NEURAL REGENERATION RESEARCH Volume 7, Issue 10, April 2012
Cite this article as: Neural Regen Res. 2012;7(10):725-730.
www.nrronline.org
Dose-dependent and combined effects of N-methyl- D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the
survival of retinal ganglion cells in adult hamsters***☆△
Yaoyu Li1, An’an Yang2, Tingting Zhu1, Zhao Liu2, Siwei You2, Kwok-Fai So3
1Department of Ophthalmology, General Hospital of Beijing Military Region, Beijing 100700, China
2Institute of Neurosciences, the Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
3Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
Abstract
This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine (L-NA) on the
survival of retinal ganglion cells (RGCs) at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 (1.0, 3.0 and 4.5 mg/kg) and L-NA (1.5, 3.0, 4.5 and 6.0 mg/kg) increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.
Key Words: retinal ganglion cells; optic nerve transection; N-methyl-D-aspartate receptor; nitric oxide synthase; neuronal survival; hamster
Abbreviations: RGCs, retinal ganglion cells; ON, optic nerve; NO, nitric oxide; NOS, nitric oxide synthase; NMDA, N-methyl-D-aspartate
Yaoyu Li☆, Ph.D., Professor, Department of
Ophthalmology, General Hospital of Beijing Military Region, Beijing 100700, China
Yaoyu Li and An’an Yang contributed equally to this work.
Corresponding author: Siwei You, Professor,
Doctoral supervisor, Institute of Neurosciences, the Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China; Kwok-Fai So, Ph.D., Chair professor, Department of Anatomy, Li Ka Shing Faculty of
Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
[email protected]; [email protected]
Received: 2012-01-15 Accepted: 2012-03-30 (N[1**********])
Li YY, Yang AA, Zhu TT, Liu Z, You SW, So KF. Dose-dependent and combined effects of N-methyl-D-aspartate
receptor antagonist (MK-801) and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters. Neural Regen Res. 2012;7(10):725-730.
www.crter.cn
www.nrronline.org
doi:10.3969/j.issn.1673-5374.2012.10.001
selective non-competitive NMDA receptor antagonist dizocilpine maleate (MK-801) and the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) could exert
neuroprotective actions in experimentally induced ischemia[4-5]. In the visual system, the excitotoxicity induced by glutamate has been thought to be one of the major reasons for the death of retinal ganglion cells (RGCs) caused by glaucoma and retinal ischemia. However, the effects of glutamate are not clear in traumatic optic nerve (ON) injury since activation/inactivation of retinal
metabotropic glutamate receptors does not play an important role for the initiation and execution of secondary RGC loss after ON transection and NMDA lesion in the adult rat [6]. Whether NMDA antagonists can decrease RGC death remains
controversial [7-10], and effects of NO/NOS on the survival of axotomized RGCs are still inconclusive [11-14]. Moreover, no studies have ever been reported to examine
dose-dependent and combined effects of a NMDA antagonist and NOS inhibitor on the
INTRODUCTION
The excitatory amino acid glutamate is one of the major excitatory transmitters in the central nervous system, and its interactions with specific membrane receptors are responsible for many neurobiological
functions. Glutamate has been implicated in delayed brain injury after cerebral ischemia and hypoglycemia, and traumatic brain injury also caused a marked elevation in extracellular glutamate adjacent to the
trauma site. The effect of excitotoxic insult is caused by an increased formation of nitric oxide (NO)[1-2] catalysed by nitric oxide
synthase (NOS) which is induced by excess calcium influx via N-methyl-D-aspartate (NMDA) subtype of glutamate receptor operated channels. NO released from
injured neurons results in a direct massive neuronal death[3]. The stimulation of glutamate receptors by excitatory amino acids and released NO has been linked to neuronal degeneration and toxicity. The
725
survival of RGCs after traumatic ON injury. In this study we examined dose-dependent and combined effects of MK-801 and nitro-L-arginine (L-NA) on RGC survival after ON transaction (Table 1).
