RGC loss and tested the sensitivity of melanopsin-expressing cells to NMDA toxicity through histology, immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and optical coherence tomography (OCT). We found that a significant decrease in the thickness of the inner plexiform layer (IPL) and an acute and severe damage in the conventional RGCs after NMDA injection. Meanwhile, melanopsin-expressing RGCs appeared to be relatively resistant to excitotoxicity induced by NMDA.
2.4. Assay of cell apoptosis To detect cell apoptosis, TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche), as per the manufacturer’s instructions. The nuclei were counterstained with DAPI. 2.5. Transmission electron microscopy Mice were sacrificed on the 21 st day after NMDA injection, and the optic nerves were harvested and fixed for 24 h in cacodylate buffer containing 2.5% paraformaldehyde and 2.5% glutaraldehyde. Further, they were rinsed with 0.1 M cacodylate buffer for 1 h, dehydrated through an ascending series of ethanol and propylene oxide, washed, and then embedded in Epon (Sigma, St Louis, MO). Semi-thin (1 mm) and ultra-thin (70 μm) sections were cut using an Ultracut microtome (Leica Microsystems, Wetzlar, Germany) fitted with an appropriate diamond knife. Ultra-thin sections were stained with uranyl acetate and lead citrate at the electron microscopy facility of the Shanghai Medical School of Fudan University. Sections were analyzed using an H700 transmission electron microscope (Hitachi, Tokyo, Japan).
2. Materials and methods 2.1. Animals Male C57BL/6J mice aged 8–12 weeks were housed in standard animal rooms with food and water ad libitum, constant temperature (22 ± 2 °C) and humidity (60% ± 10%), and a 12:12-h light/dark cycle. All experimental procedures described here were carried out in accordance with the Association for Research in Vision and Ophthalmology (ARVO) resolution on the care and use of laboratory animals.
2.6. Optical coherence tomography (OCT) After the experimental animals were anesthetized, the pupils were dilated with cyclopentolate (1%), and mice were placed on a stereotaxic frame. On the 21 st day after NMDA injection, OCT was performed as routine retinal ophthalmic examination and the OCT fundus images were captured.
2.2. Administration of NMDA NMDA was administrated in accordance with the method published previously [30]. Briefly, mice were deeply anesthetized with an intraperitoneal injection of 400 mg/kg chloral hydrate, and the pupil of eye was dilated with tropicamide drops. Further, animals were intravitreally injected with 3 μL of 10 mM NMDA (M3262, Sigma) through a postlimbus spot using a microinjector (Hamilton) under the stereoscopic microscope (SMZ 168, Motic, China). The same volume of 0.01 M sterile phosphate buffered saline (PBS) was injected into the contralateral eye, which was used as a control. There were at least 12 animals in each group. All the procedures were performed under sterile conditions.
2.7. Cell quantification Quantification of Brn3a and melanopsin positive cells was performed by counting labeled cells in eight 581 μm × 581 μm microscopic fields per retina. Two fields were captured in each retinal quadrant. Further, the cell counts for each retina were averaged and converted to cells per mm2. The positive cells were quantified using Image J software.
2.3. Histology and immunofluorescence
2.8. Statistical analyses
Animals were sacrificed on the 1st, 3rd, 7th, and 21 st day after NMDA injection. The eyes were enucleated and immersion fixed for 12 h in a specific fixative provided by the Eye & ENT Hospital of Fudan University and processed for routine paraffin-embedded sectioning protocol. Next, 4 μm serial sections were cut with a rotary microtome (Microm HM 315, Thermo) and stained with hematoxylin and eosin. For staining of whole-mount retinas, immunofluorescence was performed with Brn3a (1:200, sc-31984, Santa Cruz Biotechnology) and melanopsin antibodies (1:1000, PA1-780, Thermo Scientific Pierce, USA). First, the eyes were enucleated and fixed for 10 min in 4%
Statistical analyses were performed using GraphPad Prism 5 software (GraphPadSoftware, SanDiego, CA, USA). Unpaired two-tailed student’s t-test was used. Differences were considered statistically significant when P values were less than 0.05. 3. Results No obvious changes were observed in the retinal structures at various times after PBS injection (Fig. 1A, C, E, G). However, from the light microscopy images of transverse retinal sections on the 1st, 3rd, 7th, 369
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Fig. 1. NMDA-induced acute ganglion cell degeneration in adult mice. Compared to the control group (A, C, E, G), on the 1 st day after NMDA injection, about half of the nuclei in the GCL began to contract (B). Cells in the GCL were decreased significantly with time after intravitreal injection of NMDA (D, F, H), and the thickness of IPL was discernibly reduced. No morphological changes were observed in the control group by H.E staining. NMDA, N-methyl-D-aspartate; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; H.E., hematoxylin and eosin. Scale bar = 100 μm.
