Author Affiliations: Department of Ophthalmology, Osaka Medical College, Takatsuki City, Osaka, Japan (Drs Sugiyama, Yoshioka, Oku, and Ikeda); and Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan (Dr Mashima).
To determine whether subconjunctival injection of unoprostone isopropyl alters changes in the topography and blood flow of the optic disc induced by endothelin 1 (ET-1) in rabbits.
From April 1, 2005, to April 28, 2006, we injected ET-1 (20 pmol) intravitreally into rabbits twice per week for 4 weeks. The observation period was 8 weeks. The first group received an intravitreal injection of ET-1 followed by a subconjunctival injection of unoprostone (0.12%, 50 μL). The second group received the same amount of ET-1 followed by a subconjunctival injection of the vehicle of unoprostone. The third group received the intravitreal vehicle of ET-1. The blood flow and topography of the optic nerve head (ONH) were measured by laser speckle flowgraphy and confocal scanning ophthalmoscopy, respectively. The number of cells in the retinal ganglion cell layer and inner nuclear layer was determined histologically.
We found that ET-1 decreased the ONH blood flow, decreased the cells in the ganglion cell layer and inner nuclear layer, enlarged the cup area of the ONH, and reduced the rim area of the ONH. When unoprostone was given with ET-1, no such changes occurred.
Unoprostone can suppress the effects of ET-1 on the circulation and topography of the ONH.
Unoprostone could be a candidate for treating eyes with ischemic ONH.
It has been well established that the size of the optic disc cup enlarges as the glaucomatous stage advances and that the size of the cup can be evaluated objectively with high accuracy and reproducibility by confocal scanning ophthalmoscopy.1 It has also been shown that chronic optic disc ischemia induced by endothelin 1 (ET-1) results in an enlargement of the optic disc cup accompanied by a reduction in the number of retinal ganglion cell axons.2,3 These latter findings are significant because the results of clinical and genetic studies4- 8 have suggested that ET-1 is associated with glaucoma in humans, especially in those with normal-tension glaucoma.
Unoprostone isopropyl, the first type of prostaglandin-related antiglaucoma eyedrops marketed in Japan, has been shown to reduce the intraocular pressure (IOP) by increasing uveoscleral outflow in rabbits and conventional outflow in humans.9,10 In addition, intravitreal injections of unoprostone were found to inhibit the decrease of optic disc blood flow induced by ET-1.11 Topical application of unoprostone in eyedrops was shown to partially antagonize the ET-1–induced decrease of the choroidal blood flow in humans12 and to increase the blood flow in the optic nerve head (ONH) of humans, including patients with normal-tension glaucoma.13- 15 Some studies16- 18 have also reported that unoprostone has neuroprotective activity in vivo and in vitro.
The aim of this study was to determine whether subconjunctival injection of unoprostone alters changes in the topography and blood flow of the optic disc induced by ET-1 in rabbits. In addition, the concentration of unoprostone metabolites in the retina after subconjunctival injection was estimated using high-performance liquid chromatography.
Fifteen male Japanese albino rabbits (2.5-3.5 kg; Shimizu Laboratory Supplies, Kyoto, Japan) were housed in a climate-controlled room (mean [SD] temperature, 22°C [2°C]; mean [SD] humidity, 45% [10%]; and 12-hour lighting cycle) with free access to food and water. The experimental procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research.
Human ET-1 was purchased from the Peptide Institute, Inc. (Osaka, Japan). After ET-1 was dissolved in 0.1% aqueous acetic acid to obtain a 10−4M solution, the concentration was adjusted to 10−6M by dilution with balanced saline solution. Unoprostone (Rescula eyedrops; 0.12%) and its metabolites, M1 and M2, were provided by R-Tech Ueno, Ltd (Tokyo, Japan). M1 and M2 were used as authentic standards for quantification. As an internal standard for the quantification, 13,14-dihydro-15-keto-prostaglandin F2α (Cayman Chemical Company, Ann Arbor, Michigan) was used.
Only 1 of the eyes was randomly selected for the experiments. We injected ET-1 (10−6M, 20 μL) into the posterior vitreous of the selected eye through the pars plana with a Hamilton syringe using a 30-gauge needle with the rabbit under local anesthesia with 0.4% oxybuprocaine hydrochloride (Benoxil; Santen Pharmaceutical Co, Ltd, Osaka, Japan). This dose of ET-1 was determined by an earlier study.3 For the injections, each rabbit was placed in a holding box, its pupil was dilated with 1 drop of 0.4% tropicamide (Mydrin M; Santen Pharmaceutical Co, Ltd), and its eye was held open with a Barraquer wire speculum (Inami & Co, Ltd, Tokyo, Japan). The injections were given with the rabbit under local anesthesia with oxybuprocaine on Tuesday and Friday for 4 weeks, and the overall observation period was 8 weeks beginning from the first injection of ET-1.
