Kaohsiung Journal of Medical Sciences (2014) 30, 593e598
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
ORIGINAL ARTICLE
Nitric oxide levels in the aqueous humor vary in different ocular hypertension experimental models Da-Wen Lu a, Yi-Hao Chen a, Charn-Jung Chang b, Chiao-Hsi Chiang c, Hsin-Yu Yao a,* a
Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan b Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan c School of Pharmacy, National Defense Medical Center, Taipei, Taiwan Received 28 March 2014; accepted 3 August 2014
Available online 25 October 2014
KEYWORDS Experimental glaucoma; Fluorophotometer; Latex microspheres; Nitric oxide
Abstract This study investigated the relationships among intraocular pressure (IOP), nitric oxide (NO) levels, and aqueous flow rates in experimental ocular hypertension models. A total of 75 rabbits were used. One of four different materials [i.e., a-chymotrypsin, latex microspheres (Polybead), red blood cell ghosts, or sodium hyaluronate (Healon GV)] was injected into the eyes of the 15 animals in each experimental group; the remaining 15 rabbits were reserved for a control group. The IOP changes in the five groups were recorded on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120. On postinduction Day 7, the dynamics and NO levels in the aqueous humor were recorded. Significant IOP elevations were induced by a-chymotrypsin (p < 0.01) and Polybead (p < 0.01) on each postinduction day. In the red blood cell ghosts model, significant elevations (p < 0.01) were found on postinduction Days 1e3; Healon GV significantly elevated IOP (p < 0.01) on postinduction Day 1 and Day 2. On postinduction Day 7, the aqueous humor NO levels increased significantly in the models of a-chymotrypsin, Polybead, and red blood cell ghosts (all p < 0.01), while the aqueous flow rates were significantly reduced in the models of a-chymotrypsin and Polybead (p < 0.005). Persistent ocular hypertension models were induced with a-chymotrypsin and Polybead in the rabbits. The Polybead model exhibited the characteristic of an increased
Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. Department of Ophthalmology, Tri-Service General Hospital, Number 325, Section 2, Cheng-Kong Road, Taipei, Taiwan. E-mail addresses: [email protected], [email protected] (H.-Y. Yao). http://dx.doi.org/10.1016/j.kjms.2014.09.004 1607-551X/Copyright ª 2014, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
594
D.-W. Lu et al. aqueous humor NO level, similar to human eyes with acute angle-closure glaucoma and neovascular glaucoma. Copyright ª 2014, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction
Materials and methods
Glaucoma is a major cause of irreversible vision loss worldwide and is considered to be a disease with progressive optic neuropathy. Elevated intraocular pressure (IOP) is a major risk factor for developing glaucomatous optic neuropathy. Thus, lowering IOP is recognized as an effective glaucoma treatment [1]. In the past, various animal models have been used to investigate the pharmacology and pathophysiology of glaucoma, particularly the effects of elevated IOP [2]. The study of animal models for different types of glaucoma has not been highly successful. Most studies have utilized rabbits, or to a lesser extent subhuman primates, such as monkey species [2]. Rabbits exhibit an inherited congenital glaucoma, and normotensive rabbits and rabbits with different forms of experimental glaucoma have frequently been investigated, particularly when screening drugs to lower IOP [3]. According to Gelatt et al [2], the most common experimental models used to induce glaucoma in rabbits include the following: water loading; injection of red blood cell (RBC) ghosts; injection of a-chymotrypsin; laser-induction; and steroid injections. The water-loading model does not maintain an elevated IOP (> 30 mmHg) or increase the duration of the elevated IOP. In the models of RBC ghost-induced glaucoma, the elevated IOP does not persist for > 3 months. In the a-chymotrypsin model, a long-term period of elevated IOP (> 30 mmHg) was achieved. However, a-chymotrypsin causes intense iridocyclitis, intraocular inflammation, lens dislocation and vitreous precipitates. Laser-induced glaucoma requires additional laser equipment and carries the risk of inducing phthisis bulbi. Steroid-induced glaucoma requires frequent injections and is associated with serious systemic side effects. A novel model of experimental glaucoma or ocular hypertension in rabbits is therefore necessary. In the eyes, nitric oxide (NO) plays a vital role in the pathogenesis of glaucoma and involves regulating the IOP [4], modulating local ocular blood flow [5], and controlling retinal ganglion cell death by apoptosis [6]. The excessive production of NO in glaucomatous eyes is stimulated by the expression of the inducible isoform of NO synthase [7]. It has been demonstrated that excessive NO is produced in the retina and optic disc of rats with experimental glaucoma and in the eyes of glaucoma patients [8e11]. In our previous study, we also found that NO levels in the aqueous humor (AH) differ in patients with different types of glaucoma [10]. In the present study, one of our goals was to compare four different experimental models based on their IOP levels, AH NO levels, and aqueous flow rates. Another goal was to elucidate the relationship between NO and different ocular hypertension models.