showed no significant change. To assess the acute effects of excitotoxicity and the mode of cell death, TUNEL staining was used to label the apoptotic cells on the 1 st day after NMDA injection. We found that no positive cells in the GCL after PBS injection (Fig. 3A), while approximately half of the cells were positive in the GCL (Fig. 3B) in the NMDA-treated group. However, we could not identify whether the RGCs or displaced amacrine cells in the GCL were TUNEL positive. Though the significant loss of displaced amacrine cells after NMDA injection has been reported previously [31], the loss of RGC were not evaluated accurately. Since NMDA injection led to a substantial loss of cells in the GCL, ultrathin cross sections of the optic nerves were observed on the 21 st day after intravitreal injection of PBS (Fig. 3C) or NMDA (Fig. 3D) using transmission electron microscope. Our data demonstrated that NMDA injection resulted in a substantial decrease in the number of axons, especially some large axons. The areas where the axons had degenerated were filled with connective tissue. Degenerating axons, stained with black, were mainly observed in NMDA-treated optic nerves (Fig. 3D). The disintegrated or disintegrating myelin sheaths of the axons were clearly shown. In contrast, the axons of control eyes
and 21 st day after intravitreal injection of NMDA (Fig. 1B, D, F, H), we found that about half of the cells in the GCL showed pyknotic nuclei on the 1 st day (Fig. 1B). Furthermore, cells in the GCL decreased significantly with time after NMDA injection (Fig. 1D, F). Only 10–20% of cells in the GCL remained on the 21 st day after NMDA treatment (Fig. 1H). Moreover, the thickness of the IPL was reduced in a similar manner. Hence, we confirmed that administration of NMDA resulted in a time-dependent loss of cells in the GCL and a significant reduction of the IPL thickness. In addition, obvious alterations were not observed in the other retinal layers. To trace the changes in the retinal layers in vivo, the Micro IV retinal imaging system was used. The color fundus photographs (Fig. 2A-B) and corresponding retinal OCT images (Fig. 2C-D) were obtained from the eyes on the 21 st day after NMDA injection. Compared to the retinas in the PBS-treated group, a defective GCL appeared on the fundus photographs of the NMDA-treated mice. Furthermore, a significantly reduction of the IPL thickness was observed in the retinas of the NMDAtreated mice (30.84 ± 2.33 μm in NMDA group versus 50.6 ± 1.06 μm in PBS group). Hence, the IPL thickness declined by 40% (Fig. 2E). However, the thickness of the other retinal layers 370
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Fig. 2. OCT images simultaneously acquired from a mouse retina in vivo on the 21 st day after NMDA or PBS injection. (A, B) OCT fundus image; (C, D) OCT B-scan image. The retinal damage was quantitatively assessed by measuring the thickness of the IPL from the OCT images on the 21 st day after NMDA or PBS injection (E). Data in each column with a vertical bar represents the mean ± standard error of mean (SEM) from five animals. ***P < 0.0001. OCT, optical coherence tomography; NMDA, Nmethyl-D-aspartate; PBS, phosphate buffer saline; IPL, inner plexiform layer. In C and D, scale bar = 50 μm.
appeared to be healthy and were still filled with neurofilaments, which were surrounded by myelin sheaths (Fig. 3C). To identify and assess the loss of RGCs, two specific and reliable markers (Brn3a and melanopsin) were selected to label the conventional RGCs and melanopsin-expressing ipRGCs. The total number of remaining RGCs was quantified in the retinal whole-mounts at different time points after NMDA-injection. We found that the number of Brn3apositive cells decreased significantly (1749 ± 84.43 cells/mm2) in NMDA-treated retinas on the 1 st day after injection (Fig. 4B) compared to control retinas (3700 ± 110.9 qcells/mm2, n = 5, P < 0.0001)
(Fig. 4A). This indicated that about half of the Brn3a-positive RGCs were damaged within one day after NMDA-treatment. The reduction in cells continued on the 3rd, and 7th day after NMDA injection (Fig. 4D, F). We observed fewer Brn3a-positive cells (462.3 ± 102.9 cells/mm2 in NMDA group versus 3183 ± 89.52 cells/mm2 in PBS group) in NMDA-injected retinas (Fig. 4H) than in the control retinas (Fig. 4G) on the 21 st day after NMDA-injection. This indicated that approximately 85% of the Brn3a-positive RGCs were decreased with a significant difference (n = 5, P < 0.0001) (Fig. 4I). However, the quantification of melanopsin-positive cells showed Fig. 3. TUNEL staining of retinas and electron microscopy of ultrathin sections though the optic nerve after intravitreal injection of PBS (A, C) or NMDA (B, D). No TUNEL-positive cells were observed in the retina on the 1 st day after PBS injection (A); about half of the cells in the GCL were TUNEL-positive after NMDA injection (B). On the 21 st day, the axons from control eyes appeared to be healthy and were filled with neurofilaments, which surrounded by myelin sheaths (C). However, NMDA injection led to a substantial decrease in the number of axons (D). Asterisks: degenerated axons; arrows: healthy axons. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; NMDA, N-methyl-D-aspartate; PBS, phosphate buffer saline; GCL: ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. In A and B, sacle bar = 50 μm; in C and D, sacle bar = 2 μm.