RESULTS
Fluorogold-labeled RGCs and glial cells
RGCs of all sizes were labeled with fluorogold. They had large oval somas and very few processes could be seen with the exception of the proximal part of some primary dendrites. Due to uptake of the dye from dead RGCs, fluorogold-labeled glial cells appeared in the retina 1 week after ON transection. Glial cells had smaller somas and irregular cell borders, and their short and branched processes could be seen clearly. When RGC number decreased with the survival time, the number of glial cells increased (Figures 1 and 2).
Figure 1 Microphotographs of Fluorogold-labeled surviving retinal ganglion cells 1 week after optic nerve transection in control (A), 1.0 mg/kg MK-801 (B), 3.0 mg/kg MK-801(C), 4.5 mg/kg MK-801 (D), 1.5 mg/kg
nitro-L-arginine (L-NA) (E), 3.0 mg/kg L-NA (F), 4.5 mg/kg L-NA (G), 6.0 mg/kg L-NA (H), combinational 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA (I) groups. Retinal ganglion cells have large circular cell bodies with clear boundaries (arrow heads). A few glial cells with small irregular bodies and short branched-processes are also visible in the retina (arrows). Scale bar: 100 μm.
Surviving RGCs
When compared with the mean numbers of Fluorogold- labeled surviving RGCs at 1 week in control group, no significant difference (P > 0.05) in the RGC number could be observed in 1.0 mg/kg MK-801 subgroup. Similar RGC numbers (P > 0.05) in 3.0 and 4.5 mg/kg MK-801 subgroups were significantly higher (P = 0.001) than those in control group and 1.0 mg/kg MK-801 subgroup at 1 week (Figure 3). When survival time extended to
2 weeks, the RGC numbers in 3.0 and 4.5 mg/kg
subgroups remained significantly higher than those in 1.0 mg/kg subgroup and control group (P
726
highest RGC numbers at 1 and 2 weeks were obtained in 3.0 mg/kg MK-801 subgroup (Figure 4 and Table 1).
Figure 2 Microphotographs of Fluorogold-labeled
surviving retinal ganglion cells (arrow heads) 2 weeks after optic nerve transection in control (A), 1.0 mg/kg MK-801 (B), 3.0 mg/kg MK-801(C), 4.5 mg/kg MK-801 (D), 1.5 mg/kg nitro-L-arginine (L-NA) (E), 3.0 mg/kg L-NA (F), 4.5 mg/kg L-NA (G), 6.0 mg/kg L-NA (H), combinational 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA (I) groups. A few glial cells with small irregular bodies and short branched processes are also visible in the retina (arrows). Scale bar: 100 μm.
The RGC numbers were elevated with increased dosage of L-NA at 1 week. The highest RGC number in 4.5 mg/kg L-NA subgroup was then followed by a slightly lower but similar RGC number in 6.0 mg/kg L-NA
subgroup (P > 0.05). All these 4 numbers were
significantly higher (P 0.05) in the RGC numbers between 1.5 mg/kg L-NA subgroup and control group at 2 weeks, increased dosage of L-NA at 3.0, 4.5 and 6.0 mg/kg resulted in significantly higher RGC numbers (P = 0.000 1), with the highest RGC number also in 4.5 mg/kg L-NA subgroup (Table1 and Figure 6).
The RGC numbers in combination group were
significantly higher than those in control groups (P
727
Moreover, the effect of the combinational treatment of MK-801 and L-NA was potentiated in comparison with the added effects of MK-801 or L-NA alone because the RGC number in combination group at 2 weeks (5 900) was higher than the added number of 2 749 in 1.0 mg/kg MK-801 subgroup and 2 696 in 1.5 mg/kg L-NA subgroup (Figure 8 and Table 1).
Examination of the right retina from hamsters of all groups indicated that daily injections of MK-801 and/or L-NA, and the long term labeling of the RGCs with fluorogold for at least 11 weeks did not affect the intact RGCs, and hence the results of the present study.
