November 2014
Volume 55, Issue 11
Free
Biochemistry and Molecular Biology  |   November 2014
The Novel Rho Kinase (ROCK) Inhibitor K-115: A New Candidate Drug for Neuroprotective Treatment in Glaucoma
Author Notes
  • Department of Ophthalmology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan 
  • Correspondence: Toru Nakazawa, Tohoku University Graduate School of Medicine, Department of Ophthalmology, 1-1 Seiryo, Aoba, Sendai, Miyagi, 980-8574, Japan; ntoru@oph.med.tohoku.ac.jp
Investigative Ophthalmology & Visual Science November 2014, Vol.55, 7126-7136. doi:10.1167/iovs.13-13842
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Kotaro Yamamoto, Kazuichi Maruyama, Noriko Himori, Kazuko Omodaka, Yu Yokoyama, Yukihiro Shiga, Ryu Morin, Toru Nakazawa; The Novel Rho Kinase (ROCK) Inhibitor K-115: A New Candidate Drug for Neuroprotective Treatment in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2014;55(11):7126-7136. doi: 10.1167/iovs.13-13842.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: To investigate the effect of K-115, a novel Rho kinase (ROCK) inhibitor, on retinal ganglion cell (RGC) survival in an optic nerve crush (NC) model. Additionally, to determine the details of the mechanism of K-115's neuroprotective effect in vivo and in vitro.

Methods.: ROCK inhibitors, including K-115 and fasudil (1 mg/kg/d), or vehicle were administered orally to C57BL/6 mice. Retinal ganglion cell death was then induced with NC. Retinal ganglion cell survival was evaluated by counting surviving retrogradely labeled cells and measuring RGC marker expression with quantitative real-time polymerase chain reaction (qRT-PCR). Total oxidized lipid levels were assessed with a thiobarbituric acid-reactive substances (TBARS) assay. Reactive oxygen species (ROS) levels were assessed by co-labeling with CellROX and Fluorogold. Expression of the NADPH oxidase (Nox) family of genes was evaluated with qRT-PCR.

Results.: The survival of RGCs after NC was increased 34 ± 3% with K-115, a significantly protective effect. Moreover, a similar effect was revealed by the qRT-PCR analysis of Thy-1.2 and Brn3a, RGC markers. Levels of oxidized lipids and ROS also increased with time after NC. NC-induced oxidative stress, including oxidation of lipids and production of ROS, was significantly attenuated by K-115. Furthermore, expression of the Nox gene family, especially Nox1, which is involved in the NC-induced ROS production pathway, was dramatically reduced by K-115.

Conclusions.: The results indicated that oral K-115 administration delayed RGC death. Although K-115 may be mediated through Nox1 downregulation, we found that it did not suppress ROS production directly. Our findings show that K-115 has a potential use in neuroprotective treatment for glaucoma and other neurodegenerative diseases.

