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Neuroprotective effect of 5ɑ-androst-3β,5,6β-triol on retinal ganglion cells in a rat chronic ocular hypertension model
Neuroscience Letters 660 (2017) 90–95
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Research article
Neuroprotective effect of 5ɑ-androst-3β,5,6β-triol on retinal ganglion cells in a rat chronic ocular hypertension model
MARK
Yan-Qiu Chena, Shu-Min Zhonga, Shu-Ting Liua, Feng Gaoa,b, Fang Lia, Yuan Zhaoa,b, ⁎ ⁎ Xing-Huai Suna,b, Yanying Miaoa, , Zhongfeng Wanga,b, a Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China b Department of Ophthalmology at Eye & ENT Hospital, Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, Shanghai 200031, China
A R T I C L E I N F O
A B S T R A C T
Keywords: 5ɑ-androst-3β,5,6β-triol Chronic ocular hypertension Retinal ganglion cells Apoptosis Lipid peroxidation Mitochondrial respiratory chain complex
Previous studies have demonstrated that 5ɑ-androst-3β,5,6β-triol (Triol), a synthesized steroid compound, showed notable neuroprotective effect in cultured cortical neurons. In the present study, we explored whether and how Triol have neuroprotective effect on retinal ganglion cells (RGCs) in a chronic ocular hypertension (COH) rat model. COH model was produced by injecting superparamagnetic iron oxide micro-beads into the anterior chamber, and Triol was administrated (4.8 μg/100 g, i.p., once daily for 4 weeks). Immunohistochemistry experiments showed that in whole flat-mounted COH retinas, the number of CTB-labeled survival RGCs was progressively reduced, while TUNEL-positive signals were significantly increased from 1 to 4 weeks after the micro-bead injection. Triol administration significantly attenuated the reduction in the number of CTB-labeled RGCs, and partially reduced the increased number of TUNEL-positive signals in COH retinas. Furthermore, Triol administration partially reduced the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), and significantly rescued the activities of mitochondrial respiratory chain complex (MRCC) I/II/ III in COH retinas. Our results suggest that Triol prevents RGCs from apoptotic death in COH retinas by reducing the lipid peroxidation and enhancing the activities of MRCCs.
1. Introduction Glaucoma is an irreversible blinding neurodegenerative disease, characterized by progressive apoptotic death of retinal ganglion cells (RGCs) [1–3]. Elevated intraocular pressure (IOP) is considered to be one of the most important risk factors [4,5]. However, a subset of glaucoma patients continues to exhibit RGC death and glaucoma progression after IOP has been therapeutically well-controlled [3]. Although the exact mechanism underlying glaucoma pathogenesis remains to be elucidated, glutamate excitotoxicity, oxidative stress, mitochondrial dysfunction, and inflammatory responses have been suggested to contribute to glaucoma pathogenesis [6–8]. Therefore, it is urgently required to develop novel neuroprotective strategies for improving the survival of RGCs in glaucoma, in addition to reducing IOP. Previous studies have demonstrated that cholesterol-3β,5,6β-triol, a major metabolite of cholesterol, protected neurons from the glutamateinduced neurotoxicity, and reduced neuronal injury after spinal cord
ischemia in rabbits and transient focal cerebral ischemia in rats [9]. Ischemia preconditioning may induce an elevated level of cholesterol3β,5,6β-triol, which resulted in subsequent neuroprotection in the spinal cord of rabbits. The neuroprotective effect of cholesterol3β,5,6β-triol was mediated by attenuating Ca2+ influx through NMDA receptors [9]. 24-keto-cholest-5-en-3β, 19-diol, a synthetic steroid cholesterol-3β,5,6β-triol analog, showed similar neuroprotective effect [10]. 5ɑ-androst-3β,5,6β-triol (Triol), another synthesized steroid compound, is a liposoluble steroid compound that shares the same parental structure of endogenous cholesterol-3β,5,6β-triol [11]. Triol significantly ameliorated the reduction in cell viability, mitochondrial membrane potential and ATP production, and decreased oxidative stress induced by hypoxia/reoxygenation exposure in rat primary cultured cortical neurons [11]. These results suggest that steroid compounds could be novel endogenous neuroprotectants. In the present study, effect of Triol on RGCs and the possible mechanisms were investigated in a rat chronic ocular hypertension (COH) model.
Abbreviations: COH, chronic ocular hypertension; CTB, cholera toxin subunit B; IOP, intraocular pressure; MDA, malonaldehyde; Mn-SOD, manganese superoxide dismutase; MRCC, mitochondrial respiratory chain complex; RGCs, retinal ganglion cells; ROS, reactive oxygen species; TUNEL, terminal dUTP nick end labeling ⁎ Corresponding authors at: Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China. E-mail addresses: [email protected] (Y. Miao), [email protected], [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.neulet.2017.09.022 Received 9 June 2017; Received in revised form 21 August 2017; Accepted 11 September 2017 Available online 15 September 2017 0304-3940/ © 2017 Elsevier B.V. All rights reserved.
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2. Materials and methods
Isolation Kit (Cayman chemical, Ann Arbor, USA), and following the manufacturer instructions. The mitochondrial protein concentration was quantitated using the BCA concentration assay kit.