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that the degree of NMDA-mediated ipRGCs damage was less than that of Brn3a-positive RGCs. About one-fourth of the melanopsin-expressing ipRGCs were reduced on the 1 st day after NMDA injection (36.35 ± 4.10 cells/mm2 in NMDA group versus 48.84 ± 1.98 cells/ mm2 in PBS group) with a significant difference (n = 5, P < 0.01). In contrast, on the 3rd, 7th, and 21 st day after NMDA injection, the number of the melanopsin-expressing RGCs was constant compared to control retinas without significant difference (P > 0.05) (Fig. 4J). Furthermore, our preliminary results showed that there were no significant changes in the soma sizes and dendritic fields of melanopsinexpressing ipRGCs after NMDA injection. The melanopsin antibody we used in this study was PA1-780, and it primarily labels the M1 ipRGCs in the mouse retina [1]. This suggests that melanopsin-expressing ipRGCs have greater resistance towards the excitotoxicity induced by NMDA than Brn3a-positive RGCs. 4. Discussion In the present study, we showed that the excitotoxicity induced by NMDA causes acute and severe damage of conventional RGCs and a significant decrease in IPL thickness in mice. However, melanopsinexpressing ipRGCs were relatively resistant to NMDA excitotoxicity. We used previously published methods, and some of the results were consistent with the works of Seitz and Tamm [30]. However, the sequent RGC injury was not evaluated and other subtypes of RGCs were not mentioned in their studies. Our results are important because the dynamic changes in the cells in the GCL at different time points after NMDA injection are shown, and the two remaining subtypes of RGCs were quantified. Thus, this study provides a complete overview of the NMDA-mediated RGC damage and direct evidence of the resistance of ipRGCs. NMDA-induced cell apoptosis in murine retina has been reported several decades ago [16,31]; however, the loss of the cells in the GCL could not represent the degree of damage in RGCs due to equally numerous population of both RGCs and displaced amacrine cells located in the GCL [8,13]. Besides, a recent morphological study has shown that there are more than 20 subtypes of ganglion cells in the mouse retina [34]. It is difficult to distinguish these subtypes of RGCs with a simple method such as immunodetection. Therefore, systematic and accurate evaluation of the RGC death still needs to be studied. We labeled conventional RGCs on retinal whole-mounts using a reliable marker, Brn3a, which is convenient for quantitative analysis of conventional RGCs. Siliprandi et al. [31] found that a single intravitreal injection of different doses of NMDA (20–200 nM) in the adult rat retina resulted in a dose-dependent loss of cells in the GCL, and a dose-dependent decrease of retinal choline acetyltransferase (ChAT) activity; however, the RGCs were not identified and quantified. Our data indicate that a single injection of NMDA (30 nM) in the adult mouse retina caused a time-dependent loss of RGCs in the GCL. Counting the total number of the surviving RGC axons in the optic nerves was also a feasible way to evaluate RGC damage. However, how to identify the axons of different subtypes of RGCs, such as conventional RGCs and ipRGCs, is still a barrier for researchers. Less than 5% of the total number of RGCs in the retina are ipRGCs, a remarkably rare subpopulation of RGCs. Recent morphological and functional studies indicated that there are at least six subtypes of ipRGCs, namely M1-M6 [2,24,35]. M1 cells have a noticeably high level of melanopsin immunoreactivity among the ipRGC subtypes [1,12]. Furthermore, they are reported to be resistant to death in several injury and disease models, including axotomized mouse RGCs and chronic ocular hypertension [18,27]. Although DeParis et al. (2012) have also reported the resistance of ipRGCs against NMDA-induced excitotoxicity in mice, the changes in the density of the surviving Brn3a-positive cells and melanopsin-expressing cells were not shown. It is well known that the mRNA expression level does not represent the change in protein level. A previous study also showed that melanopsin expression was
Fig. 4. Melanopsin-positive cells are relatively resistant to excitotoxicity induced by NMDA. Representative photomicrographs from retinal whole-mounts showing double labeling for Brn3a-positive (red) and melanopsin positive (green) cells are shown at different time points after NMDA (B, D, F, H) or PBS injection (A, C, E, G). Brn3a-positive cells decreased significantly in NMDA-treated retinas compared to control retinas. I-J: quantification of I: Brn3a-positive cells (I) and J: melanopsin-positive cells (J) in retinal whole-mounts at different time points after NMDA or PBS injection. Values are mean ± standard error of mean (SEM); n = 5. *P < 0.05, ***P < 0.0001. Two tailed student’s t test was used to test statistical significance. Scale bar = 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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reduced by 83% after N-methyl-N-nitrosourea (MNU) treatment; however, about two-thirds of melanopsin-expressing cells survived [4]. Hence, it is necessary to assess the resistance of ipRGCs to excitotoxicity from the level of melanopsin protein. Our data provides morphological evidence that melanopsin-expressing RGCs were relatively resistant to NMDA-induced excitotoxicity, compared to the conventional RGCs. Currently, we have no clear explanation for the mechanism of the resistance. The PI3K/AKT pathway may play a role in ipRGC survival after optic nerve transection [19], whereas the resistance to NMDAinduced excitotoxicity does not appear to depend on PI3K/AKT or JAK/ STAT signaling [7]. We believe that ipRGCs might have their own intrinsic strength to survive. It is unlikely that a single molecular mechanism is responsible for the resistance of ipRGCs to several pathological situations. NMDA-induced excitotoxicity is predominately mediated by various glutamate receptors [11,16,20]; hence, in the future, investigating the differential expression of different types of glutamate receptors in ipRGCs could be helpful to reveal the mechanisms that underlie in vivo protection of ipRGCs against NMDA excitotoxicity. In conclusion, we showed that a single injection of NMDA-mediated acute and severe damage of conventional RGCs in a time-dependent manner. Furthermore, a significant reduction was found in the thickness of the IPL after NMDA injection. Moreover, our results suggested that compared to conventional RGCs, melanopsin-expressing RGCs are relatively resistant to excitotoxicity for a long duration after NMDA injection. Conflict of interest The authors declare no financial conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (31571238). We gratefully acknowledge the assistance of Dr. Min Wang at Eye & ENT Hospital of Fudan University for offering the fixtive. We also thank Professor Xiangtian Zhou and Shijun Weng for their technical assistance. References [1] S.B. Baver, G.E. Pickard, P.J. Sollars, G.E. Pickard, Two types of melanopsin retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic nucleus and the olivary pretectal nucleus, Eur. J. Neurosci. 