Introduction
Glaucoma is well known as one of the world's major causes of secondary blindness,1 and in Japan in particular, glaucoma is quickly becoming the most common cause of secondary blindness. Maintenance of low intraocular pressure (IOP) is the classic treatment for glaucoma and is the only therapy that has been shown to be effective in large-scale clinical studies. The primary method of reducing IOP is generally medication, mainly topical eye drops, although filtration surgery is also used. These are the only current treatments for glaucoma. Increased IOP is the most well-known risk factor for the progression of glaucoma, and IOP reduction is usually effective in slowing the progress of the disease. However, the majority of glaucoma patients in Asia are affected by normal tension glaucoma (NTG), and recent epidemiological studies have revealed that IOP reduction alone cannot prevent the progression of visual field loss in these patients.2,3 In addition to reducing IOP, reduction of damage to retinal ganglion cells (RGCs) caused by IOP-independent risk factors such as mechanical stress on the axons in the lamina cribrosa might be useful for treating NTG.4 Novel treatment strategies have therefore recently been explored, such as protecting RGCs or increasing retinal or choroidal blood flow. In particular, the neuroprotection of RGCs has drawn attention as a new approach to glaucoma therapy because it is thought that the ultimate cause of vision loss in glaucoma is RGC apoptosis.5 
Several potential mechanisms of RGC death in glaucoma have been hypothesized, including compromised blood flow in the optic nerve,6,7 nitric oxide–induced injury to the optic nerve,810 and glutamate excitotoxicity.1113 In addition to these primary mechanisms, other studies have provided evidence that oxidative stress contributes to the degeneration of RGCs in glaucoma.1417 However, the precise nature of the damage caused to RGCs by oxidative stress remains unclear. Moreover, treatments for oxidative stress in glaucoma patients have not been established. 
Rho kinase (ROCK) is a serine/threonine (Ser/Thr) protein kinase and a key downstream effector of Rho.18,19 ROCK controls multiple signaling pathways and many cellular processes such as cytoskeletal rearrangement and cell movement.20 Thus, it has recently been suggested that the Rho/ROCK pathway is involved in a number of disorders. Indeed, abnormal activation of ROCK has been observed in diabetic nephropathy,2126 cardiovascular disease,19,24,2731 and central nervous system (CNS) diseases including Alzheimer's disease,3234 spinal cord injury,32,3538 stroke,3946 multiple sclerosis,32 and glaucoma.4756 In particular, a recent study reported that the protein level of RhoA increased in the optic nerve head of patients with primary open-angle glaucoma (POAG),57 an effect that might lead to excessive activation of ROCK. Many studies using models such as hypertension, hyperlipidemia, and diabetes have demonstrated that ROCK activation caused elevated oxidative stress levels via NADPH oxidase (Nox), and that this was eliminated by oral administration of the ROCK inhibitor fasudil.22 
ROCK inhibitors are thought to be one of the most promising candidates for the treatment of glaucoma. Previously, the targeting of small Rho GTPase has been shown to increase regeneration in models of optic nerve lesions.58 Specifically, pharmacological inhibition of ROCK had a dose-dependent regenerative effect on RGCs after an optic nerve crush (NC) injury.59 Moreover, selective ROCK inhibitors have also been shown to lower IOP in rabbits,48 rats,60 and monkeys.61 This compound had a direct effect on the trabecular meshwork and the cells in Schlemm's canal. Recent research had provided a great deal of data on the multiple potential therapeutic uses of ROCK inhibitors in glaucoma, including both IOP maintenance48,53,55,6265 and RGC neuroprotection.66 
K-115, an isoquinolinesulfonamide derivative, shows high selectivity for ROCK inhibition, especially ROCK 2. The 50% inhibitory concentration (IC50) of K-115 for ROCK 1, ROCK 2, PKACα, PKC, and CaMKIIα was 0.051, 0.019, 2.1, 27, and 0.37 μM, respectively.67 In contrast, the IC50 of other ROCK inhibitors such as Y-27632 and fasudil was 2 to 18 times higher than that of K-115. This high selectivity contributes to the safety profile of K-115 because different protein kinases have structurally similar active binding sites yet regulate diverse signaling pathways.32,68 Indeed, phase 1 and 2 clinical trials have indicated that K-115 is a safe topical agent for IOP reduction over an 8-week course of treatment in healthy volunteers and patients with POAG.69,70 
Methods
Materials
Fluorogold (FG) was purchased from Fluorochrome (Denver, CO, USA). All chemicals used in this study's thiobarbituric acid-reactive substances (TBARS) assays were purchased from Wako Pure Chemicals (Osaka, Japan), except for the protease inhibitor cocktails, which were purchased from Sigma-Aldrich (Tokyo, Japan). K-115, a ROCK inhibitor, was kindly provided free of charge by Kowa Company, Ltd. (Nagoya, Japan). Fasudil was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 
Animals
Nine- to 12-week-old male C57BL/6 mice (SLC, Shizuoka, Japan) were used in this study. The animals in these experiments were used in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guidelines for Animal Experiments of Tohoku University. All animal experiments were conducted with the approval of the Animal Research Committee, Graduate School of Medicine, Tohoku University. Every assay was conducted on a separate set of retinas. 
Retrograde Labeling of RGCs and Optic Nerve Surgery
To identify RGCs in the ganglion cell layer (GCL), retrograde labeling was performed 7 days before optic nerve surgery. Labeling was performed by injecting 1 μL of 2% aqueous FG containing 1% dimethylsulfoxide (DMSO) into the superior colliculus, using a Hamilton syringe with a 32-gauge needle. Seven days after retrograde labeling with FG, NC was performed as described previously.71,72 Briefly, 15 minutes after administration of K-115 or fasudil, the optic nerve was crushed approximately 1 mm posterior to the eyeball without damage to the retinal blood supply. Beginning the day after surgery, K-115 or fasudil (1 mg/kg) was then administered orally once a day for 7 days. 
Quantitative Real-Time RT-PCR
The retinas were directly lysed in Qiagen RNeasy RLT Lysis buffer. Subsequent RNA extraction was performed with the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA (50 ng) was reverse transcribed using a SuperScript III First Strand Synthesis kit (Life Technologies, Inc., MD, USA) to synthesize cDNA. Real-time quantitative RT-PCR was carried out with a 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) using TaqMan probes (Life Technologies, Inc.). The catalog numbers of the predesigned TaqMan probes were as follows: Thy-1.2 (Mm00493681_m1), Brn3a (Mm02343791_m1), Nox1 (Mm00549170_m1), Nox2 (Mm01287743_m1), Nox3 (Mm01339132_m1), Nox4 (Mm00479246_m1), and GAPDH (Mm99999915_g1). Relative gene expression levels were calculated using the delta-delta Ct method. 
TBARS Assay
The TBARS assay was carried out according to previously reported methods73,74 with minor modifications. TBARS assays measure the total level of oxidized lipids based on the reaction of malondialdehyde (MDA), one of the end products of lipid peroxidation, with thiobarbituric acid (TBA).75 Briefly, the retinal homogenate, in 1.15% KCl containing 1% protease inhibitor cocktail and 0.5 mM butylated hydroxytoluene (BHT), was added to a reaction mixture (0.81% SDS, 0.36% TBA, and 9% acetic acid) on ice. After heating the reaction mixture to 100°C for 1 hour, it was centrifuged at 20,000g for 10 minutes at 4°C. The supernatant was collected and its fluorescence was measured at 530 nm excitation and 550 nm emission. The results were normalized to protein concentration, which was determined with the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, MA, USA). 
In Vitro Lipid Peroxidation Inhibition Assay
Docosahexaenoic acid (DHA) was oxidized in a linoleic acid model system to measure antioxidant activity, following the method by Osawa and Namiki76 with minor modifications. Briefly, K-115 was dissolved in PBS and added into a mixture of 5 mM DHA and an oxidizing agent (5 mM FeSO4/10 mM ascorbic acid). In a parallel experiment, the sample was replaced with a standard antioxidant, BHT, as a positive control. The mixed solution was induced at 37°C for 2 hours. The oxidized DHA solution (200 μL) was added to 500 μL of a reaction mixture (0.81% SDS, 0.36% TBA, and 9% acetic acid) on ice. After heating the reaction mixture to 100°C for 1 hour, it was centrifuged at 20,000g for 10 minutes at 4°C. The supernatant was collected and its fluorescence was measured at 530 nm excitation and 550 nm emission. 
In Situ Detection of ROS Production
FG-labeled mice were injected intravitreally with 1 μL 50 μM CellROX Green Reagent (Life Technologies, Inc.). A Hamilton syringe with a 32-gauge needle was used. Two hours after injection, the mice were perfused with ice-cold saline, followed by 4% paraformaldehyde (PFA). The eyes of the mice were collected and fixed in 4% PFA for 1 hour on ice. Following fixation, the eyes were cryopreserved with increasing concentrations of sucrose and frozen in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan). For nuclear staining, 14-μm-thick cryosections were incubated in propidium iodide (PI) solution for 10 minutes. FG- and CellROX-positive cells in the GCL were counted in complete retinal sections taken through the optic nerve. To avoid fluorescence bleed-through caused by FG, fluorescence microscopy was carried out without Vectashield mounting medium. 
Measurement of ROS Levels in the Retina
Two hours after the intravitreal injection of 1 μL 50 μM CellROX Green Reagent, the retinas were dissected in ice-cold Dulbecco's phosphate-buffered saline (DPBS) and frozen in liquid nitrogen. The retinas were then homogenized in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing 1% protease inhibitor cocktail on ice, and centrifuged at 15,000g for 10 minutes at 4°C. The supernatant was collected and its fluorescence was measured at 485 nm excitation and 538 nm emission. The results were normalized to protein concentration, which was determined with BCA protein assay kit. 
Statistical Analysis
We used an unpaired t-test to evaluate statistical differences in the two samples. An ANOVA followed by Dunnett's test was used to compare the mean in the three groups. Data are presented as means ± standard deviation. The level of statistical significance was set at P < 0.05. 
Results
K-115 Exerts a Neuroprotective Effect on RGCs After NC
There are several ROCK inhibitors that have been reported to attenuate neuronal cell death after optic nerve injury.49,59,7779 We used a mouse NC model to determine whether K-115, a novel ROCK inhibitor, exhibited the same neuroprotective effect on RGCs. The density of RGCs in the control and PBS treatment groups was 3815 ± 430 RGCs/mm2 and 1730 ± 196 RGCs/mm2, respectively. Seven days after NC, the density of surviving RGCs in the K-115 and fasudil treatment groups decreased to 3022 ± 306 RGCs/mm2 and 2846 ± 89 RGCs/mm2, respectively (Figs. 1B–J). We also performed qRT-PCR to evaluate the neuroprotective effects of K-115 and fasudil on RGCs. This revealed that after NC, the mRNA level of Thy-1.2, an early marker of RGC stress, fell by approximately 70, 50, and 40% in the PBS, K-115, and fasudil treatment groups, respectively (Fig. 1K). Similarly, the mRNA level of Brn3a, another marker of RGC, fell by approximately 90, 80, and 70%, respectively. Our results thus demonstrated a significantly increased RGC survival rate in the K-115- and fasudil-treated group, compared to the PBS-treated group. The neuroprotective effect of K-115 was transient, as it did not promote significant RGC protection at 14 or 28 days after NC (Supplementary Fig. S1). 
Figure 1
 
K-115 and fasudil exerted a neuroprotective effect on RGCs after NC. (A) Chemical structures of K-115 and fasudil. (BI) Representative images of retrogradely labeled RGCs. (BE) Higher-magnification versions of the upper panels. Scale bars: 200 μm (BE) 50 μm (FI). (J) Oral administration of K-115 or fasudil (1 mg/kg daily) for 7 days significantly delayed cell death in post-NC RGCs (n = 6 in each group). (K) Treatment with K-115 or fasudil also delayed a reduction in mRNA of Thy-1.2 and Brn3a, RGC markers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard (**P < 0.01; error bars, SD; n = 4 in each group).
Figure 1
 
K-115 and fasudil exerted a neuroprotective effect on RGCs after NC. (A) Chemical structures of K-115 and fasudil. (BI) Representative images of retrogradely labeled RGCs. (BE) Higher-magnification versions of the upper panels. Scale bars: 200 μm (BE) 50 μm (FI). (J) Oral administration of K-115 or fasudil (1 mg/kg daily) for 7 days significantly delayed cell death in post-NC RGCs (n = 6 in each group). (K) Treatment with K-115 or fasudil also delayed a reduction in mRNA of Thy-1.2 and Brn3a, RGC markers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard (**P < 0.01; error bars, SD; n = 4 in each group).
Inhibitory Effects of K-115 on Axonal Injury-Induced Lipid Peroxidation In Vivo
We previously found that 4-hydroxynonenal (4-HNE)- and 8-hydroxy-2′-deoxyguanosine (8-OHdG)-immunostained cells increased in the GCL after NC.72 However, these markers do not always reflect the overall oxidative status of the retina. Therefore, we measured the total level of oxidized lipids with a TBARS assay. We found that the level of TBARS in the retina increased with time after NC. As shown in Figure 2A, 4 and 7 days after NC, oxidized lipids had increased significantly in comparison with the non-NC group (2.0 ± 0.6 vs. 3.4 ± 1.1 and 4.8 ± 0.7 nmol/mg protein). We next investigated whether K-115 inhibits lipid peroxidation in the retina after NC. We found that administration of 1 mg/kg/d of K-115, which qRT-PCR analysis of RGC markers such as Thy-1.2 and Brn3a revealed was an effective concentration (data not shown), significantly attenuated the oxidation of lipids in the retina after NC (Fig. 2B). This result suggests that K-115 can inhibit the oxidative stress induced by axonal injury. 
Figure 2
 