2.1. Animals and rat COH model
2.6. Detection of lipid peroxidation and superoxide dismutase
All experimental procedures described in this study were carried out in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals, and approved by the institutes of Brain Science at Fudan University. Male Sprague–Dawley rats (weighing 200 ± 10 g), were obtained from SLAC Laboratory Animal Co. Ltd (Shanghai, China) and housed under a 12 h light/dark cycle with standard food and water provided ad libitum. Rat COH model was produced by injecting superparamagnetic iron oxide into anterior chamber according to the procedure described in mouse model [12] with some modifications. Briefly, after the rats were anesthetized, the micro-magnetic beads (7 μl, diameter ≈ 9 μm, BioMag®Superparamagnetic Iron Oxide, Bangs Laboratories, Ins) were slowly injected into the anterior chamber under an OPMI VISU 140 microscope (Carl Zeiss, Jena, Germany). Sham-injection (0.9% NaCl solution) was conventionally done on the eyes of other rats. IOP was measured using a handheld Tonolab tonometer (Icare, Finland). The average value of five consecutive measurements with a deviation of less than 5% was accepted. All measurements were performed in the morning to avoid possible circadian difference [13,14]. The IOPs of both eyes were measured before surgery as a baseline (0 d), and then on the second (2 d), fourth (4 d) and seventh days (1 w) after the operation, and weekly afterwards (2 w, 3 w, and 4 w).
Reactive oxygen species (ROS) levels in retinal tissues were detected using the OxiSelect ™ In Vitro ROS/RNS Assay Kit (Cell biolabs, INC., San Diego, USA), as previously described [8]. The ROS/RNS content was converted according to the standard curve prepared by the standard samples. Malondialdehyde (MDA) content in retinal tissues was detected using the OxiSelect ™ MDA Adduct ELISA Kit (Cell biolabs, INC., San Diego, USA), as previously described [15]. The MDA content was converted according to the standard curve prepared by the standard samples. The activity of Mn-SOD was detect using the OxiSelect™ Superoxide Dismutase Activity Assay (Cell biolabs, INC., San Diego, USA), following the procedure described previously [16]. The activity of MnSOD was calculated according to the formula: SOD activity = (ODblank − ODsample)/(ODblank) × 100. 2.7. Mitochondrial function assay The activities of mitochondrial respiratory chain complex (MRCC) I, II and III were assayed using the MitoCheck® Complex I, II, and II/III Activity Assay Kit (Cayman chemical, Ann Arbor, USA), respectively, following the manufacturer instructions. MRCC I activity was detected by the change in the absorbance of the NADH oxidation rate at 340 nm [17]. MRCC II activity was detected by the changes of DCPIP absorbance value at 600 nm [18]. MRCC II/III activity was detected by the changes of MRCC III catalyzed excess cytochrome C (absorbance) at 550 nm [19]. The MRCC III activity was calculated by subtracting MRCC II from MRCC II/III.
2.2. Triol administration From 1d onwards, the COH rats received intraperitoneally injection of Triol (C27H48O3, Sigma-Aldrich, St. Louis, MO, USA) once daily at a dose of 4.8 μg/100 g till 4w. Triol was dissolved in dimethyl sulfoxide (DMSO) at concentration of 0.06 mg/ml. Glaucoma rats receiving injections of equal volume of DMSO were served as vehicle control animals.
2.8. Data analysis 2.3. Labeling and quantification of RGCs All data are presented as mean ± SEM. The data analysis was performed using GraphPad Prism 6 (La Jolla, USA). A one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test and two-way ANOVA with Tukey’s multiple comparisons test were used as appropriate. A value of P < 0.05 was considered significant.
Chorea toxin subunit B (CTB) (List Biological Laboratories, USA) was used as a retrograde tracer to label RGCs, which was performed as previously described in detail [13]. CTB immunohistochemistry in whole flat-mounted retinas were performed 5 days after CTB injection [14]. The number of CTB-positive RGCs was counted following the procedure previously described in detail [14]. Briefly, three fields (approximately 716, 2150 and 3583 μm distant from the optic nerve head) were selected at each of four angles of the retina (0, 90, 180 and 270°) (Fig. 1D). Therefore, total 12 fields were chosen from one retina, and the numbers of CTB-positive cells were counted. All the images were photographed under an Olympus confocal laser scanning microscope (Fluoview 1000, Tokyo, Japan) and assembled using Adobe Photoshop.
3. Results 3.1. Changes of IOP in COH rats The rat COH model was successfully produced with the average IOP of micro-magnetic bead injected eyes being kept at higher levels (15.7 ± 0.7 mmHg to 19.8 ± 1.1 mmHg, n = 45 to 162), which were significantly higher than that of unoperated eyes (10.0 ± 0.1 mmHg, n = 45 to 84) and sham-operated eyes (9.7 ± 0.6 mmHg, n = 45 to 84) (P all < 0.001). In addition, Triol or DMSO administration had no significant effect on IOPs (Fig. 1C).
2.4. Cell apoptosis assay To detect cell apoptosis, terminal dUTP nick end labeling (TUNEL) assay [14] was performed on whole flat-mounted retina, using the DeadEnd Fluorometric TUNEL System G3250 kit (Promega, Madison, WI, USA) and following the manufacturer's instructions. TUNEL signals were visualized with the confocal laser scanning microscope (Fluroview 1000).
3.2. Triol administration increases the number of survival RGCs in COH rats The number of CTB-labeled RGCs in COH retinas with DMSO administration (G-DMSO) was progressively reduced from 1 w to 4 w (Fig. 1A, a2-a5), compared to the control retina (Fig. 1Aa1). In Triol administration retinas (G-Triol), IOP elevation still resulted in loss of RGCs, however, the reduction was significantly attenuated (Fig. 1B, b2b5). We further counted the number of CTB-labeled RGCs in the chosen 12 fields of whole flat-mounted retina (Fig. 1D). The average number was 132.8 ± 2.9/field at 1 w in G-DMSO group (n = 7, P < 0.05 vs.