DISCUSSION
Neuronal production of NO is triggered when glutamate binds to and activates a NMDA receptor on an adjacent
728
neuron. A channel in the receptor is therefore opened, admitting Ca2+ into the cell where it binds to calmodulin. The Ca2+/calmodulin complex then binds to and activates NOS so that, in the presence of oxygen and
5,10-nicotinamide-adenine dinucleotide phosphate reductase, NOS converts L-arginine to L-citrulline and NO which is then released from the cell. It has been reported that NMDA-receptor blockade with the NMDA receptor antagonist MK-801 enhances cell apoptosis in the developing retina of the postnatal rat[4] and a
noncompetitive NMDA receptor antagonist, Bis(7)-tacrine, can prevent glutamate-induced rat RGCs damage in vitro and in vivo [9]. However, MK-801 at 0-100 µM can not significantly attenuate the H2O 2-induced cytotoxicity[10]. We have demonstrated in the present study that daily intraperitoneal injections of MK-801 increased neuronal survival of RGCs following ON transection in a
dose-dependent manner. At both 1 and 2 weeks, the numbers of surviving RGCs were not promoted
significantly with the treatment of 1.0 mg/kg MK-801 when compared to control groups. However, RGC
numbers increased significantly with a higher dosage of 3.0 or 4.5 mg/kg. Both the highest RGC numbers at 1 and 2 weeks were obtained with the treatment of MK-801 at 3.0 mg/kg.
It is also interesting that either increased or decreased NOS expression has been shown after neuronal
injury [11-16]. NOS expression can also remain unchanged within the adult rodent retina up to 4 months following intraorbital ON transection[11, 17]. The finding that
lesion-induced NOS within injured neurons is coincident with death of these neurons which suggests a role of NO in neuronal degeneration. If NO, produced by
lesion-induced NOS, is responsible for the death of injured neurons, inhibition of NOS should therefore prevent the death of these neurons. However, although this hypothesis has been widely supported by a large number of studies using NOS inhibitors[18-19], the effects of NO on neuronal survival can be either protective or destructive [18]. Such paradoxical phenomena may probably be interpreted as differences in distinct
response to axonal injury among NOS-containing neuron groups. In two main lines of investigations, NOS
expression is markedly augmented in spinal neurons after spinal root avulsion[20-21] or spinal cord transection[21] and in the hypothalamic supraoptic nuclei and
paraventricular following hypophysectomy[19, 22]. Inhibition of NOS protected RGCs against glaucomatous
damage [14]. L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of a selective inhibitor of inducible NOS, prevented the loss of retinal ganglion cells in eyes with chronic, moderately elevated intraocular pressure in an animal model of glaucoma[13]. Daily intraperitoneal injection of L-NA in the present study also retarded the degeneration of axotomized RGCs in a dose-dependent fashion. This protective effect may be achieved by the inhibition of NO production in adjacent amacrine cells with L-NA.
In this experiment, RGC loss after ON transection was reduced, but not completely prevented, by NMDA
receptor antagonist and NOS inhibitor. There are several explanations for this incomplete neuroprotective effect of NOS inhibitors: (1) the lesion-induced NOS was not completely inhibited by MK-801 and L-NA at the dose level used; (2) lesion was not the only stimulus to induce NOS/NO responsible for the death of injured RGCs; (3) the neurotoxicity of glutamate could not be overcome completely with MK-801; the antagonist of the NMDA subtype of glutamate receptors, as it has been reported recently that low doses of glutamate can activate α-amino-3-digydro-5-methyl-isoxazol-4-propionic
acid-kainate receptors in cultured RGCs, which causes increases in intracellular calcium and decreases in cell survival [23]. Also, there might be other than NMDA and NOS death mechanisms activated after ON transection. The mechanisms for the protective effects of these NOS inhibitors might be both indirect, reducing the production of NO which is synthesized in and released from
adjacent amacrine cells in either the inner nuclear layer or the ganglion cell layer, and direct, blocking the
neurotoxicity of glutamate with MK-801 on injured RGCs. We have also shown that combinational injections of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA, neither of the dosages are the optimal ones, enhanced the survival of RGCs 1 and 2 weeks after ON transection, suggesting the potentiative action of these two chemicals, which influence different phases of NO biosynthesis on the same biochemical pathway, on the survival of
axotomized RGCs. NO synthesis can be inhibited by MK-801 as a NMDA antagonist or L-NA as a competitive NOS inhibitor, thus inhibiting NOS more effectively. Our findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these two chemicals possesses a potentiative and protective effect.