Inhibitory effects of K-115 on axonal injury-induced lipid peroxidation in vivo. (A) After NC, the level of lipid peroxidation increased in a time-dependent manner in the retina compared with the nontreated control group (“Cont”). (B) Daily administration of K-115 for 7 days significantly inhibited lipid peroxidation in mice after NC (*P < 0.05, **P < 0.01; error bars, SD; n = 6 in each group).
Figure 2
 
Inhibitory effects of K-115 on axonal injury-induced lipid peroxidation in vivo. (A) After NC, the level of lipid peroxidation increased in a time-dependent manner in the retina compared with the nontreated control group (“Cont”). (B) Daily administration of K-115 for 7 days significantly inhibited lipid peroxidation in mice after NC (*P < 0.05, **P < 0.01; error bars, SD; n = 6 in each group).
Antioxidant Effects of K-115 on the Free Radical-Mediated Oxidative System
To further determine the inhibitory effect of K-115 on lipid peroxidation after NC, we measured the TBARS level induced by free radical-mediated oxidative stress. Since it is well known that DHA is the major polyunsaturated fatty acid (PUFA) in the retina,80 we assessed the oxidative level of DHA in an in vitro system. Our in vivo system clearly indicated that K-115 had an inhibitory effect on NC-induced lipid peroxidation. However, as the functional mechanism behind this effect remained unclear, we tried to determine if K-115 functions directly as an antioxidant. Butylated hydroxytoluene, a well-known synthetic antioxidant, efficiently delayed lipid peroxidation in comparison to an untreated group, whereas the delay in peroxidation after the administration of K-115 was significantly lower (Fig. 3). This indicated that K-115 did not, in fact, act as an antioxidant reagent in this system. 
Figure 3
 
Antioxidative effects of K-115 on the free radical-mediated oxidative system. The synthetic antioxidant BHT (50 mM) delayed lipid peroxidation efficiently, but K-115 (1–1000 μM) did not show an antioxidative effect (**P < 0.01; error bars, SD; n = 3 in each group).
Figure 3
 
Antioxidative effects of K-115 on the free radical-mediated oxidative system. The synthetic antioxidant BHT (50 mM) delayed lipid peroxidation efficiently, but K-115 (1–1000 μM) did not show an antioxidative effect (**P < 0.01; error bars, SD; n = 3 in each group).
Identification of ROS-Generating Cells in the GCL After NC
Previously, our group found oxidative stress markers such as 8-OHdG and 4-HNE in the GCL after NC,72 clearly indicating that NC induces oxidative stress. Since ROS, including free radicals such as superoxide anions and hydroxyl radicals, are one of the main contributors to oxidative stress, we attempted to identify the major source of ROS production in the GCL by performing double labeling with the retrograde tracers FG and CellROX, since these accumulate in the mitochondria and nucleus. As shown in Figure 4A, the cells in the GCL that were positive for the CellROX fluorescence signal were mostly RGCs. Interestingly, however, some cells in the inner nuclear layer (INL) also produced ROS 4 days after NC. The percentages of FG/CellROX double-positive cells among the GCL cells 1, 4, and 7 days after NC were 58, 88, and 73%, respectively (Fig. 4B). The percentage of non-RGC cells positive for CellROX cells in the GCL reached a maximum of 11% on day 4. These results indicate that production of ROS after NC occurs mainly in RGCs. 
Figure 4
 
Identification of ROS-generating cells in the GCL after NC injury. (A) Representative fluorescence images of frozen sections showing ROS-producing cells (shown in green) in the GCL on day 4 after NC. Scale bars: 50 μm. (B) Quantification of the number of FG-positive (shown in blue) or -negative cells among the CellROX-labeled cells on days 1, 4, and 7 after NC injury (day 1 n = 4, days 4 and 7 n = 6). Cell counting results are shown as the ratio of FG- and CellROX-positive cells (i.e., RGCs) or FG-negative and CellROX-positive cells (i.e., other cells) to the number of PI-positive cells (red) in each section.
Figure 4
 
Identification of ROS-generating cells in the GCL after NC injury. (A) Representative fluorescence images of frozen sections showing ROS-producing cells (shown in green) in the GCL on day 4 after NC. Scale bars: 50 μm. (B) Quantification of the number of FG-positive (shown in blue) or -negative cells among the CellROX-labeled cells on days 1, 4, and 7 after NC injury (day 1 n = 4, days 4 and 7 n = 6). Cell counting results are shown as the ratio of FG- and CellROX-positive cells (i.e., RGCs) or FG-negative and CellROX-positive cells (i.e., other cells) to the number of PI-positive cells (red) in each section.
K-115 Suppressed the Time-Dependent Production of ROS in RGCs After NC Injury
As shown in Figures 4A and 4B, CellROX labeling identified the location of ROS production, indicating that oxidative stress is mainly induced in RGCs after NC. Furthermore, as shown in Figure 1, K-115 dramatically altered the RGC death rate after NC. This prompted an investigation of K-115's role in suppressing ROS production in RGCs using an in vivo model, by first inducing ROS production with NC, and then assessing the level of CellROX fluorescence. We found that after NC, the percentage of FG/CellROX double-positive cells gradually increased, reaching a maximum of 94% on day 4 before decreasing to 78% on day 7 (Figs. 5A, 5B). With K-115 treatment, although the percentage of CellROX-positive RGCs decreased in a time-dependent manner for 7 days, at all time points the percentage of double-positive cells was at least 40% lower than with PBS treatment. We also found that CellROX fluorescence intensity in the retinal lysate was significantly increased after NC, and that this elevated intensity was almost completely suppressed by administration of K-115 (Fig. 5C). These results indicate that K-115 treatment, rather that inhibiting downstream ROS production after NC, such as lipid peroxidation, inhibits ROS production itself through an indirect mechanism. 
Figure 5
 
K-115 suppressed the time-dependent production of ROS in RGCs. (A) Representative fluorescence images of frozen sections confirming ROS production (green) in FG-labeled RGCs (blue) on day 4 after NC. K-115 treatment attenuated the NC-induced increase in CellROX fluorescence in RGCs. Scale bar: 100 μm. (B) Quantification of the number of CellROX positive cells among FG-labeled RGCs on days 1, 4, and 7 after NC injury. K-115 significantly reduced the number of CellROX-labeled cells among the retrogradely labeled RGCs at each time point (*P < 0.05, **P < 0.01; error bars, SD; PBS group: day 1 n = 4, days 4 and 7 n = 6; K-115 group: day 1, 4 n = 6, day 7 n = 5). (C) Measurement of ROS levels using a fluorophotometer. Fluorescence intensity (RFU) was normalized to protein concentration (mg/mL), which was determined with a BCA protein assay kit (*P < 0.05, **P < 0.01; error bars, SD; n = 6 in each group).
Figure 5
 