2.5. Whole retinal and mitochondrial protein extraction Whole retinal proteins were extracted following a previously described procedure [13]. Mitochondria protein extraction was performed as previously described in detailed [8] using the Mitochondrial (Tissue) 91
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Fig. 1. Triol administration promotes RGC survival in COH retinas. (A) Representative confocal images of CTB-labeled RGCs taken from control whole-flat mounted retina (a1), and retinas of COH rats with DMSO administration (G-DMSO) at 1 w (a2), 2 w (a3), 3 w (a4) and 4 w (a5) after operation, respectively. (B) Representative confocal images of CTB-labeled RGCs taken from control (b1), and G-Triol at 1 w (b2), 2 w (b3), 3 w (b4) and 4 w (b5) after operation, respectively. Scale bar: 50 μm for all images in A and B panels. (C) Changes in IOP under different conditions. ***P < 0.001 vs. control at the same time point, & & & P < 0.001 vs. 0d. (D) Schematic diagram showing the selected areas for counting the numbers of CTB-labeled RGCs. Scale bar: 500 μm. (E) Bar chart showing the average number of CTB-labeled RGCs in each field under different conditions. **P < 0.01 and ***P < 0.001 vs. control; & & & P < 0.001 vs. G-DMSO.
peroxidation and antioxidants. We first detected the effect of Triol on MDA, one of lipid peroxidation, content in COH rats. Since Triol showed remarkable neuroprotection at 2 w in COH rats, the following experiments were all carried out at 2 w after the microbead injection. MDA content in retinas of G-DMSO group was increased to 23.8 ± 1.8 pmol/mg protein (n = 8, P < 0.01 vs. control) from the control value of 18.0 ± 1.4 pmol/mg protein (n = 7), while it was significantly reduced to 16.6 ± 1.7 pmol/mg protein (n = 7, P < 0.01 vs. G-DMSO) In G-Triol group (Fig. 3A). In G-DMSO retinas, ROS content was significantly increased to 1404 ± 36 μmol/mg protein (n = 13, P < 0.05 vs. control) from the control value of 1312 ± 46 μmol/mg protein (n = 5). Triol administration significantly reduced the ROS content to 1349 ± 55 μmol/mg protein (n = 6, P < 0.01) in COH retinas (Fig. 3B). The activity of Mn-SOD, one of important antioxidants, exhibited a decreasing tendency in G-DMSO rats, and Triol administration slightly increased it, but not statistically (Fig. 3C). These results suggest that Triol may reduce lipid peroxidation in COH rats through decreasing ROS levels, but not affecting Mn-SOD activity. Mitochondrial dysfunction has been documented as a key component of many neurodegenerative diseases, including glaucoma [8,20]. During oxidative phosphorylation, the MRCCs carry out reactions that drive the production of ATP. We examined whether Triol administration may change activities of MRCC I/II/III in COH rats. MRCC I activity in retinas obtained from G-DMSO group was reduced to 46.0 ± 7.0 μmol/min/mg (n = 6, P < 0.01 vs. the control value of
the control value of 158.1 ± 8.2/field, n = 10), and declined to 40.8 ± 3.2 (n = 46, P < 0.001) at 4 w. Triol administration significantly attenuated the IOP elevation-induced reduction in the number of CTB-positive RGCs, with the average number being 142.1 ± 1.6/field from 1 w (n = 5, P > 0.05 vs. control and P > 0.05 vs. G-DMSO) to 83.7 ± 4.1/field at 4 w (n = 5, P < 0.001 vs. control and P < 0.001 vs. G-DMSO), respectively (Fig. 1E). These results suggest that Triol may protect RGC against glaucoma injurys. 3.3. Triol administration decreases RGC apoptosis in COH rats As shown in Fig. 2 as representative images, sparse TUNEL-positive signals were detected in retina obtained from G-DMSO group at 1 w (a1). Numerous TUNEL-positive signals were observed at 2 w and 3 w (a2 and a3), and then slightly reduced at 4 w (a5). Triol administration significantly reduced the number of TUNEL-positive signals (b1-b5). On average, Triol administration significantly decreased the total number of TUNEL-positive signals in whole flat-mounted COH retinas (Fig. 2C). These results strongly suggest that Triol rescues RGCs in rat COH model through inhibiting the apoptosis of RGCs. 3.4. Triol administration reduces MDA and ROS contents, and rescues MRCC activities in COH rats Oxidative stress is one of the main mechanisms in pathogenesis of glaucoma, which is resulted from the imbalance between lipid 92
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(3.8 ± 0.3 pmol/min/mg, n = 8, P < 0.001 vs. G-DMSO, and P < 0.001 vs. control) (Fig. 4B). Furthermore, MRCC III activity decreased to 30.9 ± 4.5 pmol/min/mg (n = 6, P < 0.01 vs. control) in G-DMSO group from the control value of 46.9 ± 4.1 pmol/min/mg (n = 8), while it was partially rescued to 35.4 ± 4.4 pmol/min/mg (n = 8, P < 0.05 vs. control) (Fig. 4C). These results suggest that Triol may protect RGCs in COH retinas by inhibiting the reduction of MRCC I/II/III activities. 4. Discussion In the present study, we demonstrate that Triol provides neuroprotective effect on RGCs in experimental glaucoma rats, which is mediated by reducing RGC apoptosis through inhibiting lipid peroxidation and rescuing activities of MRCCs. Since elevated IOP is one of the major risk factors in glaucomatous pathogenesis [4,5], various COH animal models have been employed to study glaucoma, such as ligation of episcleral veins [13,14], trabecular mesh laser photocoagulation [21], polystyrene microspheres anterior chamber injection [22], etc. The advantage and disadvantage of these experimental glaucoma models have been widely discussed [23–25]. Among these models, the polystyrene microbead injection offers a relatively easy method, and the elevated IOP could be maintained with subsequent injections of the microbeads. However, the major disadvantage of this model is that polystyrene microspheres is not easy to be retained in the anterior chamber angle after injection [24]. In our rat COH model, superparamagnetic iron oxide beads were used instead of polystyrene microbeads. It was clearly showed that micro-magnetic beads could be evenly distributed around the