Notes
GYY4137, an Extended-Release Hydrogen Sulfide Donor, Reduces NMDA-Induced Neuronal Injury in the Murine Retina
2018 Volume 41 Issue 4 Pages 657-660
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2018 Volume 41 Issue 4 Pages 657-660
We previously reported that systemic administration with sodium hydrogen sulfide, a rapid-release donor compound of hydrogen sulfide (H2S), protected retinal neurons against N-methyl-D-aspartic acid (NMDA)-induced injury. For clinical application of H2S donors for retinal neurodegeneration, topical administration with an extended-release donor compound will be better. In the present study, we histologically investigated whether GYY4137, an extended-release hydrogen sulfide donor, had a protective effect on NMDA-induced retinal injury in the mice in vivo. Male and female B6.Cg-Tg(Thy1-CFP)23Jrs/J and C57BL/6J mice anesthetized with a mixture of ketamine and xylazine were subjected to intravitreal NMDA injection (80 nmol/eye). GYY4137 was intravitreally administered with NMDA simultaneously. Morphometric evaluation was carried out seven days after NMDA injection. Intravitreal NMDA induced retinal ganglion cell loss. GYY4137 (1, 10 and 100 nmol/eye) significantly reduced retinal ganglion cell loss seven days after NMDA injection. GYY4137 (10 nmol/eye) decreased the numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive and 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive cells 12 h after NMDA injection. These results suggest that extended release donor compounds of H2S protect retinal neurons against excitotoxicity induced by intravitreal NMDA in the mice in vivo through its anti-oxidative activity.
The death of retinal ganglion cell (RGC) leads to optic nerve degeneration, eventually visual loss. But the underlying mechanism is not completely understood. Although hydrogen sulfide (H2S) is famous as a toxic gas in general,1) various cytoprotective effects of H2S has been reported.2–5) It has been shown that 3-mercaptopyruvate sulfurtransferase and cysteine aminotransferase, H2S-producing enzymes produce H2S also in retinal neurons.5) In addition, we recently showed that systemic treatment with sodium hydrogen sulfide (NaHS), a rapid-release donor compound of H2S reduced N-methyl-D-aspartic acid (NMDA)-induced retinal injury in the rat in vivo.6) These results suggest that administration of donor compounds of H2S is a promising way for treatment of retinal excitotoxicity caused by glaucoma and retinal artery occlusion.
Although we previously demonstrated that systemic treatment with NaHS did not induce any toxic effects,6) at the thought of the toxicity of H2S, topical administration with minimum amounts of the donor is preferred for clinical application of H2S donors for retinal neurodegeneration. In addition, to prevent frequent administration of the drug, an extended-release donor compound will be suitable for treatment. However, it has not been clarified whether an extended-release donor compound of H2S has neuroprotective effect in the retina. GYY4137 (morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate) is a water-soluble, extended-release H2S donor, and one of the most widely used extended-release H2S donor for biological studies.7,8) GYY4137 has been reported to release lower level of H2S for at least seven days, whereas NaHS has been reported to release high level of H2S for up to one hour.8)
Recently, GYY4137 was reported to protect against RGC death induced by chronic elevation of intraocular pressure, optic nerve crash and ischemia–reperfusion (I/R) in the rats in vivo, and cell death induced by H2O2 in the cultured RGC.9) Because multiple factors, such as excitotoxicity, infiltration of leukocytes, inflammation, and so on, are involved in the RGC death induced by chronic elevation of intraocular pressure, optic nerve crash and I/R, it is still unknown whether GYY4137 is protective against excitoneurotoxicity in the retina in vivo. In addition, it has not been directly clarified whether anti-oxidative activity of GYY4137 is involved in its neuroprotective effect in the retina in vivo. In the present study, we clarified whether intravitreal GYY4137, an extended-release donor compound of H2S, protected against NMDA-induced retinal injury in the mice in vivo.