K-115 suppressed the time-dependent production of ROS in RGCs. (A) Representative fluorescence images of frozen sections confirming ROS production (green) in FG-labeled RGCs (blue) on day 4 after NC. K-115 treatment attenuated the NC-induced increase in CellROX fluorescence in RGCs. Scale bar: 100 μm. (B) Quantification of the number of CellROX positive cells among FG-labeled RGCs on days 1, 4, and 7 after NC injury. K-115 significantly reduced the number of CellROX-labeled cells among the retrogradely labeled RGCs at each time point (*P < 0.05, **P < 0.01; error bars, SD; PBS group: day 1 n = 4, days 4 and 7 n = 6; K-115 group: day 1, 4 n = 6, day 7 n = 5). (C) Measurement of ROS levels using a fluorophotometer. Fluorescence intensity (RFU) was normalized to protein concentration (mg/mL), which was determined with a BCA protein assay kit (*P < 0.05, **P < 0.01; error bars, SD; n = 6 in each group).
Involvement of the Nox Family in NC-Induced ROS Production
As previous reports indicated that the Rho/ROCK pathway is involved in inducing the Nox family,22,81,82 we also performed an investigation of the involvement of this family with ROS production after NC. After NC, the mRNA levels of Nox1, Nox2, and Nox4 in the PBS treatment group increased 5.6-, 3.2-, and 5.7-fold relative to the control group (Fig. 6A). While K-115 had an inhibitory effect on NC-induced up-regulation of Nox1, it had no effect on Nox2 and Nox4. We next considered the direct involvement of the Nox family in NC-induced ROS production. We found that within 5 minutes of treatment with the Nox inhibitor VAS2870 after NC, CellROX fluorescence was almost completely eliminated. This suggests that almost all ROS production is derived from Nox1
Figure 6
 
Involvement of the Nox family in NC-induced ROS production. (A) Treatment with K-115 suppressed induction of the Nox family, Nox1-4 mRNA, at day 4 after NC. GAPDH was used as an internal standard (**P < 0.01; error bars, SD; n = 8 in each group). (B) Representative fluorescence images of frozen sections showing that axonal injury-induced ROS production was greatly reduced by treatment with the Nox inhibitor VAS2870 (10 pmol/eye) at day 4. VAS2870 was intravitreally injected within 5 minutes of NC (n = 4 in each group). Scale bar: 100 μm.
Figure 6
 
Involvement of the Nox family in NC-induced ROS production. (A) Treatment with K-115 suppressed induction of the Nox family, Nox1-4 mRNA, at day 4 after NC. GAPDH was used as an internal standard (**P < 0.01; error bars, SD; n = 8 in each group). (B) Representative fluorescence images of frozen sections showing that axonal injury-induced ROS production was greatly reduced by treatment with the Nox inhibitor VAS2870 (10 pmol/eye) at day 4. VAS2870 was intravitreally injected within 5 minutes of NC (n = 4 in each group). Scale bar: 100 μm.
Discussion
The results of the present study strongly suggest that K-115, a ROCK inhibitor, can prolong RGC cell survival by suppressing oxidative stress through pathways involving the Nox family (Fig. 7). 
Figure 7
 
K-115 inhibited oxidative stress via the Rho/ROCK pathway in RGCs. Axonal injury resulted in Nox-mediated ROS production via the Rho/ROCK pathway, which was attenuated by K-115.
Figure 7
 