anterior chamber angle by the hand-held magnet, and the IOP was steadily maintained at higher levels, similar to the report in mouse [12]. Our present work provides direct evidence showing that Triol protected RGCs against glaucomatous injury, which was due to inhibiting RGC apoptosis based on the following facts. Firstly, Triol administration significantly attenuated the reduction in the number of CTB-labeled RGCs in COH rats. Secondly, Triol administration significantly reduced the number of TUNEL-positive signals in COH rats, which was consistent with the changes in number of CTB-labeled RGCs on time course. It should be noted that the number of TUNEL-positive signals was slightly decreased at 4 w in COH retinas. We speculated that a large number of RGCs has been died at that time. Growing evidence has shown that oxidative stress is proposed as an etiologic factor in the pathophysiology of glaucomatous RGC death. RGCs are especially susceptible to oxidative stress because of their tremendous oxygen consumption and high proportion of polyunsaturated fatty acids [26,27]. Oxidative stresses are induced through the formation of ROS. ROS may decrease mitochondrial membrane potential, then induce cytochrome C release, thus triggering the mitochondria-mediated RGC apoptosis [28,29]. In addition, ROS may also activate caspase cascades, which result in RGC apoptosis [29]. MDA, one of major lipid peroxidation products, is another indicator of oxidative stress, which may reflect cell damage to certain extent. Our results showed that MDA and ROS levels were indeed significantly increased in COH retinas, and Triol administration completely rescued MDA level and partially inhibited ROS level, suggesting that Triol may mainly inhibit lipid peroxidation in COH retinas. It should be noted that in our COH model, we did not detect significant change in the activity of Mn-SOD, an antioxidant, in COH retinas with or without Triol administration. We speculate that the change of Mn-SOD activity in COH retinas may be transient and occurred in an early stage after IOP elevation, which remains to be examined in our future study. Previous study has shown that activities of MRCC I and III were significantly reduced in an experimental glaucoma model [8]. Consistently, in the present study, we observed the decreased activities of MRCC I/II/III in our rat glaucoma model. Considering the fact that MRCC activity is closely related to ATP production, decreased MRCC I/
Fig. 2. Triol administration reduces the number of RGC apoptosis in COH rats. (A) Microphotographs show representative confocal images of TUNEL signals (green) in whole flat-mounted retinas taken from control(a1) and G-DMSO groups at 1 w (a2), 2 w (a3), 3 w (a4) and 4 w (a5) after operation, respectively. (B) Microphotographs show representative confocal images of TUNEL signals (green) in whole flat-mounted retinas taken from control (b1) and G-Triol groups at 1 w (b2), 2 w (b3), 3 w (b4) and 4 w (b5) after operation, respectively. Scale bar: 50 μm for all images. (C) Bar chart showing the average total number of TUNEL-positive signals in whole flat-mounted retinas under different conditions. *P < 0.05 and ***P < 0.001 vs. control; & & & P < 0.001 vs. GDMSO.
79.8 ± 8.6 μmol/min/mg, n = 8). Triol administration partially rescued the reduced activity of MRCC I in COH rats (58.3 ± 8.5 μmol/ min/mg, n = 8, P < 0.05 vs. control) (Fig. 4A). Tremendous decrease in MRCC II activity was observed in G-DMSO retinas (0.9 ± 0.2 pmol/ min/mg, n = 5, P < 0.001 vs. the control value of 6.1 ± 0.3 pmol/ min/mg, n = 8) (Fig. 4B). Triol administration significantly and partially rescued the reduction of MRCC II activity in COH rats 93
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Fig. 3. Triol administration partially reverses changes in MDA and ROS contents in COH retinas. (A, B) Bar chart showing changes in the average contents of MDA (A) and ROS (B) in G-DMSO and G-Triol groups, assayed by ELISA. MDA content was expressed as pmol/mg protein. (C) Bar chart showing changes in the average activity of Mn-SOD in G-DMSO and G-Triol groups. *P < 0.05 and **P < 0.01 vs. control; & & P < 0.01 vs. G-DMSO.
Fig. 4. Triol administration partially reverses changes in activities of MRCC I, II, and III in COH retinas. (A, B, C) Bar chart showing changes in the average activity of MRCC I (A), II (B), and III (C) in G-DMSO and G-Triol groups. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control; & & & P < 0.001 vs. G-DMSO.
References
II/III activities may result in mitochondrial dysfunction, thus contributing to RGC injury and apoptosis. Triol administration partially reversed the reduced MRCC I/II/III activities, suggesting the neuroprotective role of Triol on RGCs in glaucoma retinas is at least partially by improving the mitochondrial function, which is consistent with the observations in rat primary cultured cortical neurons with hypoxia/ reoxygenation exposure [11]. Numerous studies have demonstrated that endogenous steroid compounds inhibited neuronal apoptosis by reducing Ca2+ influx through NMDA receptors, improving antioxidant capacity, and increasing anti-apoptotic protein Bcl-2 level [9–11,30,31]. Our present work showed that Triol significantly reduced MDA level and partially reversed the reduction of MRCC activities in COH rats, suggesting that Triol may be a potential neuroprotectant in treatment of glaucoma. Triol is a lipsoluble compound and may pass through the bloodbrain or blood-retinal barriers, thus acting at brain or retinal cells. In addition, the concentration of endogenous cholesterol-3β,5α,6β-triol, a major metabolite of cholesterol, in rat blood plasma is around 0.5 μM [9]. In the present study, the estimated maximal plasma concentration of Triol is around 1 μM. At lower concentration, Triol has no significant side effects on neurological and histopathological outcomes [9]. Whether Triol at lower concentration may affect vision behavior remains to be addressed in our future study.