Experimental procedures conformed to the Regulations for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Kitasato University. Male and female B6.Cg-Tg (Thy1-CFP) 23Jrs/J (11–12 weeks) and C57BL/6J mice were used in the present study. The environment of the animal room was kept at 25°C with a 12 h : 12 h light–dark cycle. All animals were fed and watered ad libitum.
Intravitreal InjectionThe mice were anesthetized with intraperitoneal ketamine (90 mg/kg, Daiichi-Sankyo, Tokyo, Japan) and xylazine (10 mg/kg, Tokyo Kasei, Tokyo, Japan). NMDA (Nacalai Tesque, Kyoto, Japan) and GYY4137 (Dojindo Laboratories, Kumamoto, Japan) were dissolved in phosphate-buffered saline (PBS). Intravitreal injection was performed as previously described6,10–13) with a slight modification. Two microliters of the mixed drug solution were administered into one eye, and the vehicle was administered into the contralateral eye, which served as the control.
Evaluation of the Death of the Retinal Ganglion CellsB6.Cg-Tg(Thy1-CFP)23Jrs/J mice were euthanized with an overdose of sodium pentobarbital 7 d after intravitreal injection of 80 nmol/eye NMDA and 1, 10 and 100 nmol/eye GYY4137. Retinas were removed from the eyecup, fixed in 4% paraformaldehyde for 1 h at room temperature. Retinal whole mount was made, and the images were collected on a confocal laser microscope (LSM 700; Carl Zeiss, Jena, Germany). The number of CFP-positive cells in the area of 0.3 mm2 just beside the optic nerve head were counted. The cell number of the NMDA-injected eye was normalized to that of the contralateral eye and presented as the percentage.14)
Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNEL) Staining and ImmunohistochemistryC57BL/6J mice were euthanized with an overdose of sodium pentobarbital 12 h after intravitreal injection of 80 nmol/eye NMDA and 10 nmol/eye GYY4137. Retinal horizontal sections were obtained as described previously.10–13) TUNEL staining was performed to detect apoptotic cells using ApopTag® Plus Fluorescein In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA, U.S.A.). Immunohistochemical analysis using mouse monoclonal anti-8-hydroxy-deoxyguanosine monoclonal antibody (1 : 10; Japan Institute for the Control of Aging, Nikken Seil, Tokyo, Japan) was performed as previously described.6) The images of TUNEL staining and immunohistochemical staining were obtained with confocal laser microscopy (LSM710, Zeiss, Jena, Germany).
Statistical AnalysesThe data are represented as mean±standard error of the mean (S.E.M.). Student’s t-test was used to compare the means of the two groups. Dunnett’s test was used for multiple comparisons. Differences were considered to be statistically significant if p<0.05.
We first investigated the effects of GYY4137 on the loss of RGC induced by NMDA in the murine retina. Representative photomicrographs of the retinal whole mounts obtained 7 d after intravitreal NMDA injection (80 nmol/eye) are shown in Fig. 1A. In the NMDA-injected eyes, the marked loss of RGCs, which were labeled with enhanced cyan fluorescent protein (ECFP), were observed. GYY4137 (1, 10, 100 nmol/eye) prevented the loss of RGC. The result of the cell count from 4–5 independent experiments are summarized in Fig. 1B. GYY4137 (1, 10, 100 nmol/eye) itself did not significantly alter the number of RGCs (Fig. 1C).