K-115 inhibited oxidative stress via the Rho/ROCK pathway in RGCs. Axonal injury resulted in Nox-mediated ROS production via the Rho/ROCK pathway, which was attenuated by K-115.
Both our present study and previous studies by others have shown that oxidative stress is involved in RGC death after axonal injury.17,72,8389 In order to evaluate the efficacy of K-115, we compared the density of FG-labeled RGCs in mice treated with either PBS or ROCK inhibitors such as K-115 and fasudil 7 days after NC, in addition to the qRT-PCR analysis of RGC markers. We found that the neuroprotective effects of K-115 and fasudil after NC were similar, but that the specificity of the effect of K-115 on ROCK was 2 to 18 times higher than that of fasudil. This might have been related to our finding that the concentration-dependent neuroprotective effect of K-115 and fasudil against active ROCK in the retina after NC reached a plateau at 1 mg/kg/d. We therefore speculate that both ROCK inhibitors have a similar protective effect against axonal injury. Additionally, we found that K-115 dramatically suppressed oxidative stress, including ROS production by the RGCs themselves. Although the precise mechanism by which K-115 suppresses ROS production after NC has not yet been adequately determined, our present results strongly indicate that K-115 does not directly function as an antioxidant (Fig. 3). Moreover, we confirmed that expression of Nox1, which is strongly related to ROS production after NC, decreased with K-115 treatment. Based on previous findings that fasudil, a compound whose structure is similar to K-115, had an indirect antioxidant effect in various disease models including hypercholesterolemia,90 diabetes,91 and ischemia,92 it is possible that K-115 also had a similar antioxidant effect in our NC model. 
Oxidative stress is implicated in neuronal cell death in many neurodegenerative diseases, such as Alzheimer's disease,34 amyotrophic lateral sclerosis,93 and Parkinson's disease.94,95 In glaucoma it has been reported that increases in oxidative stress markers can be found in a patient's aqueous humor and plasma.86,87 Our research to date, using an experimental glaucoma model, strongly suggests that glaucoma should also be considered as a chronic neurodegenerative disease associated with oxidative stress. One widely accepted measurement of oxidative stress is the TBARS assay. Lipid peroxides, unstable indicators of oxidative stress in cells, decompose to form more complex and reactive compounds. Measurements of these end products indicate the level of oxidative damage. Our study demonstrated that the TBARS level in the entire retina increased with time after NC, and was highest on the seventh day. However, as indicated in our previous report, oxidative stress appears to be higher in RGCs than in any other layer of the retina.72 Therefore, we suggest that oxidative stress-induced RGC death directly affects the survival of RGCs after NC. To protect RGCs from oxidative stress, we examined the effects of the ROCK inhibitor K-115, and found that it could inhibit increases in the TBARS level. In other words, K-115 can suppress oxidative stress after NC. 
ROCK inhibitors have been widely used as treatments for various neurological disorders, including spinal cord injuries,32,3538 stroke,3946 multiple sclerosis,32 and Alzheimer's disease.3234 It has previously been reported that targeting small Rho GTPase has a positive dose-dependent effect on the regeneration of RGCs after injuries such as NC.49,59,7779 In the axonal injury model, it has been reported that ROCK activity increased in the RGC layer, reaching a maximum on the fourth day after injury.59 
Moreover, the increase in ROCK activity was not observed in other retinal layers, implying that this response to axonal lesions is specific to RGCs. However, we had difficulty at first finding evidence directly indicating which oxidative stress pathway was suppressed by the ROCK inhibitor K-115. Figure 3 shows our assessment of the ability of ROCK inhibitors to directly suppress oxidative stress. The antioxidant effect of K-115 was significantly lower than that of BHT. This suggests that K-115 did not act as an antioxidant reagent in this system, and led us to believe that ROCK inhibitors increase antioxidant activity through another mechanism. Looking to test this hypothesis, we assessed the gene expression pattern after NC of the Nox family, including Nox1, 2, 3, and 4, with K-115 treatment. Previously, it was reported that the Nox family is expressed downstream of ROCK activity,22,81,82,96 and indeed, we did find that the expression of Nox1, 2, and 4 increased after NC, particularly Nox1 and 2 in the RGCs. Previous findings that Nox1, 2, and 4 were expressed in surviving RGCs, and that RGCs had a significantly higher level of Nox1 than other members of the Nox family,96 support our results. It has also been reported that Nox2 is particularly expressed in the microglial cells.97,98 The final results of the experiments reported here showed that only Nox1 was suppressed after NC with K-115 treatment. This decrease in Nox1 expression after K-115 treatment supported our hypothesis. Indeed, Nox1 was found to play a critical role in ischemia-induced oxidative stress and RGC death in experiments using the Nox inhibitor VAS2870.96 Furthermore, since K-115 also significantly inhibits RGC death in ischemia-reperfusion models (Mizuno K, et al. IOVS 2007;48:ARVO E-Abstract 4805), this protective effect might have a similar mechanism that modulates Nox family expression. 
It is widely accepted that mitochondrial ROS cause oxidative damage to nuclear DNA. Mitochondrial-derived death signaling has previously been reported to be an important pathway for RGC death induced by axonal damage.99,100 Mitochondria are also known to be abundant in the optic nerve.101 Previously, we detected ROS in the mitochondria of RGCs, suggesting that axonal damage affects mitochondrial function, which in turn triggers RGC death.72 In the present study, we have confirmed that oxidative stress is also involved in NC-induced RGC death. In this study we present evidence, using CellROX staining, that NC-induced apoptosis in RGCs produced high amounts of ROS. Indeed, the reduction in the number of ROS-producing cells with K-115 treatment was confirmed by both counting the cells (Fig. 5B) and by a fluorophotometric analysis (Fig. 5C). However, the difference between the PBS and K-115 treatment groups, shown in Figure 5C, were smaller than in the groups shown in Figure 5B. The different results obtained from these two analyses raise the possibility that our fluorophotometric evaluation of the suppressive effect of K-115 on ROS production after NC may have been affected by the difficulty of measuring the fluorescence intensity of retinal lysates. ROS are generated by the process of isolating the retina itself, even if the retinas are immediately dissected in ice-cold DPBS. Therefore, we consider that counting the number of cells was the most suitable method to evaluate K-115's effect on ROS production. The metabolic processes involving the mitochondrial electron transport chain are known to contribute to the formation of harmful ROS. Furthermore, our results indicated that K-115 significantly suppressed NC-induced oxidative stress by inhibiting ROS production in RGCs. 
Our results thus strongly suggest that the prevention of oxidative stress in the mitochondria or nucleus should be regarded as candidates for the treatment of glaucoma. Furthermore, we believe that we have shown that suppression of Rho activity also has the potential to be a new neuroprotective treatment for glaucoma, particularly NTG (the main type of glaucoma in Asian countries).102 
Acknowledgments
We thank Tim Hilts for reviewing and editing the language of the manuscript, and we thank Junko Sato for the technical assistance. 
Supported by Grants-in-Aid from the Ministry of Education, Science and Technology of Japan (23592613; KM) and in part by grants from Kowa Company, Ltd. 
Disclosure: K. Yamamoto, None; K. Maruyama, None; N. Himori, None; K. Omodaka, None; Y. Yokoyama, None; Y. Shiga, None; R. Morin, None; T. Nakazawa, None 
References
Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol. 1996; 80: 389–393. [CrossRef] [PubMed]
Iwase A Suzuki Y Araie M The prevalence of primary open-angle glaucoma in Japanese: the Tajimi Study. Ophthalmology. 2004; 111: 1641–1648. [PubMed]
Kass MA Heuer DK Higginbotham EJ The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch Ophthalmol. 2002; 120: 701–713, discussion 829–830. [CrossRef] [PubMed]
Shiga Y Omodaka K Kunikata H Waveform analysis of ocular blood flow and the early detection of normal tension glaucoma. Invest Ophthalmol Vis Sci. 2013; 54: 7699–7706. [CrossRef] [PubMed]
Yücel YH Zhang Q Weinreb RN Kaufman PL Gupta N. Effects of retinal ganglion cell loss on magno-, parvo-, koniocellular pathways in the lateral geniculate nucleus and visual cortex in glaucoma. Prog Retin Eye Res. 2003; 22: 465–481. [CrossRef] [PubMed]
Hayreh SS. Factors influencing blood flow in the optic nerve head. J Glaucoma. 1997; 6: 412–425. [PubMed]
Kerr J Nelson P O'Brien C. A comparison of ocular blood flow in untreated primary open-angle glaucoma and ocular hypertension. Am J Ophthalmol. 1998; 126: 42–51. [CrossRef] [PubMed]
Neufeld AH Sawada A Becker B. Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma. Proc Natl Acad Sci U S A. 1999; 96: 9944–9948. [CrossRef] [PubMed]
Shareef S Sawada A Neufeld AH. Isoforms of nitric oxide synthase in the optic nerves of rat eyes with chronic moderately elevated intraocular pressure. Invest Ophthalmol Vis Sci. 1999; 40: 2884–2891. [PubMed]
Liu B Neufeld AH. Nitric oxide synthase-2 in human optic nerve head astrocytes induced by elevated pressure in vitro. Arch Ophthalmol. 2001; 119: 240–245. [PubMed]
Sullivan RK Woldemussie E Macnab L Ruiz G Pow DV. Evoked expression of the glutamate transporter GLT-1c in retinal ganglion cells in human glaucoma and in a rat model. Invest Ophthalmol Vis Sci. 2006; 47: 3853–3859. [CrossRef] [PubMed]
Vorwerk CK Gorla MS Dreyer EB. An experimental basis for implicating excitotoxicity in glaucomatous optic neuropathy. Surv Ophthalmol. 1999; 43( suppl 1): S142–S 150. [CrossRef] [PubMed]
Seki M Lipton SA. Targeting excitotoxic/free radical signaling pathways for therapeutic intervention in glaucoma. Prog Brain Res. 2008; 173: 495–510. [PubMed]
Izzotti A Bagnis A Saccà SC. The role of oxidative stress in glaucoma. Mutat Res. 2006; 612: 105–114. [CrossRef] [PubMed]
Bagnis A Izzotti A Saccà SC. Helicobacter pylori, oxidative stress and glaucoma. Dig Liver Dis. 2012; 44: 963–964. [CrossRef] [PubMed]
Ferreira SM Lerner SF Brunzini R Reides CG Evelson PA Llesuy SF. Time course changes of oxidative stress markers in a rat experimental glaucoma model. Invest Ophthalmol Vis Sci. 2010; 51: 4635–4640. [CrossRef] [PubMed]
Engin KN Yemişci B Yiğit U Ağaçhan A Coşkun C. Variability of serum oxidative stress biomarkers relative to biochemical data and clinical parameters of glaucoma patients. Mol Vis. 2010; 16: 1260–1271. [PubMed]
Riento K Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003; 4: 446–456. [CrossRef] [PubMed]
Olson MF. Applications for ROCK kinase inhibition. Curr Opin Cell Biol. 2008; 20: 242–248. [CrossRef] [PubMed]
Hall A. Rho GTPases and the control of cell behaviour. Biochem Soc Trans. 2005; 33: 891–895. [CrossRef] [PubMed]
Bach LA. Rho kinase inhibition: a new approach for treating diabetic nephropathy? Diabetes. 2008; 57: 532–533. [CrossRef] [PubMed]
Gojo A Utsunomiya K Taniguchi K The Rho-kinase inhibitor, fasudil, attenuates diabetic nephropathy in streptozotocin-induced diabetic rats. Eur J Pharmacol. 2007; 568: 242–247. [CrossRef] [PubMed]
Gupta J Gaikwad AB Tikoo K. Hepatic expression profiling shows involvement of PKC epsilon, DGK eta, Tnfaip, and Rho kinase in type 2 diabetic nephropathy rats. J Cell Biochem. 2010; 111: 944–954. [CrossRef] [PubMed]
Komers R. Rho kinase inhibition in diabetic nephropathy. Curr Opin Nephrol Hypertens. 2011; 20: 77–83. [CrossRef] [PubMed]
Komers R Oyama TT Beard DR Rho kinase inhibition protects kidneys from diabetic nephropathy without reducing blood pressure. Kidney Int. 2011; 79: 432–442. [CrossRef] [PubMed]
Matoba K Kawanami D Okada R Rho-kinase inhibition prevents the progression of diabetic nephropathy by downregulating hypoxia-inducible factor 1α. Kidney Int. 2013; 84: 545–554. [CrossRef] [PubMed]
Budzyn K Marley PD Sobey CG. Targeting Rho and Rho-kinase in the treatment of cardiovascular disease. Trends Pharmacol Sci. 2006; 27: 97–104. [CrossRef] [PubMed]
Cosentino F. Statins in cardiovascular disease. Role of Rho/Rho kinase inhibition and of Akt activation [in Italian]. Recenti Prog Med. 2003; 94: 444–450. [PubMed]
Jalil J Lavandero S Chiong M Ocaranza MP. Rho/Rho kinase signal transduction pathway in cardiovascular disease and cardiovascular remodeling [in Spanish]. Rev Esp Cardiol. 2005; 58: 951–961. [CrossRef] [PubMed]
Lai A Frishman WH. Rho-kinase inhibition in the therapy of cardiovascular disease. Cardiol Rev. 2005; 13: 285–292. [CrossRef] [PubMed]
Surma M Wei L Shi J. Rho kinase as a therapeutic target in cardiovascular disease. Future Cardiol. 2011; 7: 657–671. [CrossRef] [PubMed]