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Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31671078; 81430007), the Natural Science Foundation of Shanghai, China (14ZR1402200), and the International Science & Technology Cooperation Program of China (2015DFA31340). 94
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Neuroprotective effect of 5ɑ-androst-3β,5,6β-triol on retinal ganglion cells in a rat chronic ocular hypertension model
MARK
Yan-Qiu Chena, Shu-Min Zhonga, Shu-Ting Liua, Feng Gaoa,b, Fang Lia, Yuan Zhaoa,b, ⁎ ⁎ Xing-Huai Suna,b, Yanying Miaoa, , Zhongfeng Wanga,b, a Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China b Department of Ophthalmology at Eye & ENT Hospital, Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, Shanghai 200031, China
A R T I C L E I N F O
A B S T R A C T
Keywords: 5ɑ-androst-3β,5,6β-triol Chronic ocular hypertension Retinal ganglion cells Apoptosis Lipid peroxidation Mitochondrial respiratory chain complex
Previous studies have demonstrated that 5ɑ-androst-3β,5,6β-triol (Triol), a synthesized steroid compound, showed notable neuroprotective effect in cultured cortical neurons. In the present study, we explored whether and how Triol have neuroprotective effect on retinal ganglion cells (RGCs) in a chronic ocular hypertension (COH) rat model. COH model was produced by injecting superparamagnetic iron oxide micro-beads into the anterior chamber, and Triol was administrated (4.8 μg/100 g, i.p., once daily for 4 weeks). Immunohistochemistry experiments showed that in whole flat-mounted COH retinas, the number of CTB-labeled survival RGCs was progressively reduced, while TUNEL-positive signals were significantly increased from 1 to 4 weeks after the micro-bead injection. Triol administration significantly attenuated the reduction in the number of CTB-labeled RGCs, and partially reduced the increased number of TUNEL-positive signals in COH retinas. Furthermore, Triol administration partially reduced the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), and significantly rescued the activities of mitochondrial respiratory chain complex (MRCC) I/II/ III in COH retinas. Our results suggest that Triol prevents RGCs from apoptotic death in COH retinas by reducing the lipid peroxidation and enhancing the activities of MRCCs.
1. Introduction Glaucoma is an irreversible blinding neurodegenerative disease, characterized by progressive apoptotic death of retinal ganglion cells (RGCs) [1–3]. Elevated intraocular pressure (IOP) is considered to be one of the most important risk factors [4,5]. However, a subset of glaucoma patients continues to exhibit RGC death and glaucoma progression after IOP has been therapeutically well-controlled [3]. Although the exact mechanism underlying glaucoma pathogenesis remains to be elucidated, glutamate excitotoxicity, oxidative stress, mitochondrial dysfunction, and inflammatory responses have been suggested to contribute to glaucoma pathogenesis [6–8]. Therefore, it is urgently required to develop novel neuroprotective strategies for improving the survival of RGCs in glaucoma, in addition to reducing IOP. Previous studies have demonstrated that cholesterol-3β,5,6β-triol, a major metabolite of cholesterol, protected neurons from the glutamateinduced neurotoxicity, and reduced neuronal injury after spinal cord
ischemia in rabbits and transient focal cerebral ischemia in rats [9]. Ischemia preconditioning may induce an elevated level of cholesterol3β,5,6β-triol, which resulted in subsequent neuroprotection in the spinal cord of rabbits. The neuroprotective effect of cholesterol3β,5,6β-triol was mediated by attenuating Ca2+ influx through NMDA receptors [9]. 24-keto-cholest-5-en-3β, 19-diol, a synthetic steroid cholesterol-3β,5,6β-triol analog, showed similar neuroprotective effect [10]. 5ɑ-androst-3β,5,6β-triol (Triol), another synthesized steroid compound, is a liposoluble steroid compound that shares the same parental structure of endogenous cholesterol-3β,5,6β-triol [11]. Triol significantly ameliorated the reduction in cell viability, mitochondrial membrane potential and ATP production, and decreased oxidative stress induced by hypoxia/reoxygenation exposure in rat primary cultured cortical neurons [11]. These results suggest that steroid compounds could be novel endogenous neuroprotectants. In the present study, effect of Triol on RGCs and the possible mechanisms were investigated in a rat chronic ocular hypertension (COH) model.
Abbreviations: COH, chronic ocular hypertension; CTB, cholera toxin subunit B; IOP, intraocular pressure; MDA, malonaldehyde; Mn-SOD, manganese superoxide dismutase; MRCC, mitochondrial respiratory chain complex; RGCs, retinal ganglion cells; ROS, reactive oxygen species; TUNEL, terminal dUTP nick end labeling ⁎ Corresponding authors at: Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China. E-mail addresses: [email protected] (Y. Miao), [email protected], [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.neulet.2017.09.022 Received 9 June 2017; Received in revised form 21 August 2017; Accepted 11 September 2017 Available online 15 September 2017 0304-3940/ © 2017 Elsevier B.V. All rights reserved.
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2. Materials and methods
Isolation Kit (Cayman chemical, Ann Arbor, USA), and following the manufacturer instructions. The mitochondrial protein concentration was quantitated using the BCA concentration assay kit.