Whole mount retinal images of the eyes treated with the vehicle, 1 nmol/eye GYY4137 (GYY), 10 nmol/eye GYY, 100 nmol/eye GYY, 80 nmol/eye NMDA, 80 nmol/eye NMDA+1 nmol/eye GYY, 80 nmol/eye NMDA+10 nmol/eye GYY, and 80 nmol/eye NMDA+100 nmol/eye GYY were taken with the confocal laser microscope. In the NMDA-injected eye, The numbers of RGC expressing ECFP decreased in the NMDA-injected eye. GYY4137 significantly reduced this deleterious effect of NMDA. The counted cells were surrounded with yellow line. Scale bar=100 µm. Original magnification is ×200. (B) The survival rate of RGCs were shown. Data are presented as mean±S.E.M. of 4 (GYY) or 5 (vehicle) independent experiments. * p<0.05, vs. the vehicle-injected group. (C) The effect of GYY itself on the cell number of RGCs were shown. Data are presented as mean±S.E.M. of 4 (GYY) or 5 (vehicle) independent experiments.
To determine whether GYY4137 has an anti-apoptotic effect, we tested the effects of GYY4137 (10 nmol/eye) on the NMDA-induced apoptosis in the retina using TUNEL staining. Typical photomicrographs of TUNEL staining examined 12 h after NMDA injection (80 nmol/eye) were shown in Fig. 2A. The summary of the number of the TUNEL-positive cells from 6 independent experiments are shown in Fig. 2B. GYY4137 significantly decreased the number of the NMDA-induced TUNEL-positive cells in the ganglion cell layer (GCL) and the inner nuclear layer (INL). No TUNEL-positive cells were observed in the retinas of the GYY4137-injected eyes and the vehicle-injected eyes without NMDA (data not shown).
All images were taken 12 h after intravitreal injection. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Arrowheads represent the TUNEL-positive cells. Scale bar=25 µm. Original magnification, ×200. (B) Effect of intravitreal 10 nmol/eye GYY on the number of TUNEL-positive cells induced by 80 nmol/eye NMDA, examined 12 h after intravitreal injection. The number of the TUNEL-positive cells in the ganglion cell layer (GCL) and the inner nuclear layer (INL) in one retinal section are shown. Data are presented as mean±S.E.M. of 6 independent experiments. * p<0.05, vs. the NMDA+PBS-injected group. (C) Immunohistochemical analysis using mouse anti-8-OHdG antibody in the retina of the eye intravitreally injected 80 nmol/eye NMDA+PBS and 80 nmol/eye NMDA+10 nmol/eye GYY. All images were taken 12 h after intravitreal injection. Nuclei were counterstained with DAPI. Arrowheads represent the 8-OHdG-positive cells. Scale bar=25 µm. Original magnification, ×200. (D). Effect of intravitreal 10 nmol/eye GYY on the number of the 8-OHdG-possitive cells induced by 80 nmol/eye NMDA, examined 12 h after intravitreal injection. The number of the 8-OHdG-positive cells in GCL and INL in one retinal section are shown. Data are presented as mean±S.E.M. of 6 independent experiments. * p<0.05, the NMDA+PBS-injected group. IPL, inner plexiform layer; ONL, outer nuclear layer. (E) Scheme of the proposed mechanism of the protective effect of GYY4137.
Reduction of oxidative stress is thought to be involved in the mechanism of the protective effect of H2S. In the present study, we clarified whether GYY4137 (10 nmol/eye) reduced oxidative stress in the cells in GCL and INL by immunohistochemistry using anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) antibody. Typical photomicrographs of the retinal sections obtained 12 h after intravitreal injection of NMDA are shown in Fig. 2C. In the NMDA-injected group, many 8-OHdG positive nuclei were observed in GCL and INL. In the vehicle-injected group, few positive signals of 8-OHdG were found in the retina. Treatment with GYY4137 (10 nmol/eye) significantly reduced the number of the 8-OHdG positive cells induced by NMDA (n=6) (Fig. 2D). No 8-OHdG-positive cells were observed in the retinas of the GYY4137-injected eyes and the vehicle-injected eyes without NMDA (data not shown).