27 (2008) 1763–1770. [2] D.M. Berson, A.M. Castrucci, I. Provencio, Morphology and mosaics of melanopsinexpressing retinal ganglion cell types in mice, J. Comp. Neurol. 518 (2010) 2405–2422. [3] D.M. Berson, F.A. Dunn, M. Takao, Phototransduction by retinal ganglion cells that set the circadian clock, Science 295 (2002) 1070–1073. [4] D.L. Boudard, J. Mendoza, D. Hicks, Loss of photic entrainment at low illuminances in rats with acute photoreceptor degeneration, Eur. J. Neurosci. 30 (2009) 1527–1536. [5] G.H. Bresnick, Excitotoxins: a possible new mechanism for the pathogenesis of ischemic retinal damage, Arch. Ophthalmol. 107 (1989) 339–341. [6] Q. Cui, C. Ren, P.J. Sollars, G.E. Pickard, K.F. So, The injury resistant ability of melanopsin-expressing intrinsically photosensitive retinal ganglion cells, Neuroscience 284 (2015) 845–853. [7] S. DeParis, C. Caprara, C. Grimm, Intrinsically photosensitive retinal ganglion cells are resistant to N-methyl-D-aspartic acid excitotoxicity, Mol. Vis. 18 (2012) 2814–2827. [8] U.C. Drager, J.F. Olsen, Ganglion cell distribution in the retina of the mouse, Invest. Ophthalmol. Vis. Sci. 20 (1981) 285–293. [9] E.B. Dreyer, D. Zurakowski, R.A. Schumer, S.M. Podos, S.A. Lipton, Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma, Arch.
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Research article
Melanopsin-expressing retinal ganglion cells are relatively resistant to excitotoxicity induced by N-methyl-D-aspartate Songtao Wanga, Dandan Gua, Peng Zhanga, Jing Chena, Yan Lia, Honglei Xiaoa, Guomin Zhoua,b, a b
T ⁎
Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, PR China Key Laboratory of Medical Imaging Computing and Computer Assisted Intervention of Shanghai, Shanghai 200032, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Melanopsin Retinal ganglion cell Intrinsically photosensitive retinal ganglion cell N-methyl-D-aspartate Excitotoxicity
Excitotoxicity plays an important role in neuronal loss in glaucoma. Previous studies indicate melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are more injury-resistant. However, whether melanopsin-expressing ipRGCs are resistant to N-methyl-D-aspartate (NMDA) induced excitotoxicity is not well understood. In the present study, we investigated the effects of NMDA-induced excitotoxicity in conventional retinal ganglion cells (RGCs) and melanopsin-expressing ipRGCs in adult mice. The loss of RGCs and the reduction of the thickness of inner plexiform layer (IPL) were studied by histology, immunofluorescence, TUNEL assay and optical coherence tomography (OCT). The remaining conventional RGCs and ipRGCs were quantified on the 1st, 3rd, 7th, and 21 st day after NMDA injection using immunofluorescence. NMDA mediated acute and severe damage of conventional RGCs damage in a time-dependent manner, and approximately 85% of the conventional RGCs were lost on the 21 st days. Furthermore, a significant reduction of the IPL thickness was observed. Moreover, compared to the PBS-injected eyes, the density of total melanopsin-positive RGCs decreased by 25% on the 1 st day after NMDA injection, and then the density was constant at other time points. Our results suggest that melanopsin-expressing ipRGCs are relatively resistant to excitotoxicity induced by NMDA.
1. Introduction Excitotoxicity, caused by excessive release of excitatory neurotransmitters such as glutamate and N-methyl-D-aspartate (NMDA), is known to induce neuronal cell death. It is reportedly involved in various diseases of the central nervous system (CNS) including Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's disease, and Alzheimer’s disease (AD) [21,28]. Excitotoxicity is known to play an important role in retinal ischemia/reperfusion injury [17,22] and neuronal loss in glaucoma [9]. NMDA-induced excitotoxicity is thought to be underlie in the pathogenesis of ischemic retinal damage characterized with the death of retinal ganglion cells (RGCs) [5]. Death of RGCs is a common feature of many ophthalmic disorders, such as glaucoma, diabetic retinopathy, and central retinal artery or vein occlusions. Therefore, the animal model of NMDA-induced excitotoxic damage is widely used to study the molecular mechanisms of RGC apoptosis and/or its prevention using
neuroprotective agents. Although different methods are applied to label RGCs including Nissl-staining [31], retrograde tracing technique [29,33] and immunodetection [14,32], each method has its own disadvantages. Retrograde tracing technique is one of the most common methods to label RGCs, and many retrograde tracers have been used such as DiI and Fluoro-Gold (FG). However a critical pitfall of this method is the underestimation of RGC number, and incomplete uptake the tracer resulting in incomplete labeling [30]. Another method is Nissl-staining; however it is difficult to distinguish two main types of retinal neurons in the ganglion cell layer (GCL), the RGCs and displaced amacrine cells. The third method for identifying RGCs is the immunodetection of proteins specifically expressed in RGCs. However, many RGC-specific antigens (Thy-1, γ-synuclein, and Bex1/2) are not suitable for quantification analysis because of the expression sites. Recently, transgenic approaches have been used successfully to identify RGCs. However, this technology is not widely applied in mice [25,26], since there are about
Abbreviations: PD, Parkinson’s disease; MS, multiple sclerosis; ALS, amyotrophic lateral sclerosis; CNS, central nervous system; NMDA, N-methyl-D-aspartate; TUNEL, terminal-deoxynucleoitidyl transferase mediated nick end labeling; ChAT, choline acetyltransferase; RT, room temperature; DAPI, 4 6-diamidino-2-phenylindole; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; TEM, transmission electron microscope; RGC, retinal ganglion cell; OCT, optical coherence tomography; PBS, phosphate buffered saline; MNU, N-methyl-N-nitrosourea; RGCs, retinal ganglion cells; FG, fluoro-gold; ipRGCs, intrinsically photosensitive retinal ganglion cells ⁎ Corresponding author at: Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Shanghai Medical college, Fudan University Shanghai 200032, PR China. E-mail addresses: [email protected], [email protected] (G. Zhou). http://dx.doi.org/10.1016/j.neulet.2017.10.055 Received 9 September 2016; Received in revised form 18 March 2017; Accepted 27 October 2017 Available online 02 November 2017 0304-3940/ © 2017 Elsevier B.V. All rights reserved.