2.1. Animals and rat COH model
2.6. Detection of lipid peroxidation and superoxide dismutase
All experimental procedures described in this study were carried out in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals, and approved by the institutes of Brain Science at Fudan University. Male Sprague–Dawley rats (weighing 200 ± 10 g), were obtained from SLAC Laboratory Animal Co. Ltd (Shanghai, China) and housed under a 12 h light/dark cycle with standard food and water provided ad libitum. Rat COH model was produced by injecting superparamagnetic iron oxide into anterior chamber according to the procedure described in mouse model [12] with some modifications. Briefly, after the rats were anesthetized, the micro-magnetic beads (7 μl, diameter ≈ 9 μm, BioMag®Superparamagnetic Iron Oxide, Bangs Laboratories, Ins) were slowly injected into the anterior chamber under an OPMI VISU 140 microscope (Carl Zeiss, Jena, Germany). Sham-injection (0.9% NaCl solution) was conventionally done on the eyes of other rats. IOP was measured using a handheld Tonolab tonometer (Icare, Finland). The average value of five consecutive measurements with a deviation of less than 5% was accepted. All measurements were performed in the morning to avoid possible circadian difference [13,14]. The IOPs of both eyes were measured before surgery as a baseline (0 d), and then on the second (2 d), fourth (4 d) and seventh days (1 w) after the operation, and weekly afterwards (2 w, 3 w, and 4 w).
Reactive oxygen species (ROS) levels in retinal tissues were detected using the OxiSelect ™ In Vitro ROS/RNS Assay Kit (Cell biolabs, INC., San Diego, USA), as previously described [8]. The ROS/RNS content was converted according to the standard curve prepared by the standard samples. Malondialdehyde (MDA) content in retinal tissues was detected using the OxiSelect ™ MDA Adduct ELISA Kit (Cell biolabs, INC., San Diego, USA), as previously described [15]. The MDA content was converted according to the standard curve prepared by the standard samples. The activity of Mn-SOD was detect using the OxiSelect™ Superoxide Dismutase Activity Assay (Cell biolabs, INC., San Diego, USA), following the procedure described previously [16]. The activity of MnSOD was calculated according to the formula: SOD activity = (ODblank − ODsample)/(ODblank) × 100. 2.7. Mitochondrial function assay The activities of mitochondrial respiratory chain complex (MRCC) I, II and III were assayed using the MitoCheck® Complex I, II, and II/III Activity Assay Kit (Cayman chemical, Ann Arbor, USA), respectively, following the manufacturer instructions. MRCC I activity was detected by the change in the absorbance of the NADH oxidation rate at 340 nm [17]. MRCC II activity was detected by the changes of DCPIP absorbance value at 600 nm [18]. MRCC II/III activity was detected by the changes of MRCC III catalyzed excess cytochrome C (absorbance) at 550 nm [19]. The MRCC III activity was calculated by subtracting MRCC II from MRCC II/III.
2.2. Triol administration From 1d onwards, the COH rats received intraperitoneally injection of Triol (C27H48O3, Sigma-Aldrich, St. Louis, MO, USA) once daily at a dose of 4.8 μg/100 g till 4w. Triol was dissolved in dimethyl sulfoxide (DMSO) at concentration of 0.06 mg/ml. Glaucoma rats receiving injections of equal volume of DMSO were served as vehicle control animals.
2.8. Data analysis 2.3. Labeling and quantification of RGCs All data are presented as mean ± SEM. The data analysis was performed using GraphPad Prism 6 (La Jolla, USA). A one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test and two-way ANOVA with Tukey’s multiple comparisons test were used as appropriate. A value of P < 0.05 was considered significant.
Chorea toxin subunit B (CTB) (List Biological Laboratories, USA) was used as a retrograde tracer to label RGCs, which was performed as previously described in detail [13]. CTB immunohistochemistry in whole flat-mounted retinas were performed 5 days after CTB injection [14]. The number of CTB-positive RGCs was counted following the procedure previously described in detail [14]. Briefly, three fields (approximately 716, 2150 and 3583 μm distant from the optic nerve head) were selected at each of four angles of the retina (0, 90, 180 and 270°) (Fig. 1D). Therefore, total 12 fields were chosen from one retina, and the numbers of CTB-positive cells were counted. All the images were photographed under an Olympus confocal laser scanning microscope (Fluoview 1000, Tokyo, Japan) and assembled using Adobe Photoshop.
3. Results 3.1. Changes of IOP in COH rats The rat COH model was successfully produced with the average IOP of micro-magnetic bead injected eyes being kept at higher levels (15.7 ± 0.7 mmHg to 19.8 ± 1.1 mmHg, n = 45 to 162), which were significantly higher than that of unoperated eyes (10.0 ± 0.1 mmHg, n = 45 to 84) and sham-operated eyes (9.7 ± 0.6 mmHg, n = 45 to 84) (P all < 0.001). In addition, Triol or DMSO administration had no significant effect on IOPs (Fig. 1C).
2.4. Cell apoptosis assay To detect cell apoptosis, terminal dUTP nick end labeling (TUNEL) assay [14] was performed on whole flat-mounted retina, using the DeadEnd Fluorometric TUNEL System G3250 kit (Promega, Madison, WI, USA) and following the manufacturer's instructions. TUNEL signals were visualized with the confocal laser scanning microscope (Fluroview 1000).
3.2. Triol administration increases the number of survival RGCs in COH rats The number of CTB-labeled RGCs in COH retinas with DMSO administration (G-DMSO) was progressively reduced from 1 w to 4 w (Fig. 1A, a2-a5), compared to the control retina (Fig. 1Aa1). In Triol administration retinas (G-Triol), IOP elevation still resulted in loss of RGCs, however, the reduction was significantly attenuated (Fig. 1B, b2b5). We further counted the number of CTB-labeled RGCs in the chosen 12 fields of whole flat-mounted retina (Fig. 1D). The average number was 132.8 ± 2.9/field at 1 w in G-DMSO group (n = 7, P < 0.05 vs.