Mueller BK Mack H Teusch N. Rho kinase, a promising drug target for neurological disorders. Nat Rev Drug Discov. 2005; 4: 387–398. [CrossRef] [PubMed]
Pedrini S Carter TL Prendergast G Petanceska S Ehrlich ME Gandy S. Modulation of statin-activated shedding of Alzheimer APP ectodomain by ROCK. PLoS Med. 2005; 2: e18. [CrossRef] [PubMed]
Ramamoorthy M Sykora P Scheibye-Knudsen M Sporadic Alzheimer disease fibroblasts display an oxidative stress phenotype. Free Radic Biol Med. 2012; 53: 1371–1380. [CrossRef] [PubMed]
Chiba Y Kuroda S Shichinohe H Synergistic effects of bone marrow stromal cells and a Rho kinase (ROCK) inhibitor, fasudil on axon regeneration in rat spinal cord injury. Neuropathology. 2010; 30: 241–250. [CrossRef] [PubMed]
Furuya T Hashimoto M Koda M Treatment of rat spinal cord injury with a Rho-kinase inhibitor and bone marrow stromal cell transplantation. Brain Res. 2009; 1295: 192–202. [CrossRef] [PubMed]
Nishio Y Koda M Kitajo K Delayed treatment with Rho-kinase inhibitor does not enhance axonal regeneration or functional recovery after spinal cord injury in rats. Exp Neurol. 2006; 200: 392–397. [CrossRef] [PubMed]
Sung JK Miao L Calvert JW Huang L. Louis Harkey H Zhang JH. A possible role of RhoA/Rho-kinase in experimental spinal cord injury in rat. Brain Res. 2003; 959: 29–38. [CrossRef] [PubMed]
Cheng CI Lin YC Tsai TH The prognostic values of leukocyte rho kinase activity in acute ischemic stroke. Biomed Res Int. 2014; 2014: 214587. [PubMed]
Ding J Yu JZ Li QY Wang X Lu CZ Xiao BG. Rho kinase inhibitor Fasudil induces neuroprotection and neurogenesis partially through astrocyte-derived G-CSF. Brain Behav Immun. 2009; 23: 1083–1088. [CrossRef] [PubMed]
Nishikimi T Koshikawa S Ishikawa Y Inhibition of Rho-kinase attenuates nephrosclerosis and improves survival in salt-loaded spontaneously hypertensive stroke-prone rats. J Hypertens. 2007; 25: 1053–1063. [CrossRef] [PubMed]
Okamura N Saito M Mori A Vasodilator effects of fasudil, a Rho-kinase inhibitor, on retinal arterioles in stroke-prone spontaneously hypertensive rats. J Ocul Pharmacol Ther. 2007; 23: 207–212. [CrossRef] [PubMed]
Rikitake Y Kim HH Huang Z Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke. 2005; 36: 2251–2257. [CrossRef] [PubMed]
Savoia C Tabet F Yao G Schiffrin EL Touyz RM. Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats. J Hypertens. 2005; 23: 1037–1045. [CrossRef] [PubMed]
Shin HK Salomone S Ayata C. Targeting cerebrovascular Rho-kinase in stroke. Expert Opin Ther Targets. 2008; 12: 1547–1564. [CrossRef] [PubMed]
Vesterinen HM Currie GL Carter S Systematic review and stratified meta-analysis of the efficacy of RhoA and Rho kinase inhibitors in animal models of ischaemic stroke. Syst Rev. 2013; 2: 33. [CrossRef] [PubMed]
Davis RL Kahraman M Prins TJ Benzothiophene containing Rho kinase inhibitors: efficacy in an animal model of glaucoma. Bioorg Med Chem Lett. 2010; 20: 3361–3366. [CrossRef] [PubMed]
Honjo M Tanihara H Inatani M Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility. Invest Ophthalmol Vis Sci. 2001; 42: 137–144. [PubMed]
Tokushige H Waki M Takayama Y Tanihara H. Effects of Y-39983, a selective Rho-associated protein kinase inhibitor, on blood flow in optic nerve head in rabbits and axonal regeneration of retinal ganglion cells in rats. Curr Eye Res. 2011; 36: 964–970. [CrossRef] [PubMed]
Chen HH Namil A Severns B In vivo optimization of 2,3-diaminopyrazine Rho Kinase inhibitors for the treatment of glaucoma. Bioorg Med Chem Lett. 2014; 24: 1875–1879. [CrossRef] [PubMed]
Demiryürek S Okumus S Bozgeyik I Investigation of the Rho-kinase gene polymorphism in primary open-angle glaucoma [ published online ahead of print March 11, 2014]. Ophthalmic Genet. doi:10.3109/13816810.2014.895016.
Williams RD Novack GD van Haarlem T Kopczynski C; AR-12286 Phase 2A Study Group . Ocular hypotensive effect of the Rho kinase inhibitor AR-12286 in patients with glaucoma and ocular hypertension. Am J Ophthalmol. 2011; 152: 834–841.e831. [CrossRef] [PubMed]
Honjo M Tanihara H Kameda T Kawaji T Yoshimura N Araie M. Potential role of Rho-associated protein kinase inhibitor Y-27632 in glaucoma filtration surgery. Invest Ophthalmol Vis Sci. 2007; 48: 5549–5557. [CrossRef] [PubMed]
Rao VP Epstein DL. Rho GTPase/Rho kinase inhibition as a novel target for the treatment of glaucoma. BioDrugs. 2007; 21: 167–177. [CrossRef] [PubMed]
Inoue T Tanihara H. Rho-associated kinase inhibitors: a novel glaucoma therapy. Prog Retin Eye Res. 2013; 37: 1–12. [CrossRef] [PubMed]
Challa P Arnold JJ. Rho-kinase inhibitors offer a new approach in the treatment of glaucoma. Expert Opin Investig Drugs. 2014; 23: 81–95. [CrossRef] [PubMed]
Goldhagen B Proia AD Epstein DL Rao PV. Elevated levels of RhoA in the optic nerve head of human eyes with glaucoma. J Glaucoma. 2012; 21: 530–538. [CrossRef] [PubMed]
Bertrand J Winton MJ Rodriguez-Hernandez N Campenot RB McKerracher L. Application of Rho antagonist to neuronal cell bodies promotes neurite growth in compartmented cultures and regeneration of retinal ganglion cell axons in the optic nerve of adult rats. J Neurosci. 2005; 25: 1113–1121. [CrossRef] [PubMed]
Lingor P Tönges L Pieper N ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells. Brain. 2008; 131: 250–263. [PubMed]
Yu M Chen X Wang N H-1152 effects on intraocular pressure and trabecular meshwork morphology of rat eyes. J Ocul Pharmacol Ther. 2008; 24: 373–379. [CrossRef] [PubMed]
Tian B Kaufman PL Volberg T Gabelt BT Geiger B. H-7 disrupts the actin cytoskeleton and increases outflow facility. Arch Ophthalmol. 1998; 116: 633–643. [CrossRef] [PubMed]
Tokushige H Inatani M Nemoto S Effects of topical administration of y-39983, a selective rho-associated protein kinase inhibitor, on ocular tissues in rabbits and monkeys. Invest Ophthalmol Vis Sci. 2007; 48: 3216–3222. [CrossRef] [PubMed]
Tanihara H Inatani M Honjo M Tokushige H Azuma J Araie M. Intraocular pressure-lowering effects and safety of topical administration of a selective ROCK inhibitor, SNJ-1656, in healthy volunteers. Arch Ophthalmol. 2008; 126: 309–315. [CrossRef] [PubMed]
Koga T Awai M Tsutsui J Yue BY Tanihara H. Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells. Exp Eye Res. 2006; 82: 362–370. [CrossRef] [PubMed]
Fujimoto T Inoue T Kameda T Involvement of RhoA/Rho-associated kinase signal transduction pathway in dexamethasone-induced alterations in aqueous outflow. Invest Ophthalmol Vis Sci. 2012; 53: 7097–7108. [CrossRef] [PubMed]
Kitaoka Y Kumai T Lam TT Involvement of RhoA and possible neuroprotective effect of fasudil, a Rho kinase inhibitor, in NMDA-induced neurotoxicity in the rat retina. Brain Res. 2004; 1018: 111–118. [CrossRef] [PubMed]
Isobe T Mizuno K Kaneko Y Ohta M Koide T Tanabe S. Effects of K-115, a Rho-kinase inhibitor, on aqueous humor dynamics in rabbits. Curr Eye Res. 2014; 39: 813–822. [CrossRef] [PubMed]
Jacobs M Hayakawa K Swenson L The structure of dimeric ROCK I reveals the mechanism for ligand selectivity. J Biol Chem. 2006; 281: 260–268. [CrossRef] [PubMed]
Tanihara H Inoue T Yamamoto T Phase 1 clinical trials of a selective Rho kinase inhibitor, K-115. JAMA Ophthalmol. 2013; 131: 1288–1295. [CrossRef] [PubMed]
Tanihara H Inoue T Yamamoto T Phase 2 randomized clinical study of a Rho kinase inhibitor, K-115, in primary open-angle glaucoma and ocular hypertension. Am J Ophthalmol. 2013; 156: 731–736. [CrossRef] [PubMed]
Ryu M Yasuda M Shi D Critical role of calpain in axonal damage-induced retinal ganglion cell death. J Neurosci Res. 2012; 90: 802–815. [CrossRef] [PubMed]
Himori N Yamamoto K Maruyama K Critical role of Nrf2 in oxidative stress-induced retinal ganglion cell death. J Neurochem. 2013; 127: 669–680. [CrossRef] [PubMed]
Takemura N Takahashi K Tanaka H Dietary, but not topical, alpha-linolenic acid suppresses UVB-induced skin injury in hairless mice when compared with linoleic acids. Photochem Photobiol. 2002; 76: 657–663. [CrossRef] [PubMed]
Aitken RJ Harkiss D Buckingham DW. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev. 1993; 35: 302–315. [CrossRef] [PubMed]
Esterbauer H Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 1990; 186: 407–421. [PubMed]
Osawa T Namiki M. Natural antioxidants isolated from eucalyptus leaf waxes. J Agric Food Chem. 1985; 33: 777–780. [CrossRef]
Bermel C Tönges L Planchamp V Combined inhibition of Cdk5 and ROCK additively increase cell survival, but not the regenerative response in regenerating retinal ganglion cells. Mol Cell Neurosci. 2009; 42: 427–437. [CrossRef] [PubMed]
Sagawa H Terasaki H Nakamura M A novel ROCK inhibitor, Y-39983, promotes regeneration of crushed axons of retinal ganglion cells into the optic nerve of adult cats. Exp Neurol. 2007; 205: 230–240. [CrossRef] [PubMed]
Yang Z Wang J Liu X Cheng Y Deng L Zhong Y. Y-39983 downregulates RhoA/Rho-associated kinase expression during its promotion of axonal regeneration. Oncol Rep. 2013; 29: 1140–1146. [PubMed]
Fliesler SJ Anderson RE. Chemistry and metabolism of lipids in the vertebrate retina. Prog Lipid Res. 1983; 22: 79–131. [CrossRef] [PubMed]
Nishida M Tanabe S Maruyama Y G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes. J Biol Chem. 2005; 280: 18434–18441. [CrossRef] [PubMed]
Mita S Kobayashi N Yoshida K Nakano S Matsuoka H. Cardioprotective mechanisms of Rho-kinase inhibition associated with eNOS and oxidative stress-LOX-1 pathway in Dahl salt-sensitive hypertensive rats. J Hypertens. 2005; 23: 87–96. [CrossRef] [PubMed]
Majsterek I Malinowska K Stanczyk M Evaluation of oxidative stress markers in pathogenesis of primary open-angle glaucoma. Exp Mol Pathol. 2011; 90: 231–237. [CrossRef] [PubMed]
Levkovitch-Verbin H Harris-Cerruti C Groner Y Wheeler LA Schwartz M Yoles E. RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci. 2000; 41: 4169–4174. [PubMed]
Ghanem AA Arafa LF El-Baz A. Oxidative stress markers in patients with primary open-angle glaucoma. Curr Eye Res. 2010; 35: 295–301. [CrossRef] [PubMed]
Ferreira SM Lerner SF Brunzini R Evelson PA Llesuy SF. Oxidative stress markers in aqueous humor of glaucoma patients. Am J Ophthalmol. 2004; 137: 62–69. [CrossRef] [PubMed]
Bagnis A Izzotti A Centofanti M Saccà SC. Aqueous humor oxidative stress proteomic levels in primary open angle glaucoma. Exp Eye Res. 2012; 103: 55–62. [CrossRef] [PubMed]
Hoegger MJ Lieven CJ Levin LA. Differential production of superoxide by neuronal mitochondria. BMC Neurosci. 2008; 9: 4. [CrossRef] [PubMed]
Lieven CJ Hoegger MJ Schlieve CR Levin LA. Retinal ganglion cell axotomy induces an increase in intracellular superoxide anion. Invest Ophthalmol Vis Sci. 2006; 47: 1477–1485. [CrossRef] [PubMed]
Ma Z Zhang J Ji E Cao G Li G Chu L. Rho kinase inhibition by fasudil exerts antioxidant effects in hypercholesterolemic rats. Clin Exp Pharmacol Physiol. 2011; 38: 688–694. [CrossRef] [PubMed]
Guo R Liu B Zhou S Zhang B Xu Y. The protective effect of fasudil on the structure and function of cardiac mitochondria from rats with type 2 diabetes induced by streptozotocin with a high-fat diet is mediated by the attenuation of oxidative stress. Biomed Res Int. 2013; 2013: 430791. [PubMed]
Gibson CL Srivastava K Sprigg N Bath PM Bayraktutan U. Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions. J Neurochem. 2014; 129: 816–826. [CrossRef] [PubMed]
Lee J Kannagi M Ferrante RJ Kowall NW Ryu H. Activation of Ets-2 by oxidative stress induces Bcl-xL expression and accounts for glial survival in amyotrophic lateral sclerosis. FASEB J. 2009; 23: 1739–1749. [CrossRef] [PubMed]
Clements CM McNally RS Conti BJ Mak TW Ting JP. DJ-1, a cancer- and Parkinson's disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2. Proc Natl Acad Sci U S A. 2006; 103: 15091–15096. [CrossRef] [PubMed]
Choi DH Cristóvão AC Guhathakurta S NADPH oxidase 1-mediated oxidative stress leads to dopamine neuron death in Parkinson's disease. Antioxid Redox Signal. 2012; 16: 1033–1045. [CrossRef] [PubMed]
Dvoriantchikova G Grant J Santos AR Hernandez E Ivanov D. Neuronal NAD(P)H oxidases contribute to ROS production and mediate RGC death after ischemia. Invest Ophthalmol Vis Sci. 2012; 53: 2823–2830. [CrossRef] [PubMed]
Vilhardt F Plastre O Sawada M The HIV-1 Nef protein and phagocyte NADPH oxidase activation. J Biol Chem. 2002; 277: 42136–42143. [CrossRef] [PubMed]
Lavigne MC Malech HL Holland SM Leto TL. Genetic requirement of p47phox for superoxide production by murine microglia. FASEB J. 2001; 15: 285–287. [PubMed]
Chierzi S Strettoi E Cenni MC Maffei L. Optic nerve crush: axonal responses in wild-type and bcl-2 transgenic mice. J Neurosci. 1999; 19: 8367–8376. [PubMed]
Qi X Lewin AS Sun L Hauswirth WW Guy J. Suppression of mitochondrial oxidative stress provides long-term neuroprotection in experimental optic neuritis. Invest Ophthalmol Vis Sci. 2007; 48: 681–691. [CrossRef] [PubMed]
Barron MJ Griffiths P Turnbull DM Bates D Nichols P. The distributions of mitochondria and sodium channels reflect the specific energy requirements and conduction properties of the human optic nerve head. Br J Ophthalmol. 2004; 88: 286–290. [CrossRef] [PubMed]
Iwase A Suzuki Y Araie M The prevalence of primary open-angle glaucoma in Japanese: the Tajimi Study. Ophthalmology. 2004; 111: 1641–1648. [PubMed]
Figure 1
 