MATERIALS AND METHODS
Design
A randomized controlled animal experiment. Time and setting
This study was conducted in the Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong and Institute of Neurosciences, the Fourth Military Medical University from February to December, 2011. Materials
One hundred and fourteen adult male hamsters of 6-7 weeks old (Mesocricetus auratus, the Laboratory Animal Units of the University of Hong Kong and the Fourth Military Medical University) were divided into one control and three experimental groups (MK-801, L-NA and combination groups). Methods
Pre-labeling of RGCs and ON transection
All animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight;
Ovation Pharmaceuticals, USA) for all surgical
procedures. The RGCs of the animals were retrogradely labeled by applying small pieces of Gelfoam (Upjohn, USA) soaked in 5% fluorogold (Fluorochrome, USA) onto the surfaces of bilateral superior colliculi after the cortical tissues and pia overlying the superior colliculi was carefully removed. Six animals in control group were allowed to survive for 4 days after pre-labeling without ON transection. The left ONs of all other 108 animals were exposed 4 days later through a superior temporal intraorbital approach and transected intraorbitally 1 mm from the optic disc under an operating microscope (Olympus OME, Japan). Care was taken to avoid any damages to retinal blood supply from the ophthalmic artery [24]. Treatments
One day before the left ON transection, 84 animals
received intraperitoneal injections of different dosages of MK-801 (1.0, 3.0 or 4.5 mg/kg; ICN Pharmaceuricals, USA) and L-NA (1.5, 3.0, 4.5 or 6.0 mg/kg; Aldrich, USA) in sterile saline in MK-801 and L-NA groups (n = 12 for each dosage) and survived for 1 or 2 weeks (n = 6 for each time point). Another 12 animals in combination group received daily intraperitoneal injections of
combinational 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA until the animals were killed 1 or 2 weeks following ON transection (n = 6 for each time point). Another 12 control animals received IP injections of the same volume of saline before they were killed 1 or 2 weeks after ON transection (n = 6 for each time point).
Counting of Fluorogold-labeled surviving RGCs
All animals were sacrificed with an overdose (100 mg/kg body weight) of anesthesia at the appropriate survival time. The left retinas were removed, post-fixed in 4% paraformaldehyde (Merck, Germany) in PBS (0.1 M, pH 7.4) for 1 hour, rinsed in PBS (0.1 M, pH 7.4) for 3 times, divided into superionasal, superiotemporal, inferionasal and inferiotemporal quadrants with four radial cuts
(Figure 9), flat-mounted on gelatin-coated slides in 30% glycerol (Merck, Germany) and eventually coverslipped.
729
The total populations of Fluorogold-labeled surviving RGCs in the retinas were estimated using a sampling technique [25]. In each retina, the number of RGCs was
counted in 28-30 sample areas (each occupying 40 000 µm 2) at 7 to 8 different eccentricities from the optic disc in four different quadrants of the retina under a BX51
fluorescence microscope (Olympus, Japan) using a 355-425 nm ultraviolet filter. The data from all sample areas were pooled and the total number of RGCs counted was divided by the areas sampled, thus
obtaining the estimated mean density of surviving RGCs. Finally, the estimated number of surviving RGCs in each retina was calculated by multiplying the mean density of RGCs with the area of the retina. Statistical Analysis
One-way analysis of variance was used to compare the mean numbers of surviving RGCs using SPSS (Version 11.0, USA). All data values were presented as Mean ± SEM and differences were considered significant when P
Funding: This research work was supported by research
grants from Chinese National Key Project for Basic Research, No. 2011CB504402 and the National Natural Science Foundation of China, No. 30901649 and 30872829.
Author contributions: Yaoyu Li and An’an Yang performed the experiments. Tingting Zhu and Zhao Liu conducted the
statistical analysis and completed all figures. Siwei You guided this study, wrote the manuscript and contributed to part of the fundings. Kwok-Fai So designed this study and was in charge of part of the fundings.
Conflicts of interest: None declared.
Ethical approval: Animal Care and Use Committees at the University of Hong Kong and the Fourth Military Medical University approved all animal protocols in this study.