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paraformaldehyde. Following that, the retinas were isolated, and four radial cuts were performed. Subsequently, the retinas were pasted on the nitrocellulose membrane with the GCL upturned and washed 6 times with 0.1 M PBS to remove the paraformaldehyde. Further, the retinas were blocked for 1 h at room temperature (RT; 20–25 °C) and incubated with the appropriate antibodies diluted in blocking buffer (PBS, 5% normal donkey serum, 1% Triton X-100) for 3 days at 4 °C. After 3 washes (10 min, RT), the tissues were incubated for 2 h at RT in fluorophore-conjugated secondary antibodies (Alexa Fluor 594 Donkey anti goat, A11058, Invitrogen; Alexa Fluor 488 Donkey anti rabbit, A21206, Invitrogen) diluted at 1:1000 in the same blocking buffer. Finally, the retinas were thoroughly washed with PBS, mounted onto gelatin-subbed slides, covered with anti-fading solution (Southern biotech). Photographic documentations were accomplished using a confocal laser-scanning microscope.
17–22 morphological distinct ganglion cell populations [34]. Thus, to date, the degree of NMDA-mediated RGCs damage is not well-understood. Intrinsically photosensitive retinal ganglion cells (ipRGCs), a new subtype of RGCs that have been reported to express melanopsin and are intrinsically photosensitive, were discovered more than a decade ago [3]. Studies have shown that ipRGCs are more injury-resistant following optic nerve injury [23], in both several experimental animal models of glaucoma [10,19] and even patients with mitochondrial optic neuropathies, Leber’s hereditary optic neuropathy and dominant optic atrophy [6,15]. A recently published report further pointed out that these cells are resistant to NMDA-induced excitotoxicity [7]. Although the expression levels of Melanopsin (Opn4) mRNA were unchanged after NMDA injection, it still remains unclear whether the cell density of ipRGCs is constant and whether they are selectively spared for a long duration after NMDA injection. To assess the degree of NMDA-mediated RGC damage and the longterm resistance of ipRGCs to excitotoxicity more systematically, we investigated the effect of NMDA injection in mice on RGC loss and tested the sensitivity of melanopsin-expressing cells to NMDA toxicity through histology, immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and optical coherence tomography (OCT). We found that a significant decrease in the thickness of the inner plexiform layer (IPL) and an acute and severe damage in the conventional RGCs after NMDA injection. Meanwhile, melanopsin-expressing RGCs appeared to be relatively resistant to excitotoxicity induced by NMDA.
2.4. Assay of cell apoptosis To detect cell apoptosis, TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche), as per the manufacturer’s instructions. The nuclei were counterstained with DAPI. 2.5. Transmission electron microscopy Mice were sacrificed on the 21 st day after NMDA injection, and the optic nerves were harvested and fixed for 24 h in cacodylate buffer containing 2.5% paraformaldehyde and 2.5% glutaraldehyde. Further, they were rinsed with 0.1 M cacodylate buffer for 1 h, dehydrated through an ascending series of ethanol and propylene oxide, washed, and then embedded in Epon (Sigma, St Louis, MO). Semi-thin (1 mm) and ultra-thin (70 μm) sections were cut using an Ultracut microtome (Leica Microsystems, Wetzlar, Germany) fitted with an appropriate diamond knife. Ultra-thin sections were stained with uranyl acetate and lead citrate at the electron microscopy facility of the Shanghai Medical School of Fudan University. Sections were analyzed using an H700 transmission electron microscope (Hitachi, Tokyo, Japan).
2. Materials and methods 2.1. Animals Male C57BL/6J mice aged 8–12 weeks were housed in standard animal rooms with food and water ad libitum, constant temperature (22 ± 2 °C) and humidity (60% ± 10%), and a 12:12-h light/dark cycle. All experimental procedures described here were carried out in accordance with the Association for Research in Vision and Ophthalmology (ARVO) resolution on the care and use of laboratory animals.