Materials for glaucoma induction The following materials were used to set up four different glaucoma models: RBC ghosts, sodium hyaluronate (Healon GV), a-chymotrypsin (Leurquin Co., Neuilly-sur-Marne, France); latex microspheres (Polybead polystyrene, 2.5% Solid-Polybead; PSI, Warrington, PA, USA); 10% sodium fluorescein (NaFl) and tobramycin (Alcon Lab., Fort Worth, TX, USA); and ketamine HCl, xylazine HC1, proparacaine HC1 (Alcaine), and sodium phosphate buffer (pH 7.4; Sigma Chemical Co., St. Louis, MO, USA). The New Zealand (NZW) white rabbits were obtained from the National Taiwan University Hospital Animal Center (Taipei, Taiwan).
Animal anesthesia and preparations for glaucoma induction A total of 75 eyes from 75 NZW white rabbits (weighing 2.5e3 kg, regardless of gender) were used in this in vivo study. Each group was composed of 15 rabbits, which were used for the material induction, and the remaining 15 rabbits were reserved for the control group. Animal care and treatment complied with the guidelines of the Association for Research in Vision and Ophthalmology governing research animals. The NZW white rabbits were anesthetized with an intramuscular injection of ketamine HCl (40 mg/kg) and xylazine HCl (8 mg/kg), and then, two eye drops of 0.5% proparacaine HC1 (Alcaine) were placed in the right eyes of the rabbits. After anesthesia, using two 30-G needles, we injected the four different materials into the right eyes of the rabbits to induce glaucoma; the first injection was into the anterior chamber through the limbus, while the other drained the AH from the anterior chamber. As the surgical wounds healed, infection was prevented by administering topical tobramycin for 4 days [12]. The IOP was initially measured after the inducing materials and was recorded at regular intervals. The IOP changes in the five groups were recorded on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120. On postinduction Day 7, the dynamics and NO levels of the AH in the individuals of each group were recorded.
IOP measurements The IOP changes in the five groups were recorded on the stated postinduction days with a pneumotonometer (Langham, California, USA). Ocular hypertension induction (defined as IOP >30 mmHg) was compared between the induced eyes and the control eyes according to their IOP levels [12].