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(Edited by Zeng YS, Sanz MV/Zhao LJ/Song LP)
NEURAL REGENERATION RESEARCH Volume 7, Issue 10, April 2012
Cite this article as: Neural Regen Res. 2012;7(10):725-730.
www.nrronline.org
Dose-dependent and combined effects of N-methyl- D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the
survival of retinal ganglion cells in adult hamsters***☆△
Yaoyu Li1, An’an Yang2, Tingting Zhu1, Zhao Liu2, Siwei You2, Kwok-Fai So3
1Department of Ophthalmology, General Hospital of Beijing Military Region, Beijing 100700, China
2Institute of Neurosciences, the Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
3Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
Abstract
This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine (L-NA) on the
survival of retinal ganglion cells (RGCs) at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 (1.0, 3.0 and 4.5 mg/kg) and L-NA (1.5, 3.0, 4.5 and 6.0 mg/kg) increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.
Key Words: retinal ganglion cells; optic nerve transection; N-methyl-D-aspartate receptor; nitric oxide synthase; neuronal survival; hamster
Abbreviations: RGCs, retinal ganglion cells; ON, optic nerve; NO, nitric oxide; NOS, nitric oxide synthase; NMDA, N-methyl-D-aspartate
Yaoyu Li☆, Ph.D., Professor, Department of
Ophthalmology, General Hospital of Beijing Military Region, Beijing 100700, China
Yaoyu Li and An’an Yang contributed equally to this work.
Corresponding author: Siwei You, Professor,
Doctoral supervisor, Institute of Neurosciences, the Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China; Kwok-Fai So, Ph.D., Chair professor, Department of Anatomy, Li Ka Shing Faculty of
Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
[email protected]; [email protected]
Received: 2012-01-15 Accepted: 2012-03-30 (N[1**********])
Li YY, Yang AA, Zhu TT, Liu Z, You SW, So KF. Dose-dependent and combined effects of N-methyl-D-aspartate
receptor antagonist (MK-801) and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters. Neural Regen Res. 2012;7(10):725-730.
www.crter.cn
www.nrronline.org
doi:10.3969/j.issn.1673-5374.2012.10.001
selective non-competitive NMDA receptor antagonist dizocilpine maleate (MK-801) and the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) could exert
neuroprotective actions in experimentally induced ischemia[4-5]. In the visual system, the excitotoxicity induced by glutamate has been thought to be one of the major reasons for the death of retinal ganglion cells (RGCs) caused by glaucoma and retinal ischemia. However, the effects of glutamate are not clear in traumatic optic nerve (ON) injury since activation/inactivation of retinal
metabotropic glutamate receptors does not play an important role for the initiation and execution of secondary RGC loss after ON transection and NMDA lesion in the adult rat [6]. Whether NMDA antagonists can decrease RGC death remains
controversial [7-10], and effects of NO/NOS on the survival of axotomized RGCs are still inconclusive [11-14]. Moreover, no studies have ever been reported to examine
dose-dependent and combined effects of a NMDA antagonist and NOS inhibitor on the
INTRODUCTION
The excitatory amino acid glutamate is one of the major excitatory transmitters in the central nervous system, and its interactions with specific membrane receptors are responsible for many neurobiological
functions. Glutamate has been implicated in delayed brain injury after cerebral ischemia and hypoglycemia, and traumatic brain injury also caused a marked elevation in extracellular glutamate adjacent to the
trauma site. The effect of excitotoxic insult is caused by an increased formation of nitric oxide (NO)[1-2] catalysed by nitric oxide
synthase (NOS) which is induced by excess calcium influx via N-methyl-D-aspartate (NMDA) subtype of glutamate receptor operated channels. NO released from
injured neurons results in a direct massive neuronal death[3]. The stimulation of glutamate receptors by excitatory amino acids and released NO has been linked to neuronal degeneration and toxicity. The
725
survival of RGCs after traumatic ON injury. In this study we examined dose-dependent and combined effects of MK-801 and nitro-L-arginine (L-NA) on RGC survival after ON transaction (Table 1).