2.5. Whole retinal and mitochondrial protein extraction Whole retinal proteins were extracted following a previously described procedure [13]. Mitochondria protein extraction was performed as previously described in detailed [8] using the Mitochondrial (Tissue) 91
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Fig. 1. Triol administration promotes RGC survival in COH retinas. (A) Representative confocal images of CTB-labeled RGCs taken from control whole-flat mounted retina (a1), and retinas of COH rats with DMSO administration (G-DMSO) at 1 w (a2), 2 w (a3), 3 w (a4) and 4 w (a5) after operation, respectively. (B) Representative confocal images of CTB-labeled RGCs taken from control (b1), and G-Triol at 1 w (b2), 2 w (b3), 3 w (b4) and 4 w (b5) after operation, respectively. Scale bar: 50 μm for all images in A and B panels. (C) Changes in IOP under different conditions. ***P < 0.001 vs. control at the same time point, & & & P < 0.001 vs. 0d. (D) Schematic diagram showing the selected areas for counting the numbers of CTB-labeled RGCs. Scale bar: 500 μm. (E) Bar chart showing the average number of CTB-labeled RGCs in each field under different conditions. **P < 0.01 and ***P < 0.001 vs. control; & & & P < 0.001 vs. G-DMSO.
peroxidation and antioxidants. We first detected the effect of Triol on MDA, one of lipid peroxidation, content in COH rats. Since Triol showed remarkable neuroprotection at 2 w in COH rats, the following experiments were all carried out at 2 w after the microbead injection. MDA content in retinas of G-DMSO group was increased to 23.8 ± 1.8 pmol/mg protein (n = 8, P < 0.01 vs. control) from the control value of 18.0 ± 1.4 pmol/mg protein (n = 7), while it was significantly reduced to 16.6 ± 1.7 pmol/mg protein (n = 7, P < 0.01 vs. G-DMSO) In G-Triol group (Fig. 3A). In G-DMSO retinas, ROS content was significantly increased to 1404 ± 36 μmol/mg protein (n = 13, P < 0.05 vs. control) from the control value of 1312 ± 46 μmol/mg protein (n = 5). Triol administration significantly reduced the ROS content to 1349 ± 55 μmol/mg protein (n = 6, P < 0.01) in COH retinas (Fig. 3B). The activity of Mn-SOD, one of important antioxidants, exhibited a decreasing tendency in G-DMSO rats, and Triol administration slightly increased it, but not statistically (Fig. 3C). These results suggest that Triol may reduce lipid peroxidation in COH rats through decreasing ROS levels, but not affecting Mn-SOD activity. Mitochondrial dysfunction has been documented as a key component of many neurodegenerative diseases, including glaucoma [8,20]. During oxidative phosphorylation, the MRCCs carry out reactions that drive the production of ATP. We examined whether Triol administration may change activities of MRCC I/II/III in COH rats. MRCC I activity in retinas obtained from G-DMSO group was reduced to 46.0 ± 7.0 μmol/min/mg (n = 6, P < 0.01 vs. the control value of
the control value of 158.1 ± 8.2/field, n = 10), and declined to 40.8 ± 3.2 (n = 46, P < 0.001) at 4 w. Triol administration significantly attenuated the IOP elevation-induced reduction in the number of CTB-positive RGCs, with the average number being 142.1 ± 1.6/field from 1 w (n = 5, P > 0.05 vs. control and P > 0.05 vs. G-DMSO) to 83.7 ± 4.1/field at 4 w (n = 5, P < 0.001 vs. control and P < 0.001 vs. G-DMSO), respectively (Fig. 1E). These results suggest that Triol may protect RGC against glaucoma injurys. 3.3. Triol administration decreases RGC apoptosis in COH rats As shown in Fig. 2 as representative images, sparse TUNEL-positive signals were detected in retina obtained from G-DMSO group at 1 w (a1). Numerous TUNEL-positive signals were observed at 2 w and 3 w (a2 and a3), and then slightly reduced at 4 w (a5). Triol administration significantly reduced the number of TUNEL-positive signals (b1-b5). On average, Triol administration significantly decreased the total number of TUNEL-positive signals in whole flat-mounted COH retinas (Fig. 2C). These results strongly suggest that Triol rescues RGCs in rat COH model through inhibiting the apoptosis of RGCs. 3.4. Triol administration reduces MDA and ROS contents, and rescues MRCC activities in COH rats Oxidative stress is one of the main mechanisms in pathogenesis of glaucoma, which is resulted from the imbalance between lipid 92
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(3.8 ± 0.3 pmol/min/mg, n = 8, P < 0.001 vs. G-DMSO, and P < 0.001 vs. control) (Fig. 4B). Furthermore, MRCC III activity decreased to 30.9 ± 4.5 pmol/min/mg (n = 6, P < 0.01 vs. control) in G-DMSO group from the control value of 46.9 ± 4.1 pmol/min/mg (n = 8), while it was partially rescued to 35.4 ± 4.4 pmol/min/mg (n = 8, P < 0.05 vs. control) (Fig. 4C). These results suggest that Triol may protect RGCs in COH retinas by inhibiting the reduction of MRCC I/II/III activities. 4. Discussion In the present study, we demonstrate that Triol provides neuroprotective effect on RGCs in experimental glaucoma rats, which is mediated by reducing RGC apoptosis through inhibiting lipid peroxidation and rescuing activities of MRCCs. Since elevated IOP is one of the major risk factors in glaucomatous pathogenesis [4,5], various COH animal models have been employed to study glaucoma, such as ligation of episcleral veins [13,14], trabecular mesh laser photocoagulation [21], polystyrene microspheres anterior chamber injection [22], etc. The advantage and disadvantage of these experimental glaucoma models have been widely discussed [23–25]. Among these models, the polystyrene microbead injection offers a relatively easy method, and the elevated IOP could be maintained with subsequent injections of the microbeads. However, the major disadvantage of this model is that polystyrene microspheres is not easy to be retained in the anterior chamber angle after injection [24]. In our rat COH model, superparamagnetic iron oxide beads were used instead of polystyrene microbeads. It was clearly showed that micro-magnetic beads could be evenly distributed around the anterior chamber angle by the hand-held magnet, and the IOP was steadily maintained at higher levels, similar to the report in mouse [12]. Our present work provides