K-115 and fasudil exerted a neuroprotective effect on RGCs after NC. (A) Chemical structures of K-115 and fasudil. (BI) Representative images of retrogradely labeled RGCs. (BE) Higher-magnification versions of the upper panels. Scale bars: 200 μm (BE) 50 μm (FI). (J) Oral administration of K-115 or fasudil (1 mg/kg daily) for 7 days significantly delayed cell death in post-NC RGCs (n = 6 in each group). (K) Treatment with K-115 or fasudil also delayed a reduction in mRNA of Thy-1.2 and Brn3a, RGC markers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard (**P < 0.01; error bars, SD; n = 4 in each group).
Figure 1
 
K-115 and fasudil exerted a neuroprotective effect on RGCs after NC. (A) Chemical structures of K-115 and fasudil. (BI) Representative images of retrogradely labeled RGCs. (BE) Higher-magnification versions of the upper panels. Scale bars: 200 μm (BE) 50 μm (FI). (J) Oral administration of K-115 or fasudil (1 mg/kg daily) for 7 days significantly delayed cell death in post-NC RGCs (n = 6 in each group). (K) Treatment with K-115 or fasudil also delayed a reduction in mRNA of Thy-1.2 and Brn3a, RGC markers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard (**P < 0.01; error bars, SD; n = 4 in each group).
Figure 2
 