Twelve rabbits were divided into 3 groups. Two groups received intravitreal ET-1; 1 group was given a subconjunctival injection of 50 μL of 0.12% unoprostone after the ET-1 (ET-1 plus unoprostone group, n = 4), and the second group was given a subconjunctival injection of the vehicle for unoprostone (ET-1 plus vehicle group, n = 4). A third group was given an intravitreal injection of only the vehicle for ET-1 (sham group, n = 4). Measurements of the ONH blood flow, IOP, and ONH topography were performed before and 4 and 8 weeks after the first injection of ET-1 or vehicle.
To evaluate the changes in ocular circulation, the capillary blood flow in the ONH was measured using laser speckle flowgraphy, which permits a noninvasive, 2-dimensional measurement of circulation within tissue. The details of this instrument have been described.19 Briefly, when the ocular fundus is illuminated with a diode laser (wavelength, 808 nm), a speckle pattern appears, and the frequency of the speckles varies with blood velocity. The normalized blur (NB) obtained with laser speckle flowgraphy is equivalent to a quantitative index of the blurring of speckle patterns and was originally considered to be an indicator of blood velocity within tissue. Alterations in the NB were shown to represent changes in capillary blood flow in the ONH of rabbits because NB changes were well correlated with blood flow changes simultaneously measured by the hydrogen clearance method.20
Rabbits were placed in holding boxes, and measurements were performed with the rabbits under local anesthesia with a drop of oxybuprocaine. For the measurements of the ONH capillary blood flow, the average NB over an area of 0.72 × 0.72 mm of the ONH that was free of surface vessels was measured after mydriasis with a drop of tropicamide. We always recorded the measurement area, including characteristic vessels, to ensure that the same area was analyzed at all times of measurement of the same rabbit. It required 0.18 second to record 98 scans to obtain 1 NB value. The NB at each experimental time was calculated as the average of 5 successive measurements. The IOP was also measured with a pneumatonometer (Medtronic Solan, Jacksonville, Florida) immediately after the blood flow assessments.
To quantify the changes in the topography of the ONH, Heidelberg retina tomography (HRT2; Heidelberg Engineering, Heidelberg, Germany) was used. Previous experiments using HRT showed that the reproducibility of the topographic data in rabbits was comparable to that in humans.21 To verify the reproducibility of each parameter, the coefficient of variation, (standard deviation/mean) × 100, was calculated from 6 images of each randomly selected eye before the first injection of ET-1 or vehicle in 12 rabbits. These measurements were taken on the same day as the first measurements of blood flow and IOP.
Rabbits were placed in holding boxes, and measurement was performed after mydriasis was induced with 1 drop of tropicamide. To enhance the imaging quality, the cornea was kept moistened with artificial tears between imaging acquisitions. At each session, a series of 6 topographic images was obtained from each selected eye of the same rabbit, and then the NB data was averaged to obtain the topographic parameters for statistical analyses.
After completion of the 8-week experimental period, the animals were humanely killed with an overdose of intravenous pentobarbital sodium (Nembutal; Abbot, North Chicago, Illinois). The eyes were immediately enucleated, fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 10mM phosphate-buffered saline, rinsed with 10mM phosphate-buffered saline, and embedded in paraffin. A transverse section of each retina (3 μm) was cut parallel to the medullary rays 2 mm directly inferior to the center of the ONH and stained with hematoxylin-eosin. To evaluate the damage to the retina, cells in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the retina were counted by an examiner (Y.Y.) who was masked to the experimental procedures performed on the rabbit. For the analysis, 9 light photomicrographs taken at ×260 magnification around the center of each retinal section at a distance of approximately 5 mm were obtained in a masked fashion. The examiner counted all of the cells in the GCL and INL in these photographs, with displaced amacrine cells not excluded from the counts, as described.22 The numbers of cells in the retinal ganglion cell and INL were averaged for each eye to obtain data for the statistical analyses.