NO in ocular hypertension experimental models
595
Fluorophotometric method for determining aqueous flow rates On postinduction Day 7, we placed four drops of the NaFl tracer into the center of the cornea and scanned the pupil using fluorophotometry at regular intervals to observe the AH dynamics. According to the method devised by Yablonski [13], the measurement of the permeability (flow of the fluorescent dye from the cornea to the anterior chamber) was based upon fluorescent dynamics after topical application of the fluorescent trace [14]. Mathematically, the cornea and anterior chamber of the eye can be regarded as two compartments that are separated by a semipermeable membrane; if fluorescein is introduced into the smaller compartment (the cornea), it will quickly diffuse through the semipermeable membrane into the larger compartment. Eventually, the concentrations between the two compartments will reach equilibrium, with a constant ratio between the two compartments. The following equations describe this process. The change in the concentration of the cornea: dCc/dt Z Ka.ca (Ca e Cc)
(1)
and the change in the concentration of the anterior chamber: dCa/dt Z Ko*Ca þ Ka.ca (Cc Ca)
(2)
where Cc and Ca are the fluorescein concentrations in the cornea and the anterior chamber, respectively; Ko is the transfer coefficient for anterior chamber loss; and Kc.ca and Ka.ca are the transfer coefficients of fluorescein between the cornea and anterior chamber, and vice versa. Where Vc and Vac refer to the volume of the cornea and the volume of the anterior chamber, respectively, we have: Vac Ka.ca Z Vc Kc.ca
(3)
Measurement of aqueous flow To reach the final concentration of 0.025% NaFl, we diluted 0.25 mL of the stock solution (10% w/v) with 9.75 mL of 25 mM sodium phosphate buffer (pH 7.4). The fluorescence intensity was measured on a scanning fluorophotometer (Fluorotron Master, OcuMetrics, Mountain View, CA, USA). Fluorescence intensity was linearly related to a NaFl concentration between 1 nmol/L and 10,000 nmol/L. The NaFl concentration was calculated from the fluorescence using a standard calibration curve [15].
cold absolute alcohol, mixing thoroughly, incubating for 30 minutes at 4 C, and then centrifuging for 6 minutes at 13,000 rpm (Denver Instrument, Arizona, USA). Finally, the NO analyzer (Sievers 280 NOA, Sievers Inc., Boulder, CO, USA) was used for the chemiluminescence method of assaying the supernatants [10].
Statistical analysis The t test for paired samples was used to determine the level of statistical significance. A p value < 0.05 was considered statistically significant.
Results Among the four materials used to induce the experimental models of ocular hypertension, the a-chymotrypsin-induced eyes (15 eyes of 15 rabbits) exhibited more serious inflammatory reactions than the other groups. On postinduction Day 7, all 15 eyes in the a-chymotrypsin-induced rabbit group exhibited serious intraocular inflammation. On postinduction Day 120, six eyes in the a-chymotrypsininduced rabbit group exhibited lens dislocation. The data showed that significant IOP elevations in the eyes were induced with a-chymotrypsin (p < 0.01) and Polybead (p < 0.01) on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120 (Fig. 1 and Table 1). Successful induction of ocular hypertension was achieved in the eyes induced with a-chymotrypsin on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120 and in the eyes induced with Polybead on postinduction Days 1e3, Day 7, Day 14, Day 30, and Day 60. Significant IOP elevations were only found on postinduction Days 1e3 in the eyes induced with RBC ghosts (p < 0.01) and on Days 1 and 2 with Healon GV (p < 0.01). After postinduction Day 7, the rabbits induced with RBC ghosts and Healon GV failed to achieve significant IOP elevations (Table 1). On postinduction Day 7, the AH NO levels increased significantly in the models induced with a-chymotrypsin (p < 0.005), Polybead (p < 0.005), and RBC ghosts (p < 0.01). No significant change was observed in the Healon GV-induced models (Table 1). The aqueous flow
Detection of NO levels in the AH On postinduction Day 7, the change in NO levels was measured in the experimental and control eyes by obtaining ocular humor via clear corneal paracentesis. To avoid contact with blood vessels or damage to the iris or other intraocular structures, 1 mL syringes with 30-gauge needles were used. All samples were kept on ice and frozen at 70 C until the protein extraction. Extraction was performed in a 2-mL test tube by adding a double volume of
Figure 1. Time distribution of the average intraocular pressure values (mean SD, n Z 15) of the New Zealand white rabbits. The intraocular pressure changes in the five groups were recorded on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120. * p < 0.05.
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D.-W. Lu et al.