2.6. Optical coherence tomography (OCT) After the experimental animals were anesthetized, the pupils were dilated with cyclopentolate (1%), and mice were placed on a stereotaxic frame. On the 21 st day after NMDA injection, OCT was performed as routine retinal ophthalmic examination and the OCT fundus images were captured.
2.2. Administration of NMDA NMDA was administrated in accordance with the method published previously [30]. Briefly, mice were deeply anesthetized with an intraperitoneal injection of 400 mg/kg chloral hydrate, and the pupil of eye was dilated with tropicamide drops. Further, animals were intravitreally injected with 3 μL of 10 mM NMDA (M3262, Sigma) through a postlimbus spot using a microinjector (Hamilton) under the stereoscopic microscope (SMZ 168, Motic, China). The same volume of 0.01 M sterile phosphate buffered saline (PBS) was injected into the contralateral eye, which was used as a control. There were at least 12 animals in each group. All the procedures were performed under sterile conditions.
2.7. Cell quantification Quantification of Brn3a and melanopsin positive cells was performed by counting labeled cells in eight 581 μm × 581 μm microscopic fields per retina. Two fields were captured in each retinal quadrant. Further, the cell counts for each retina were averaged and converted to cells per mm2. The positive cells were quantified using Image J software.
2.3. Histology and immunofluorescence
2.8. Statistical analyses
Animals were sacrificed on the 1st, 3rd, 7th, and 21 st day after NMDA injection. The eyes were enucleated and immersion fixed for 12 h in a specific fixative provided by the Eye & ENT Hospital of Fudan University and processed for routine paraffin-embedded sectioning protocol. Next, 4 μm serial sections were cut with a rotary microtome (Microm HM 315, Thermo) and stained with hematoxylin and eosin. For staining of whole-mount retinas, immunofluorescence was performed with Brn3a (1:200, sc-31984, Santa Cruz Biotechnology) and melanopsin antibodies (1:1000, PA1-780, Thermo Scientific Pierce, USA). First, the eyes were enucleated and fixed for 10 min in 4%
Statistical analyses were performed using GraphPad Prism 5 software (GraphPadSoftware, SanDiego, CA, USA). Unpaired two-tailed student’s t-test was used. Differences were considered statistically significant when P values were less than 0.05. 3. Results No obvious changes were observed in the retinal structures at various times after PBS injection (Fig. 1A, C, E, G). However, from the light microscopy images of transverse retinal sections on the 1st, 3rd, 7th, 369
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Fig. 1. NMDA-induced acute ganglion cell degeneration in adult mice. Compared to the control group (A, C, E, G), on the 1 st day after NMDA injection, about half of the nuclei in the GCL began to contract (B). Cells in the GCL were decreased significantly with time after intravitreal injection of NMDA (D, F, H), and the thickness of IPL was discernibly reduced. No morphological changes were observed in the control group by H.E staining. NMDA, N-methyl-D-aspartate; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; H.E., hematoxylin and eosin. Scale bar = 100 μm.
showed no significant change. To assess the acute effects of excitotoxicity and the mode of cell death, TUNEL staining was used to label the apoptotic cells on the 1 st day after NMDA injection. We found that no positive cells in the GCL after PBS injection (Fig. 3A), while approximately half of the cells were positive in the GCL (Fig. 3B) in the NMDA-treated group. However, we could not identify whether the RGCs or displaced amacrine cells in the GCL were TUNEL positive. Though the significant loss of displaced amacrine cells after NMDA injection has been reported previously [31], the loss of RGC were not evaluated accurately. Since NMDA injection led to a substantial loss of cells in the GCL, ultrathin cross sections of the optic nerves were observed on the 21 st day after intravitreal injection of PBS (Fig. 3C) or NMDA (Fig. 3D) using transmission electron microscope. Our data demonstrated that NMDA injection resulted in a substantial decrease in the number of axons, especially some large axons. The areas where the axons had degenerated were filled with connective tissue. Degenerating axons, stained with black, were mainly observed in NMDA-treated optic nerves (Fig. 3D). The disintegrated or disintegrating myelin sheaths of the axons were clearly shown. In contrast, the axons of control eyes
and 21 st day after intravitreal injection of NMDA (Fig. 1B, D, F, H), we found that about half of the cells in the GCL showed pyknotic nuclei on the 1 st day (Fig. 1B). Furthermore, cells in the GCL decreased significantly with time after NMDA injection (Fig. 1D, F). Only 10–20% of cells in the GCL remained on the 21 st day after NMDA treatment (Fig. 1H). Moreover, the thickness of the IPL was reduced in a similar manner. Hence, we confirmed that administration of NMDA resulted in a time-dependent loss of cells in the GCL and a significant reduction of the IPL thickness. In addition, obvious alterations were not observed in the other retinal layers. To trace the changes in the retinal layers in vivo, the Micro IV retinal imaging system was used. The color fundus photographs (Fig. 2A-B) and corresponding retinal OCT images (Fig. 2C-D) were obtained from the eyes on the 21 st day after NMDA injection. Compared to the retinas in the PBS-treated group, a defective GCL appeared on the fundus photographs of the NMDA-treated mice. Furthermore, a significantly reduction of the IPL thickness was observed in the retinas of the NMDAtreated mice (30.84 ± 2.33 μm in NMDA group versus 50.6 ± 1.06 μm in PBS group). Hence, the IPL thickness declined by 40% (Fig. 2E). However, the thickness of the other retinal layers 370
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Fig. 2. OCT images simultaneously acquired from a mouse retina in vivo on the 21 st day after NMDA or PBS injection. (A, B) OCT fundus image; (C, D) OCT B-scan image. The retinal damage was quantitatively assessed by measuring the thickness of the IPL from the OCT images on the 21 st day after NMDA or PBS injection (E). Data in each column with a vertical bar represents the mean ± standard error of mean (SEM) from five animals. ***P < 0.0001. OCT, optical coherence tomography; NMDA, Nmethyl-D-aspartate; PBS, phosphate buffer saline; IPL, inner plexiform layer. In C and D, scale bar = 50 μm.