Three rabbits were used for this experiment. Rabbits were anesthetized by intramuscular injection of ketamine hydrochloride (Ketalar; Daaichi Sankyo Propharma Co, Ltd; Tokyo, Japan) and xylazine hydrochloride (Celactal; Bayer Japan Co, Ltd, Osaka, Japan) at doses of 35 and 10 mg/kg. Then 50 μL of 0.12% unoprostone was injected subconjunctivally into both eyes of 2 rabbits. At 0.5 and 1 hour after the injection, the eyes were collected after exsanguinations. Another rabbit received no treatment and served as a control.
The eyes were dissected, and retinal samples were homogenized with distilled water using Ultratarax (IKA Co, Ltd; Stauffen, Germany) to prepare 10% or 20% (wt/vol) retina homogenates. The concentrations of metabolites M1 and M2 in the retinal homogenates were determined by liquid chromatography–tandem mass spectrometry. The internal standards and acetonitrile were added to the retinal homogenate samples, and the samples were centrifuged at 10 000g for 2 minutes at room temperature. The supernatants were then evaporated to dryness by a centrifugal evaporator. The dried residues were dissolved with methanol and water (2:8, vol/vol), and then the samples were filtrated by centrifugation at 5000g for 1 minute at room temperature (Centricut Ultra-mini, 0.45 μm; Kurabo, Osaka, Japan). The filtrates were injected into a liquid chromatography–tandem mass spectrometry system.
Liquid chromatography–tandem mass spectrometry analysis was performed with a high-performance liquid chromatography system (Alliance 2795 system; Waters Corporation, Milford, Massachusetts) coupled to a guard column (Inertsil ODS-3; GL Sciences Inc, Tokyo, Japan) and an analytical column (Delovosil ODS-UG-3; 2.0 × 50 mm; Nomura Chemical, Seto, Japan). The mobile phases were composed of acetonitrile, water, and acetic acid (20:80:0.1) and acetonitrile and acetic acid (100:0.1). The gradient was as follows: 0 minutes, 100% acetonitrile, water, and acetic acid; 2 minutes, 40% acetonitrile, water, and acetic acid and 60% acetonitrile and acetic acid; 2 through 5 minutes, 40% acetonitrile, water, and acetic acid and 60% acetonitrile and acetic acid; and 5.01 through 7 minutes, 100% acetonitrile and acetic acid. The flow rate was 0.25 mL/min. The column compartment was kept at 35°C. The high-performance liquid chromatography eluate was introduced via electrospray ionization directly into a mass spectrometer (API 3000 triple quadrupole mass spectrometer; MDS Sciex, Concord, Ontario, Canada) using a Turbo Ion Spray interface. Multiple-reaction monitoring analysis of M1 and M2 was performed using the following transitions: mass/charge of 381 to 183 (M1) and mass charge of 327 to 141 (M2).
The tandem mass spectrometry data analyses were performed by Analyst (version 1.1; MDS Sciex). The concentrations of M1 and M2 were determined by the peak area ratios of M1 and M2 using an internal standard method. Calibration ranges were 0.25 to 250 ng/g for M1 and 0.5 to 250 ng/g for M2.
The data are expressed as mean (SEM). Statistical comparisons among groups were performed using analysis of variance followed by unpaired t tests. Statistical comparisons of data at different time points in each group were performed using repeated-measures analysis of variance followed by paired t tests. Differences were accepted as statistically significant at P < .05.
The mean NB values determined in the same part of the ONH are shown in Figure 1. In the ET-1 plus vehicle eyes, the NB values were significantly reduced at 4 and 8 weeks, whereas in the ET-1 plus unoprostone eyes, the reduction was not significant. The mean NB values in the sham control eyes did not change significantly (P > .05) throughout the study. The changes in the mean IOPs are shown in Figure 2. In the ET-1 plus unoprostone eyes and the ET-1 plus vehicle eyes, the IOPs were reduced significantly at 4 and 8 weeks, whereas the sham control eyes did not change significantly during the study.
Mean changes in the normalized blur (NB) values in the optic nerve head (n = 4). Repeated-measures analysis of variance showed statistically significant (P < .01) changes in the endothelin 1 (ET-1) plus vehicle group (open circle) but not in the sham control group (closed circle) and the ET-1 plus unoprostone group (open square). Significant decreases from baseline are seen in the ET-1 plus vehicle group (paired ttest, *P < .01, †P < .05). Error bars indicate SEM.
Mean changes in intraocular pressure (IOP) (n = 4). Repeated-measures analysis of variance showed statistically significant (P < .001 and P = .01) changes in the endothelin 1 (ET-1) plus vehicle group (open circle) and the ET-1 plus unoprostone group (open square) but not in the sham control group (closed circle). Statistically significant decreases from baseline can be seen in the ET-1 plus vehicle group and the ET-1 plus unoprostone group (paired t test, *P < .01). Error bars indicate SEM.