Table 1 The IOP, aqueous nitric oxide levels, and aqueous dynamics in the glaucomatous eyes of New Zealand white rabbits on postinduction Day 7 after inducing the different materials (n Z 15). Groups Aqueous humor level (mM) IOP (mmHg) Aqueous flow Flow rate (ul/min) [AC]slope [Cora]slope D (aver.) Ko (min1) K(a.ca) min1 K(c.ca) min1
a-chymotrypsin
Latex microspheres (Polybead)
135.5 14.5**
RBC ghost
Healon GV
Control eyes
149.8 15.7**
87.6 6.1*
29.1 3.7
28.1 3.7
31.3 3.9**
28.5 3.1**
15.9 2.5
15.1 2.1
15.1 2.3
0.9 0.2**
1.4 0.3**
2.5 0.5
2.5 0.6
2.6 0.5
0.0023 0.0012 5.9 0.005 0.007 0.002
0.001* 0.0002** 1.8 0.002 0.002** 0.0008*
0.003 0.003 6.9 0.008 0.009 0.003
0.001* 0.001* 1.8 0.002 0.004** 0.001*
0.0047 0.0048 6.6 0.014 0.031 0.01
0.001 0.001 1.5 0.001 0.019 0.005
0.005 0.005 4.5 0.017 0.017 0.006
0.0003 0.0001 1.1* 0.003 0.007* 0.002
0.0044 0.0044 6.6 0.015 0.031 0.01
0.001 0.001 1.5 0.003 0.019 0.005
Data are presented as mean SD. * p < 0.01. ** p < 0.005. AC Z anterior chamber; Cora Z cornea; D Z the flow equivalent to loss of fluorescein by diffusion; IOP Z intraocular pressure; Ko Z the transfer coefficient for anterior chamber loss; K(c.ca) and K(a.ca) Z the transfer coefficients of fluorescein between the cornea and anterior chamber, respectively; RBC Z red blood cell.
rates on this day were significantly reduced in those eyes induced with a-chymotrypsin (p < 0.005) and Polybead (p < 0.005). No significant change was recorded in the RBC ghost- and Healon GV-induced models (Table 1).
Discussion In the present study, we used four different materials to induce an experimental model of ocular hypertension. Significant IOP elevation was achieved in the rabbit models induced with a-chymotrypsin and Polybead from postinduction Day 1 to postinduction Day 120. Both models caused ocular hypertension for at least 60 days. Neither ghost RBCs nor Healon GV achieved significant IOP elevation after postinduction Day 7. On postinduction Day 7, the AH NO level increased significantly in the models induced with a-chymotrypsin, Polybead, and RBC ghosts. Healon GV did not cause an increased NO level in AH. The aqueous flow rates were significantly decreased with a-chymotrypsin and Polybead. Neither ghost RBCs or Healon GV resulted in a significantly decreased aqueous rate. The a-chymotrypsininduced model also had more intraocular inflammation than the other groups. AH is released from the ciliary process and returned to the venous system after passing through the trabecular meshwork [16]. In our previous study, we found that the AH NO level was higher in glaucomatous eyes than in nonglaucomatous eyes [10]. The main reason for the higher AH NO level in the glaucomatous eyes was proposed to be the inflammatory reaction in response to the elevated IOP [17]. Alternatively, Wang et al [18] believe that glaucoma is a chronic disease and any increases in AH NO levels arise to compensate for the impaired aqueous outflow that results from pathogenic conditions. In rabbit models, it has been demonstrated that NO synthase 2 is present in the ocular
tissue and that glutamate is elevated in the vitreous humor. Excessive NO may be caused by elevated IOP [19e21]. In our study, we found increased AH NO levels in a-chymotrypsin-, Polybead-, and RBC ghost-induced models. There was no significant change in the AH NO level in the Healon GV-induced model. In our previous study [10], we found that neovascular glaucoma (NVG) and acute angle-closure glaucoma (AACG) patients had the highest NO levels in the AH. Recent studies have demonstrated that NVG patients also have higher levels of vascular endothelial growth factor (VEGF) in their AH [22]. As VEGF is known to increase the release of NO from human tissues [23], it is possible that the elevation of VEGF leads to the higher levels of NO in the AH of NVG patients. In addition, these patients also suffer from breakdown of the bloodeaqueous barrier and chronic inflammation in the anterior chamber, both of which might also contribute to