RESULTS
Fluorogold-labeled RGCs and glial cells
RGCs of all sizes were labeled with fluorogold. They had large oval somas and very few processes could be seen with the exception of the proximal part of some primary dendrites. Due to uptake of the dye from dead RGCs, fluorogold-labeled glial cells appeared in the retina 1 week after ON transection. Glial cells had smaller somas and irregular cell borders, and their short and branched processes could be seen clearly. When RGC number decreased with the survival time, the number of glial cells increased (Figures 1 and 2).
Figure 1 Microphotographs of Fluorogold-labeled surviving retinal ganglion cells 1 week after optic nerve transection in control (A), 1.0 mg/kg MK-801 (B), 3.0 mg/kg MK-801(C), 4.5 mg/kg MK-801 (D), 1.5 mg/kg
nitro-L-arginine (L-NA) (E), 3.0 mg/kg L-NA (F), 4.5 mg/kg L-NA (G), 6.0 mg/kg L-NA (H), combinational 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA (I) groups. Retinal ganglion cells have large circular cell bodies with clear boundaries (arrow heads). A few glial cells with small irregular bodies and short branched-processes are also visible in the retina (arrows). Scale bar: 100 μm.
Surviving RGCs
When compared with the mean numbers of Fluorogold- labeled surviving RGCs at 1 week in control group, no significant difference (P > 0.05) in the RGC number could be observed in 1.0 mg/kg MK-801 subgroup. Similar RGC numbers (P > 0.05) in 3.0 and 4.5 mg/kg MK-801 subgroups were significantly higher (P = 0.001) than those in control group and 1.0 mg/kg MK-801 subgroup at 1 week (Figure 3). When survival time extended to
2 weeks, the RGC numbers in 3.0 and 4.5 mg/kg
subgroups remained significantly higher than those in 1.0 mg/kg subgroup and control group (P
726
highest RGC numbers at 1 and 2 weeks were obtained in 3.0 mg/kg MK-801 subgroup (Figure 4 and Table 1).
Figure 2 Microphotographs of Fluorogold-labeled
surviving retinal ganglion cells (arrow heads) 2 weeks after optic nerve transection in control (A), 1.0 mg/kg MK-801 (B), 3.0 mg/kg MK-801(C), 4.5 mg/kg MK-801 (D), 1.5 mg/kg nitro-L-arginine (L-NA) (E), 3.0 mg/kg L-NA (F), 4.5 mg/kg L-NA (G), 6.0 mg/kg L-NA (H), combinational 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA (I) groups. A few glial cells with small irregular bodies and short branched processes are also visible in the retina (arrows). Scale bar: 100 μm.
The RGC numbers were elevated with increased dosage of L-NA at 1 week. The highest RGC number in 4.5 mg/kg L-NA subgroup was then followed by a slightly lower but similar RGC number in 6.0 mg/kg L-NA
subgroup (P > 0.05). All these 4 numbers were
significantly higher (P 0.05) in the RGC numbers between 1.5 mg/kg L-NA subgroup and control group at 2 weeks, increased dosage of L-NA at 3.0, 4.5 and 6.0 mg/kg resulted in significantly higher RGC numbers (P = 0.000 1), with the highest RGC number also in 4.5 mg/kg L-NA subgroup (Table1 and Figure 6).
The RGC numbers in combination group were
significantly higher than those in control groups (P
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Moreover, the effect of the combinational treatment of MK-801 and L-NA was potentiated in comparison with the added effects of MK-801 or L-NA alone because the RGC number in combination group at 2 weeks (5 900) was higher than the added number of 2 749 in 1.0 mg/kg MK-801 subgroup and 2 696 in 1.5 mg/kg L-NA subgroup (Figure 8 and Table 1).
Examination of the right retina from hamsters of all groups indicated that daily injections of MK-801 and/or L-NA, and the long term labeling of the RGCs with fluorogold for at least 11 weeks did not affect the intact RGCs, and hence the results of the present study.