The mean (SEM) (n = 12) of the coefficients of variations for each HRT2 parameter are as follows: 0.00% (0.00%) for disc area, 16.63% (2.88%) for rim area, 9.44% (6.97%) for cup area, 35.34% (4.26%) for rim volume, 23.66% (2.88%) for cup volume, 9.41% (2.32%) for cup-disc area ratio, 8.20% (2.16%) for linear cup-disc ratio, 53.46% (12.23%) for cup shape measure, 13.28% (1.22%) for mean cup depth, 9.22% (2.25%) for maximum cup depth, 21.90% (5.82%) for height variation contour, 118.76% (39.30%) for mean retinal nerve fiber larger (RNFL) thickness, and 124.76% (38.58%) for RNFL cross-sectional area. Most of the parameters, except for the RNFL cross-sectional area, mean RNFL thickness, cup shape, and rim volume, showed good reproducibility with the coefficients of variation below 25%.
Many of the HRT2 parameters decreased after the injection of ET-1, but unoprostone inhibited the changes. In fact, the differences between sham control and ET-1 plus vehicle eyes were significant for rim area, cup area, rim volume, cup volume, cup-disc area ratio, linear cup-disc ratio, mean RNFL thickness, and RNFL cross-sectional area, although no significant difference was detected between sham control and ET-1 plus unoprostone eyes (Table 1).
The histologic changes in the retina are shown in Figure 3. The numbers of cells in the GCL and INL were decreased in the ET-1 plus vehicle eyes compared with the sham control eyes but not in the ET-1 plus unoprostone eyes. A quantitative assessment of the protective effect of unoprostone is shown in Figure 4 and Figure 5. The number of cells in the GCL and INL in the retinas of the ET-1 plus vehicle eyes was significantly fewer than in the sham control eyes and ET-1 plus unoprostone eyes.
Photomicrographs of transverse sections of the posterior retina. Sections were obtained from the sham control (A), endothelin 1 (ET-1) plus vehicle (B), and ET-1 plus unoprostone (C) eyes. The number of cells in the ganglion cell layer (GCL) and inner nuclear layer (INL) is reduced by repeated applications of ET-1 (B), whereas it is not changed significantly in the ET-1 plus unoprostone eyes (C). Bar = 50 μm. ONL indicates outer nuclear layer.
Mean number of cells in the ganglion cell layer (GCL) (n = 4). Analysis of variance revealed a statistically significant difference among the 3 groups (P = .004) (unpaired t tests, *P < .01, †P < .05). When unoprostone was given, there was no reduction in the number of GCL cells. Sham indicates sham control; vehicle, endothelin 1 plus vehicle; unoprostone, endothelin 1 plus unoprostone. Error bars indicate SEM.
Mean number of cells in the inner nuclear layer (INL) (n = 4). Analysis of variance revealed a statistically significant difference among the 3 groups (P = .009) (unpaired t tests, *P < .01, †P < .05). When unoprostone was given, there was no reduction in the number of INL cells. Sham indicates sham control; vehicle, endothelin 1 plus vehicle; unoprostone, endothelin 1 plus unoprostone. Error bars indicate SEM.
The concentrations of M1 and M2 in the retina after a subconjunctival injection of unoprostone are given in Table 2. The average concentration of M1 was 1.43 ng/g at 0.5 hour after the injection. At 1 hour, the concentration decreased below the lower limit of quantification. On the other hand, the average concentration of M2 was 1.125 ng/g at 1 hour after the injection.
The methods used in this study (laser speckle flowgraphy and HRT) have already been shown to obtain valid data in rabbits. Laser speckle flowgraphy was developed in Japan for assessing the blood flow in the ONH, choroid, and retina, and its accuracy has been demonstrated in rabbits.19,20 Orgül et al21 reported good reproducibility of topographic data obtained with a scanning laser ophthalmoscope (using HRT) in rabbits. We also confirmed the good reproducibility of data obtained by HRT2 in this study.