appeared to be healthy and were still filled with neurofilaments, which were surrounded by myelin sheaths (Fig. 3C). To identify and assess the loss of RGCs, two specific and reliable markers (Brn3a and melanopsin) were selected to label the conventional RGCs and melanopsin-expressing ipRGCs. The total number of remaining RGCs was quantified in the retinal whole-mounts at different time points after NMDA-injection. We found that the number of Brn3apositive cells decreased significantly (1749 ± 84.43 cells/mm2) in NMDA-treated retinas on the 1 st day after injection (Fig. 4B) compared to control retinas (3700 ± 110.9 qcells/mm2, n = 5, P < 0.0001)
(Fig. 4A). This indicated that about half of the Brn3a-positive RGCs were damaged within one day after NMDA-treatment. The reduction in cells continued on the 3rd, and 7th day after NMDA injection (Fig. 4D, F). We observed fewer Brn3a-positive cells (462.3 ± 102.9 cells/mm2 in NMDA group versus 3183 ± 89.52 cells/mm2 in PBS group) in NMDA-injected retinas (Fig. 4H) than in the control retinas (Fig. 4G) on the 21 st day after NMDA-injection. This indicated that approximately 85% of the Brn3a-positive RGCs were decreased with a significant difference (n = 5, P < 0.0001) (Fig. 4I). However, the quantification of melanopsin-positive cells showed Fig. 3. TUNEL staining of retinas and electron microscopy of ultrathin sections though the optic nerve after intravitreal injection of PBS (A, C) or NMDA (B, D). No TUNEL-positive cells were observed in the retina on the 1 st day after PBS injection (A); about half of the cells in the GCL were TUNEL-positive after NMDA injection (B). On the 21 st day, the axons from control eyes appeared to be healthy and were filled with neurofilaments, which surrounded by myelin sheaths (C). However, NMDA injection led to a substantial decrease in the number of axons (D). Asterisks: degenerated axons; arrows: healthy axons. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; NMDA, N-methyl-D-aspartate; PBS, phosphate buffer saline; GCL: ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. In A and B, sacle bar = 50 μm; in C and D, sacle bar = 2 μm.
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that the degree of NMDA-mediated ipRGCs damage was less than that of Brn3a-positive RGCs. About one-fourth of the melanopsin-expressing ipRGCs were reduced on the 1 st day after NMDA injection (36.35 ± 4.10 cells/mm2 in NMDA group versus 48.84 ± 1.98 cells/ mm2 in PBS group) with a significant difference (n = 5, P < 0.01). In contrast, on the 3rd, 7th, and 21 st day after NMDA injection, the number of the melanopsin-expressing RGCs was constant compared to control retinas without significant difference (P > 0.05) (Fig. 4J). Furthermore, our preliminary results showed that there were no significant changes in the soma sizes and dendritic fields of melanopsinexpressing ipRGCs after NMDA injection. The melanopsin antibody we used in this study was PA1-780, and it primarily labels the M1 ipRGCs in the mouse retina [1]. This suggests that melanopsin-expressing ipRGCs have greater resistance towards the excitotoxicity induced by NMDA than Brn3a-positive RGCs. 4. Discussion In the present study, we showed that the excitotoxicity induced by NMDA causes acute and severe damage of conventional RGCs and a significant decrease in IPL thickness in mice. However, melanopsinexpressing ipRGCs were relatively resistant to NMDA excitotoxicity. We used previously published methods, and some of the results were consistent with the works of Seitz and Tamm [30]. However, the sequent RGC injury was not evaluated and other subtypes of RGCs were not mentioned in their studies. Our results are important because the dynamic changes in the cells in the GCL at different time points after NMDA injection are shown, and the two remaining subtypes of RGCs were quantified. Thus, this study provides a complete overview of the NMDA-mediated RGC damage and direct evidence of the resistance of ipRGCs. NMDA-induced cell apoptosis in murine retina has been reported several decades ago [16,31]; however, the loss of the cells in the GCL could not represent the degree of damage in RGCs due to equally numerous population of both RGCs and displaced amacrine cells located in the GCL [8,13]. Besides, a recent morphological study has shown that there are more than 20 subtypes of ganglion cells in the mouse retina [34]. It is difficult to distinguish these subtypes of RGCs with a simple method such as immunodetection. Therefore, systematic and accurate evaluation of the RGC death still needs to be studied. We labeled conventional RGCs on retinal whole-mounts using a reliable marker, Brn3a, which is convenient for quantitative analysis of conventional RGCs. Siliprandi et al. [31] found that a single intravitreal injection of different doses of NMDA (20–200 nM) in the adult rat retina resulted in a dose-dependent loss of cells in the GCL, and a dose-dependent decrease of retinal choline acetyltransferase (ChAT) activity; however, the RGCs were not identified and quantified. Our data indicate that a single injection of NMDA (30 nM) in the adult mouse retina caused a time-dependent loss of RGCs in the GCL. Counting the total number of the surviving RGC axons in the optic nerves was also a feasible way to evaluate RGC damage. However, how to identify the axons of different