direct evidence showing that Triol protected RGCs against glaucomatous injury, which was due to inhibiting RGC apoptosis based on the following facts. Firstly, Triol administration significantly attenuated the reduction in the number of CTB-labeled RGCs in COH rats. Secondly, Triol administration significantly reduced the number of TUNEL-positive signals in COH rats, which was consistent with the changes in number of CTB-labeled RGCs on time course. It should be noted that the number of TUNEL-positive signals was slightly decreased at 4 w in COH retinas. We speculated that a large number of RGCs has been died at that time. Growing evidence has shown that oxidative stress is proposed as an etiologic factor in the pathophysiology of glaucomatous RGC death. RGCs are especially susceptible to oxidative stress because of their tremendous oxygen consumption and high proportion of polyunsaturated fatty acids [26,27]. Oxidative stresses are induced through the formation of ROS. ROS may decrease mitochondrial membrane potential, then induce cytochrome C release, thus triggering the mitochondria-mediated RGC apoptosis [28,29]. In addition, ROS may also activate caspase cascades, which result in RGC apoptosis [29]. MDA, one of major lipid peroxidation products, is another indicator of oxidative stress, which may reflect cell damage to certain extent. Our results showed that MDA and ROS levels were indeed significantly increased in COH retinas, and Triol administration completely rescued MDA level and partially inhibited ROS level, suggesting that Triol may mainly inhibit lipid peroxidation in COH retinas. It should be noted that in our COH model, we did not detect significant change in the activity of Mn-SOD, an antioxidant, in COH retinas with or without Triol administration. We speculate that the change of Mn-SOD activity in COH retinas may be transient and occurred in an early stage after IOP elevation, which remains to be examined in our future study. Previous study has shown that activities of MRCC I and III were significantly reduced in an experimental glaucoma model [8]. Consistently, in the present study, we observed the decreased activities of MRCC I/II/III in our rat glaucoma model. Considering the fact that MRCC activity is closely related to ATP production, decreased MRCC I/
Fig. 2. Triol administration reduces the number of RGC apoptosis in COH rats. (A) Microphotographs show representative confocal images of TUNEL signals (green) in whole flat-mounted retinas taken from control(a1) and G-DMSO groups at 1 w (a2), 2 w (a3), 3 w (a4) and 4 w (a5) after operation, respectively. (B) Microphotographs show representative confocal images of TUNEL signals (green) in whole flat-mounted retinas taken from control (b1) and G-Triol groups at 1 w (b2), 2 w (b3), 3 w (b4) and 4 w (b5) after operation, respectively. Scale bar: 50 μm for all images. (C) Bar chart showing the average total number of TUNEL-positive signals in whole flat-mounted retinas under different conditions. *P < 0.05 and ***P < 0.001 vs. control; & & & P < 0.001 vs. GDMSO.
79.8 ± 8.6 μmol/min/mg, n = 8). Triol administration partially rescued the reduced activity of MRCC I in COH rats (58.3 ± 8.5 μmol/ min/mg, n = 8, P < 0.05 vs. control) (Fig. 4A). Tremendous decrease in MRCC II activity was observed in G-DMSO retinas (0.9 ± 0.2 pmol/ min/mg, n = 5, P < 0.001 vs. the control value of 6.1 ± 0.3 pmol/ min/mg, n = 8) (Fig. 4B). Triol administration significantly and partially rescued the reduction of MRCC II activity in COH rats 93
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Fig. 3. Triol administration partially reverses changes in MDA and ROS contents in COH retinas. (A, B) Bar chart showing changes in the average contents of MDA (A) and ROS (B) in G-DMSO and G-Triol groups, assayed by ELISA. MDA content was expressed as pmol/mg protein. (C) Bar chart showing changes in the average activity of Mn-SOD in G-DMSO and G-Triol groups. *P < 0.05 and **P < 0.01 vs. control; & & P < 0.01 vs. G-DMSO.
Fig. 4. Triol administration partially reverses changes in activities of MRCC I, II, and III in COH retinas. (A, B, C) Bar chart showing changes in the average activity of MRCC I (A), II (B), and III (C) in G-DMSO and G-Triol groups. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control; & & & P < 0.001 vs. G-DMSO.
References
II/III activities may result in mitochondrial dysfunction, thus contributing to RGC injury and apoptosis. Triol administration partially reversed the reduced MRCC I/II/III activities, suggesting the neuroprotective role of Triol on RGCs in glaucoma retinas is at least partially by improving the mitochondrial function, which is consistent with the observations in rat primary cultured cortical neurons with hypoxia/ reoxygenation exposure [11]. Numerous studies have demonstrated that endogenous steroid compounds inhibited neuronal apoptosis by reducing Ca2+ influx through NMDA receptors, improving antioxidant capacity, and increasing anti-apoptotic protein Bcl-2 level [9–11,30,31]. Our present work showed that Triol significantly reduced MDA level and partially reversed the reduction of MRCC activities in COH rats, suggesting that Triol may be a potential neuroprotectant in treatment of glaucoma. Triol is a lipsoluble compound and may pass through the bloodbrain or blood-retinal barriers, thus acting at brain or retinal cells. In addition, the concentration of endogenous cholesterol-3β,5α,6β-triol, a major metabolite of cholesterol, in rat blood plasma is around 0.5 μM [9]. In the present study, the estimated maximal plasma concentration of Triol is around 1 μM. At lower concentration, Triol has no significant side effects on neurological and histopathological outcomes [9]. Whether Triol at lower concentration may affect vision behavior remains to be addressed in our future study.
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Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31671078; 81430007), the Natural Science Foundation of Shanghai, China (14ZR1402200), and the International Science & Technology Cooperation Program of China (2015DFA31340). 94
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