Our results showed significant changes in some of the topographical parameters (rim area, cup area, rim volume, cup volume, cup-disc area ratio, linear cup-disc ratio) of the optic disc. Similar findings have been observed in patients with glaucoma23 and in optic disc ischemia models induced by ET-1 administered via an osmotic minipump.2,24,25 In our study, the enlargement of the optic disc cup, which had previously been shown by National Institutes of Health images,3 was confirmed quantitatively using HRT2 in the same model of the ischemic ONH of rabbits. The decrease in ONH blood flow and the numbers of cells in the GCL induced by ET-1 was consistent with previous reports.3,22 In addition, the number of cells in the INL was also decreased in the ET-1 plus vehicle group. As we have stated,22 a direct comparison of this model with human glaucoma should be performed cautiously because anatomical differences exist between the vasculature and structure of the optic disc of rabbits and humans. Significant IOP reduction induced by ET-1 was not precisely consistent with the same model in previous studies,3,22 in which IOP was at a lower level in treated eyes, although the difference was not significant. However, several reports4,26,27 have found that IOP reduction is induced by ET-1 and its mechanism. One study suggests that ET-1 reduces IOP by decreasing aqueous humor production and by increasing outflow facility through ETA and ETB receptors.27
Our results demonstrated the inhibitory effect of subconjunctival injection of unoprostone on the topographical changes in the optic disc induced by ET-1. Unoprostone also suppressed the decrease in ONH blood flow and the number of cells in the GCL and INL induced by ET-1. On the other hand, unoprostone did not affect IOP-reducing action by ET-1, suggesting that IOP is not involved in the mechanism of action of the treatment in this model. In addition, it was confirmed that the unoprostone metabolites (M1 and M2) reach the retina at concentrations of approximately 1.4 and 1.1 ng/g, respectively. These concentrations corresponded to approximately 3.7nM and 3.4nM for molecular weights of 382.5 for M1 and 328.4 for M2. These concentrations can be pharmacologically effective on the microvasculature in the eye because unoprostone was reported to alter the ET-1–contracted pig retinal arterioles at 10−9M28; therefore, the effective concentration of M1 and M2 should be much lower than that of unoprostone.29 In addition, the retinal concentration of M1 was increased by approximately 3-fold after repeated (n=4) applications of unoprostone compared with a single application, and the retinal concentration of M2 was detected at least until 4 hours after a single application of unoprostone (Yosuke Kawai, MSc, R-tech Ueno Ltd, July 25, 2008, written). Given the above information, repeated subconjunctival injection of unoprostone might produce M1 and M2 at pharmacologically effective levels against ET-1 action in the eye.
It was recently reported that unoprostone augments the opening of the maxi-K potassium channels without affecting the basal (Ca2+) channels in ET-1–treated bovine trabecular meshwork and ciliary muscle cells.30 A similar action might occur in the ONH ischemia induced by ET-1. However, another study31 reported that the mechanism of the relaxation of vascular smooth muscle by unoprostone differed from that of IOP reduction and did not depend on the maxi-K potassium channels. Instead, the relaxation might be mediated by inhibition of Ca2+ entry possibly through capacitative Ca2+ channels. Moreover, M1 was shown to suppress the Ca2+ influx through Ca2+ release–activated Ca2+-current channels in trabecular meshwork and ciliary muscle cells.32 These findings are consistent with the idea that unoprostone does not increase the Ca2+ concentration in human cortical neuronal cells, a model system for studies of BK channel activator–based neuroprotective agents.33
There are several reports on the neuroprotective effects of unoprostone and its metabolites in vivo: unoprostone-protected photoreceptors against constant light-induced damage in rats,16 and M1 rescued retinal progenitor cells from apoptosis induced by serum deprivation via the phosphatidylinositol 3-hydroxyl kinase and protein kinase G pathways.18 Although the neuroprotective mechanisms of unoprostone remain to be definitively determined, these properties might be involved in the inhibitory effect of topical unoprostone on the topographical changes induced by ET-1.
In conclusion, we have demonstrated that subconjunctivally applied unoprostone can ameliorate topographical changes and blood flow reduction in the ONH of rabbits. These findings show the possibility of using unoprostone to treat optic neuropathic disease caused by ischemia, including glaucoma. However, because our experiments were performed in rabbits, the transfer of these results to human optic neuropathy should be considered cautiously.
Correspondence: Tetsuya Sugiyama, MD, PhD, Department of Ophthalmology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City 569-8686, Japan (firstname.lastname@example.org).
Submitted for Publication: May 31, 2008; final revision received July 30, 2008; accepted August 18, 2008.
Author Contributions: Dr Sugiyama had full access to all of the data in this study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Financial Disclosure: None reported.
Additional Contributions: Ryosuke Ono, MSc, Akiko Morikawa, MSc, and Yosuke Kawai, MSc, of R-Tech Ueno Ltd, and Asako Komori, BA, provided technical support.
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