subtypes of RGCs, such as conventional RGCs and ipRGCs, is still a barrier for researchers. Less than 5% of the total number of RGCs in the retina are ipRGCs, a remarkably rare subpopulation of RGCs. Recent morphological and functional studies indicated that there are at least six subtypes of ipRGCs, namely M1-M6 [2,24,35]. M1 cells have a noticeably high level of melanopsin immunoreactivity among the ipRGC subtypes [1,12]. Furthermore, they are reported to be resistant to death in several injury and disease models, including axotomized mouse RGCs and chronic ocular hypertension [18,27]. Although DeParis et al. (2012) have also reported the resistance of ipRGCs against NMDA-induced excitotoxicity in mice, the changes in the density of the surviving Brn3a-positive cells and melanopsin-expressing cells were not shown. It is well known that the mRNA expression level does not represent the change in protein level. A previous study also showed that melanopsin expression was
Fig. 4. Melanopsin-positive cells are relatively resistant to excitotoxicity induced by NMDA. Representative photomicrographs from retinal whole-mounts showing double labeling for Brn3a-positive (red) and melanopsin positive (green) cells are shown at different time points after NMDA (B, D, F, H) or PBS injection (A, C, E, G). Brn3a-positive cells decreased significantly in NMDA-treated retinas compared to control retinas. I-J: quantification of I: Brn3a-positive cells (I) and J: melanopsin-positive cells (J) in retinal whole-mounts at different time points after NMDA or PBS injection. Values are mean ± standard error of mean (SEM); n = 5. *P < 0.05, ***P < 0.0001. Two tailed student’s t test was used to test statistical significance. Scale bar = 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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reduced by 83% after N-methyl-N-nitrosourea (MNU) treatment; however, about two-thirds of melanopsin-expressing cells survived [4]. Hence, it is necessary to assess the resistance of ipRGCs to excitotoxicity from the level of melanopsin protein. Our data provides morphological evidence that melanopsin-expressing RGCs were relatively resistant to NMDA-induced excitotoxicity, compared to the conventional RGCs. Currently, we have no clear explanation for the mechanism of the resistance. The PI3K/AKT pathway may play a role in ipRGC survival after optic nerve transection [19], whereas the resistance to NMDAinduced excitotoxicity does not appear to depend on PI3K/AKT or JAK/ STAT signaling [7]. We believe that ipRGCs might have their own intrinsic strength to survive. It is unlikely that a single molecular mechanism is responsible for the resistance of ipRGCs to several pathological situations. NMDA-induced excitotoxicity is predominately mediated by various glutamate receptors [11,16,20]; hence, in the future, investigating the differential expression of different types of glutamate receptors in ipRGCs could be helpful to reveal the mechanisms that underlie in vivo protection of ipRGCs against NMDA excitotoxicity. In conclusion, we showed that a single injection of NMDA-mediated acute and severe damage of conventional RGCs in a time-dependent manner. Furthermore, a significant reduction was found in the thickness of the IPL after NMDA injection. Moreover, our results suggested that compared to conventional RGCs, melanopsin-expressing RGCs are relatively resistant to excitotoxicity for a long duration after NMDA injection. Conflict of interest The authors declare no financial conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (31571238). We gratefully acknowledge the assistance of Dr. Min Wang at Eye & ENT Hospital of Fudan University for offering the fixtive. We also thank Professor Xiangtian Zhou and Shijun Weng for their technical assistance. References [1] S.B. Baver, G.E. Pickard, P.J. Sollars, G.E. Pickard, Two types of melanopsin retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic nucleus and the olivary pretectal nucleus, Eur. J. Neurosci. 27 (2008) 1763–1770. [2] D.M. Berson, A.M. Castrucci, I. Provencio, Morphology and mosaics of melanopsinexpressing retinal ganglion cell types in mice, J. Comp. Neurol. 518 (2010) 2405–2422. [3] D.M. Berson, F.A. Dunn, M. Takao, Phototransduction by retinal ganglion cells that set the circadian clock, Science 295 (2002) 1070–1073. [4] D.L. Boudard, J. Mendoza, D. Hicks, Loss of photic entrainment at low illuminances in rats with acute photoreceptor degeneration, Eur. J. Neurosci. 30 (2009) 1527–1536. [5] G.H. Bresnick, Excitotoxins: a possible new mechanism for the pathogenesis of ischemic retinal damage, Arch. Ophthalmol. 107 (1989) 339–341. [6] Q. Cui, C. Ren, P.J. Sollars, G.E. Pickard, K.F. So, The injury resistant ability of melanopsin-expressing intrinsically photosensitive retinal ganglion cells, Neuroscience 284 (2015) 845–853. [7] S. DeParis, C. Caprara, C. Grimm, Intrinsically photosensitive retinal ganglion cells are resistant to N-methyl-D-aspartic acid excitotoxicity, Mol. Vis. 18 (2012) 2814–2827. [8] U.C. Drager, J.F. Olsen, Ganglion cell distribution in the retina of the mouse, Invest. Ophthalmol. Vis. Sci. 20 (1981) 285–293. [9] E.B. Dreyer, D. Zurakowski, R.A. Schumer, S.M. Podos, S.A. Lipton, Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma, Arch.
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