In the present study, we showed that GYY4137, an extended-release donor compound of H2S, had a histologically protective effect against NMDA-induced retinal injury in murine. Intravitreal injection of GYY4137 significantly decreased the number of TUNEL-positive cells and 8-OHdG-positive cells in the NMDA-injected retina. GYY4137 itself did not show the neurotoxic effect on RGCs at the dose used in the present study. These results suggest that GYY4137 prevented the NMDA-induced retinal injury via its anti-oxidative activity. The results in the present study are consistent with those of the previous report showing that GYY4137 protected RGCs against the injuries induced by chronic elevation of intraocular pressure, optic nerve crash and I/R in the rats in vivo.9) However, the estimated intravitreal concentration of GYY4137 in the present study was approximately 1 mM and 1000 folds higher than that in the previous report.9) Although we assume that the reason of the different effective dose is mainly species difference between the mice and the rats, we cannot explain the reason clearly at the present time. Further studies are needed to clarify this issue.
We previously reported that systemic treatment of NaHS, a rapid-release donor compound of H2S, reduced the NMDA-induced neuronal cell death in the retina in vivo.6) In addition, inhalation of H2S before ischemia was also reported to prevent the loss of retinal ganglion cells induced by retinal I/R.3) These reports indicated that systemic treatment with H2S protected retinal neurons against excitotoxicity in the retina. Although these previous reports demonstrated that systemic treatment with H2S and a H2S donor did not induce any toxic effects, at the thought of the toxicity of H2S, topical administration with minimum amounts of the donor is preferred for clinical application of H2S donors. The present study demonstrated that the topically-applied extended-release donor compound of H2S could reduce retinal injury induced by intravitreal NMDA in the mice in vivo. In addition, recently, Liu et al.9) also reported that intravitreal GYY4137 protected against RGC death induced by chronic elevation of intraocular pressure, optic nerve crash and I/R in the rats in vivo. In the future, ophthalmic drop comprised of an improved H2S donor compound may be put into practical use for treatment of the diseases related to excitotoxicity, such as glaucoma and retinal artery occlusion.
Intravitreal GYY4137 as well as intraperitoneal NaHS6) was reduced the number of the 8-OHdG-positive cells induced by intravitreal NMDA, indicating that H2S prevented NMDA-induced retinal injury via its anti-oxidative activity. Scheme of the proposed mechanism of the protective effect of GYY4137 was shown in Fig. 2E. Previous reports also showed that NaHS4) and ACS67,15) an analogue of latanoprost that also acts as a fast-release H2S donor, reduced neuronal apoptosis induced by reactive oxygen species in the light-induced damage of photoreceptor cells and RGC-5, a mammalian cell line, respectively.
GYY4137 is degraded to morpholine and 4-phosphonylanisole when the compound releases H2S.16) It is not clear whether these degraded compounds have protective effects against excitotoxicity in the retina. Further studies are clearly needed to clarify whether these degraded compounds and/or structural analogues of GYY4137 prevent the NMDA-induced retinal injury.
Salvi et al.17) recently reported that ophthalmic drop of H2S donors, including L-cysteine (5%) and GYY4137 (0.1–2%) produced a long-lasting decrease of the intraocular pressure for seven hours after treatment in the rabbit. They also reported that ACS67, a latanoprost analogue that also acts as a fast-release H2S donor reduced the intraocular pressure for four hours after treatment in the rabbit. At the present, a decrease of intraocular pressure is the best way to reduce the defect of field of vision in glaucomatous patients. Because donor compounds of H2S not only protect retinal neurons against excitotoxicity but also decrease intraocular pressure, we strongly believe that H2S donor compounds will be a relevant therapy for glaucomatous patients.
In conclusion, we first demonstrate that GYY4137, a slow-release H2S donor, has neuroprotective effects on the NMDA-induced retinal injury via its anti-oxidative activity in the mice in vivo. The present study suggests that slow-release H2S donors may be good candidates for a for retinal diseases related to excitotoxicity, such as glaucoma and retinal artery occlusion.
This work is supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research (C) #24590329 (KS) and #15K07971 (KS).
The authors declare no conflict of interest.