Inhibitory effects of K-115 on axonal injury-induced lipid peroxidation in vivo. (A) After NC, the level of lipid peroxidation increased in a time-dependent manner in the retina compared with the nontreated control group (“Cont”). (B) Daily administration of K-115 for 7 days significantly inhibited lipid peroxidation in mice after NC (*P < 0.05, **P < 0.01; error bars, SD; n = 6 in each group).
Figure 2
 
Inhibitory effects of K-115 on axonal injury-induced lipid peroxidation in vivo. (A) After NC, the level of lipid peroxidation increased in a time-dependent manner in the retina compared with the nontreated control group (“Cont”). (B) Daily administration of K-115 for 7 days significantly inhibited lipid peroxidation in mice after NC (*P < 0.05, **P < 0.01; error bars, SD; n = 6 in each group).
Figure 3
 
Antioxidative effects of K-115 on the free radical-mediated oxidative system. The synthetic antioxidant BHT (50 mM) delayed lipid peroxidation efficiently, but K-115 (1–1000 μM) did not show an antioxidative effect (**P < 0.01; error bars, SD; n = 3 in each group).
Figure 3
 
Antioxidative effects of K-115 on the free radical-mediated oxidative system. The synthetic antioxidant BHT (50 mM) delayed lipid peroxidation efficiently, but K-115 (1–1000 μM) did not show an antioxidative effect (**P < 0.01; error bars, SD; n = 3 in each group).
Figure 4
 
Identification of ROS-generating cells in the GCL after NC injury. (A) Representative fluorescence images of frozen sections showing ROS-producing cells (shown in green) in the GCL on day 4 after NC. Scale bars: 50 μm. (B) Quantification of the number of FG-positive (shown in blue) or -negative cells among the CellROX-labeled cells on days 1, 4, and 7 after NC injury (day 1 n = 4, days 4 and 7 n = 6). Cell counting results are shown as the ratio of FG- and CellROX-positive cells (i.e., RGCs) or FG-negative and CellROX-positive cells (i.e., other cells) to the number of PI-positive cells (red) in each section.
Figure 4
 
Identification of ROS-generating cells in the GCL after NC injury. (A) Representative fluorescence images of frozen sections showing ROS-producing cells (shown in green) in the GCL on day 4 after NC. Scale bars: 50 μm. (B) Quantification of the number of FG-positive (shown in blue) or -negative cells among the CellROX-labeled cells on days 1, 4, and 7 after NC injury (day 1 n = 4, days 4 and 7 n = 6). Cell counting results are shown as the ratio of FG- and CellROX-positive cells (i.e., RGCs) or FG-negative and CellROX-positive cells (i.e., other cells) to the number of PI-positive cells (red) in each section.
Figure 5
 
K-115 suppressed the time-dependent production of ROS in RGCs. (A) Representative fluorescence images of frozen sections confirming ROS production (green) in FG-labeled RGCs (blue) on day 4 after NC. K-115 treatment attenuated the NC-induced increase in CellROX fluorescence in RGCs. Scale bar: 100 μm. (B) Quantification of the number of CellROX positive cells among FG-labeled RGCs on days 1, 4, and 7 after NC injury. K-115 significantly reduced the number of CellROX-labeled cells among the retrogradely labeled RGCs at each time point (*P < 0.05, **P < 0.01; error bars, SD; PBS group: day 1 n = 4, days 4 and 7 n = 6; K-115 group: day 1, 4 n = 6, day 7 n = 5). (C) Measurement of ROS levels using a fluorophotometer. Fluorescence intensity (RFU) was normalized to protein concentration (mg/mL), which was determined with a BCA protein assay kit (*P < 0.05, **P < 0.01; error bars, SD; n = 6 in each group).
Figure 5
 
K-115 suppressed the time-dependent production of ROS in RGCs. (A) Representative fluorescence images of frozen sections confirming ROS production (green) in FG-labeled RGCs (blue) on day 4 after NC. K-115 treatment attenuated the NC-induced increase in CellROX fluorescence in RGCs. Scale bar: 100 μm. (B) Quantification of the number of CellROX positive cells among FG-labeled RGCs on days 1, 4, and 7 after NC injury. K-115 significantly reduced the number of CellROX-labeled cells among the retrogradely labeled RGCs at each time point (*P < 0.05, **P < 0.01; error bars, SD; PBS group: day 1 n = 4, days 4 and 7 n = 6; K-115 group: day 1, 4 n = 6, day 7 n = 5). (C) Measurement of ROS levels using a fluorophotometer. Fluorescence intensity (RFU) was normalized to protein concentration (mg/mL), which was determined with a BCA protein assay kit (*P < 0.05, **P < 0.01; error bars, SD; n = 6 in each group).
Figure 6
 
Involvement of the Nox family in NC-induced ROS production. (A) Treatment with K-115 suppressed induction of the Nox family, Nox1-4 mRNA, at day 4 after NC. GAPDH was used as an internal standard (**P < 0.01; error bars, SD; n = 8 in each group). (B) Representative fluorescence images of frozen sections showing that axonal injury-induced ROS production was greatly reduced by treatment with the Nox inhibitor VAS2870 (10 pmol/eye) at day 4. VAS2870 was intravitreally injected within 5 minutes of NC (n = 4 in each group). Scale bar: 100 μm.
Figure 6
 
Involvement of the Nox family in NC-induced ROS production. (A) Treatment with K-115 suppressed induction of the Nox family, Nox1-4 mRNA, at day 4 after NC. GAPDH was used as an internal standard (**P < 0.01; error bars, SD; n = 8 in each group). (B) Representative fluorescence images of frozen sections showing that axonal injury-induced ROS production was greatly reduced by treatment with the Nox inhibitor VAS2870 (10 pmol/eye) at day 4. VAS2870 was intravitreally injected within 5 minutes of NC (n = 4 in each group). Scale bar: 100 μm.
Figure 7
 
K-115 inhibited oxidative stress via the Rho/ROCK pathway in RGCs. Axonal injury resulted in Nox-mediated ROS production via the Rho/ROCK pathway, which was attenuated by K-115.
Figure 7
 
K-115 inhibited oxidative stress via the Rho/ROCK pathway in RGCs. Axonal injury resulted in Nox-mediated ROS production via the Rho/ROCK pathway, which was attenuated by K-115.
Supplementary Figure S1
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×