elevation of NO levels. The excessively high NO levels in the AHs of NVG patients may indicate that the pathology of NVG is usually complicated in diabetic retinopathy, which is related to the development of neovascularization in the iris and in the anterior chamber angle of the eyes [24]. In AACG patients, inflammatory reactions are usually present in the anterior chamber, which might partially explain the elevated NO levels in the AH. Chiou et al [11] have demonstrated significantly higher NO levels in the AHs of AACG patients than in cataract patients. An acute attack of glaucoma supposedly exacerbates the inflammation, thereby inducing a higher NO level in the AH. Thus, in NVG and AACG patients, we hypothesized that the increased NO levels in the AH may be a manifestation of the inflammatory status of the anterior chamber [10]. In our study, both a-chymotrypsin and Polybead caused significant IOP elevations and increased AH NO levels. The reason for the increased AH NO levels in these models may be that both materials caused various degrees of intraocular
NO in ocular hypertension experimental models inflammation. However, Polybead, a type of latex microsphere, induced the experimental model with less intraocular inflammation and tissue destruction than achymotrypsin. In the current study, the Polybead model exhibited the characteristics of high AH NO levels in the AH and fewer complications; therefore, it may be an acceptable experimental ocular hypertension model for the further investigation of NVG and AACG. In the past, many materials have shown the blockage of the trabecular meshwork and elevated IOP in the eyes of rabbit models, mimicking human glaucoma. However, disadvantages have existed in each of the various glaucoma and ocular hypertension models, with major disadvantages including a shorter period of IOP elevation and a lesser degree of IOP elevation (< 30 mmHg). In our study, a-chymotrypsin and Polybead resulted in significant IOP elevations from postinduction Day 1 to Day 120, whereas RBC ghosts and Healon GV did not achieve significant IOP elevations after postinduction Day 7. Bead microspheres were used to induce experimental glaucoma by obstructing aqueous outflow, and acute glaucoma was induced after the intracameral or intravitreous injection of polystyrene beads in the various animal models [25]. Weber et al [26] have proposed that IOP elevation most likely results from the bead obstruction of aqueous outflow, bead-induced swelling within the trabecular meshwork, or infiltration of the drainage channels by inflammatory cells. We found that the Polybead-induced experimental model had significant IOP elevations lasting for 120 days and IOP > 30 mmHg lasting for 60 days. a-Chymotrypsin is a commonly used material for inducing experimental glaucoma. However, induction with a-chymotrypsin destroys the fibrin of the zonular fibers in the anterior chamber, causing lens dislocation and serious intraocular inflammation [21]. Such effects complicate the intraocular condition and interfere with the investigation of the pathology of glaucoma or drug mechanisms in experimental models. Although the a-chymotrypsin-induced model caused a longer period of IOP elevation than the Polybead model, it also caused more severe intraocular inflammation. Healon GV, a common material for inducing IOP elevation in ocular surgery, was not a good experimental model, according to our results. After injecting Healon GV into the eyes, the IOP was elevated but slowly declined to normal within 3 days. On Day 7 after the Healon GV induction, the experimental model showed no significant IOP change. Compared to the other models, Healon GV resulted in a poor effect on experimental ocular hypertension. The RBC ghost model has been addressed previously, and it confirmed the possibility of secondary glaucoma due to trabecular obstruction [27,28]. According to the previous study, the model easily produced IOP elevation for 7e36 days and was not associated with intraocular inflammation. However, our study revealed that the IOP elevation induced with the RBC ghost did not last for 7 days. This fact most likely contributed to the variable results of the RBC ghostinduced models. Interestingly, the AH NO levels were significantly elevated in the RBC ghost-induced eyes despite the lack of significant changes in IOP or aqueous flow rate. We believe that RBC ghosts can still induce transient IOP elevation in experimental eyes, while the effect of transient IOP elevation caused the subsequent responses observed as elevated AH NO levels on postinduction Day 7. In the present