DISCUSSION
Neuronal production of NO is triggered when glutamate binds to and activates a NMDA receptor on an adjacent
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neuron. A channel in the receptor is therefore opened, admitting Ca2+ into the cell where it binds to calmodulin. The Ca2+/calmodulin complex then binds to and activates NOS so that, in the presence of oxygen and
5,10-nicotinamide-adenine dinucleotide phosphate reductase, NOS converts L-arginine to L-citrulline and NO which is then released from the cell. It has been reported that NMDA-receptor blockade with the NMDA receptor antagonist MK-801 enhances cell apoptosis in the developing retina of the postnatal rat[4] and a
noncompetitive NMDA receptor antagonist, Bis(7)-tacrine, can prevent glutamate-induced rat RGCs damage in vitro and in vivo [9]. However, MK-801 at 0-100 µM can not significantly attenuate the H2O 2-induced cytotoxicity[10]. We have demonstrated in the present study that daily intraperitoneal injections of MK-801 increased neuronal survival of RGCs following ON transection in a
dose-dependent manner. At both 1 and 2 weeks, the numbers of surviving RGCs were not promoted
significantly with the treatment of 1.0 mg/kg MK-801 when compared to control groups. However, RGC
numbers increased significantly with a higher dosage of 3.0 or 4.5 mg/kg. Both the highest RGC numbers at 1 and 2 weeks were obtained with the treatment of MK-801 at 3.0 mg/kg.
It is also interesting that either increased or decreased NOS expression has been shown after neuronal
injury [11-16]. NOS expression can also remain unchanged within the adult rodent retina up to 4 months following intraorbital ON transection[11, 17]. The finding that
lesion-induced NOS within injured neurons is coincident with death of these neurons which suggests a role of NO in neuronal degeneration. If NO, produced by
lesion-induced NOS, is responsible for the death of injured neurons, inhibition of NOS should therefore prevent the death of these neurons. However, although this hypothesis has been widely supported by a large number of studies using NOS inhibitors[18-19], the effects of NO on neuronal survival can be either protective or destructive [18]. Such paradoxical phenomena may probably be interpreted as differences in distinct
response to axonal injury among NOS-containing neuron groups. In two main lines of investigations, NOS
expression is markedly augmented in spinal neurons after spinal root avulsion[20-21] or spinal cord transection[21] and in the hypothalamic supraoptic nuclei and
paraventricular following hypophysectomy[19, 22]. Inhibition of NOS protected RGCs against glaucomatous
damage [14]. L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of a selective inhibitor of inducible NOS, prevented the loss of retinal ganglion cells in eyes with chronic, moderately elevated intraocular pressure in an animal model of glaucoma[13]. Daily intraperitoneal injection of L-NA in the present study also retarded the degeneration of axotomized RGCs in a dose-dependent fashion. This protective effect may be achieved by the inhibition of NO production in adjacent amacrine cells with L-NA.
In this experiment, RGC loss after ON transection was reduced, but not completely prevented, by NMDA
receptor antagonist and NOS inhibitor. There are several explanations for this incomplete neuroprotective effect of NOS inhibitors: (1) the lesion-induced NOS was not completely inhibited by MK-801 and L-NA at the dose level used; (2) lesion was not the only stimulus to induce NOS/NO responsible for the death of injured RGCs; (3) the neurotoxicity of glutamate could not be overcome completely with MK-801; the antagonist of the NMDA subtype of glutamate receptors, as it has been reported recently that low doses of glutamate can activate α-amino-3-digydro-5-methyl-isoxazol-4-propionic
acid-kainate receptors in cultured RGCs, which causes increases in intracellular calcium and decreases in cell survival [23]. Also, there might be other than NMDA and NOS death mechanisms activated after ON transection. The mechanisms for the protective effects of these NOS inhibitors might be both indirect, reducing the production of NO which is synthesized in and released from
adjacent amacrine cells in either the inner nuclear layer or the ganglion cell layer, and direct, blocking the
neurotoxicity of glutamate with MK-801 on injured RGCs. We have also shown that combinational injections of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA, neither of the dosages are the optimal ones, enhanced the survival of RGCs 1 and 2 weeks after ON transection, suggesting the potentiative action of these two chemicals, which influence different phases of NO biosynthesis on the same biochemical pathway, on the survival of
axotomized RGCs. NO synthesis can be inhibited by MK-801 as a NMDA antagonist or L-NA as a competitive NOS inhibitor, thus inhibiting NOS more effectively. Our findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these two chemicals possesses a potentiative and protective effect.
MATERIALS AND METHODS
Design
A randomized controlled animal experiment. Time and setting
This study was conducted in the Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong and Institute of Neurosciences, the Fourth Military Medical University from February to December, 2011. Materials
One hundred and fourteen adult male hamsters of 6-7 weeks old (Mesocricetus auratus, the Laboratory Animal Units of the University of Hong Kong and the Fourth Military Medical University) were divided into one control and three experimental groups (MK-801, L-NA and combination groups). Methods
Pre-labeling of RGCs and ON transection
All animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight;
Ovation Pharmaceuticals, USA) for all surgical
procedures. The RGCs of the animals were retrogradely labeled by applying small pieces of Gelfoam (Upjohn, USA) soaked in 5% fluorogold (Fluorochrome, USA) onto the surfaces of bilateral superior colliculi after the cortical tissues and pia overlying the superior colliculi was carefully removed. Six animals in control group were allowed to survive for 4 days after pre-labeling without ON transection. The left ONs of all other 108 animals were exposed 4 days later through a superior temporal intraorbital approach and transected intraorbitally 1 mm from the optic disc under an operating microscope (Olympus OME, Japan). Care was taken to avoid any damages to retinal blood supply from the ophthalmic artery [24]. Treatments
One day before the left ON transection, 84 animals
received intraperitoneal injections of different dosages of MK-801 (1.0, 3.0 or 4.5 mg/kg; ICN Pharmaceuricals, USA) and L-NA (1.5, 3.0, 4.5 or 6.0 mg/kg; Aldrich, USA) in sterile saline in MK-801 and L-NA groups (n = 12 for each dosage) and survived for 1 or 2 weeks (n = 6 for each time point). Another 12 animals in combination group received daily intraperitoneal injections of
combinational 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA until the animals were killed 1 or 2 weeks following ON transection (n = 6 for each time point). Another 12 control animals received IP injections of the same volume of saline before they were killed 1 or 2 weeks after ON transection (n = 6 for each time point).
Counting of Fluorogold-labeled surviving RGCs
All animals were sacrificed with an overdose (100 mg/kg body weight) of anesthesia at the appropriate survival time. The left retinas were removed, post-fixed in 4% paraformaldehyde (Merck, Germany) in PBS (0.1 M, pH 7.4) for 1 hour, rinsed in PBS (0.1 M, pH 7.4) for 3 times, divided into superionasal, superiotemporal, inferionasal and inferiotemporal quadrants with four radial cuts
(Figure 9), flat-mounted on gelatin-coated slides in 30% glycerol (Merck, Germany) and eventually coverslipped.
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The total populations of Fluorogold-labeled surviving RGCs in the retinas were estimated using a sampling technique [25]. In each retina, the number of RGCs was
counted in 28-30 sample areas (each occupying 40 000 µm 2) at 7 to 8 different eccentricities from the optic disc in four different quadrants of the retina under a BX51
fluorescence microscope (Olympus, Japan) using a 355-425 nm ultraviolet filter. The data from all sample areas were pooled and the total number of RGCs counted was divided by the areas sampled, thus
obtaining the estimated mean density of surviving RGCs. Finally, the estimated number of surviving RGCs in each retina was calculated by multiplying the mean density of RGCs with the area of the retina. Statistical Analysis
One-way analysis of variance was used to compare the mean numbers of surviving RGCs using SPSS (Version 11.0, USA). All data values were presented as Mean ± SEM and differences were considered significant when P
Funding: This research work was supported by research
grants from Chinese National Key Project for Basic Research, No. 2011CB504402 and the National Natural Science Foundation of China, No. 30901649 and 30872829.
Author contributions: Yaoyu Li and An’an Yang performed the experiments. Tingting Zhu and Zhao Liu conducted the
statistical analysis and completed all figures. Siwei You guided this study, wrote the manuscript and contributed to part of the fundings. Kwok-Fai So designed this study and was in charge of part of the fundings.
Conflicts of interest: None declared.
Ethical approval: Animal Care and Use Committees at the University of Hong Kong and the Fourth Military Medical University approved all animal protocols in this study.
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(Edited by Zeng YS, Sanz MV/Zhao LJ/Song LP)