597 study, the RBC ghost initially caused IOP elevation in the experimental eyes, but the effect of IOP elevation did not persist after 7 days. Hence, a longer period of higher IOP is difficult for the RBC ghosts to achieve. We believe that the RBC ghost may not impede the trabecular meshwork entirely, and a longer period of elevated IOP may not be easily achieved. In the present study, Healon GV- and RBCinduced rabbit models initially caused elevated IOPs, but the period of elevated IOP was shorter, leading us to suppose that neither material caused a sufficient period of elevated IOP or subsequent pathological changes. In conclusion, we provided comparable results for the IOP and AH NO levels and the aqueous flow rate for each of the four different ocular hypertension models. According to the present results, RBC ghost- and Healon GV- induced models initially caused IOP elevation, but the change was not significant after 7 days postinduction. a-Chymotrypsin and Polybead were the best experimental models. In addition, the Polybead-induced model exhibited fewer complications than the a-chymotrypsin induced model. Based on previous studies, Polybead exhibited similar features (increased AH NO levels) in both NVG and AACG patients, and it may constitute a suitable model for investigating these types of glaucoma.
Acknowledgments This research work was partially supported by National Science Council grants (89-2314-B-016-011 and 89-2314-B016-070) and was utilized to fulfill the dissertation requirement for C.J.C.
References [1] Alwerd WL. Medical management of glaucoma. N Engl J Med 1998;339:1298e307. [2] Gelatt KN, Brooks DE, Samuelson DA. Comparative glaucomatology II: the experimental glaucomas. J Glaucoma 1998;7: 282e94. [3] Gelatt KN, Brooks DE, Samuelson DA. Comparative glaucomatology I: the spontaneous glaucomas. J Glaucoma 1998;7: 187e201. [4] Behar-Cohen FF, Goureau OD, D’Hermies F, Courtois Y. Decreased intraocular pressure induced by nitric oxide donors is correlated to nitrite production in the rabbit eye. Invest Ophthhalmol Vis Sci 1996;37:1711e5. [5] Haefliger IO, Meyer P, Flammer J, Lu ¨scher TF. The vascular endothelium as a regulator of the ocular circulation: a new concept in ophthalmology? Surv Ophthalmol 1994;39:123e32. [6] Neufeld AH. Nitric oxide: a potential mediator of retinal ganglion cell damage in glaucoma. Surv Ophthalmol 1999;43: S129e35. [7] Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J 1994;298:249e58. [8] Siu AW, Leung MC, To CH, Siu FK, Ji JZ, So KF. Total retinal nitric oxide production is increased in intraocular pressureelevated rats. Exp Eye Res 2002;75:401e6. [9] Neufeld AH, Hernandez MR, Gonzalez M. Nitric oxide synthase in the human glaucomatous optic nerve head. Arch Ophthalmol 1997;115:497e503. [10] Chang CJ, Chiang CH, Chow JC, Lu DW. Aqueous humor nitric oxide levels differ in patients with different types of glaucoma. J Ocul Pharmacol Ther 2000;16:399e406.
598 [11] Chiou SH, Chang CJ, Hsu WM, Kao CL, Liu JH, Chen WL, et al. Elevated nitric oxide level in aqueous humor of patients with acute angle-closure glaucoma. Ophthalmologica 2001;215: 113e6. [12] Lin CH, Chiang CH, Lee AR. Effects of quinoxalinocarboxylic acids on intraocular pressure and ocular blood flow in rabbits. Chin Pharm J 1995;47:135e46. [13] Yablonski ME, Zimmerman TJ, Waltman SR, Becker B. A fluorophotometric study of the effect of topical timolol on aqueous humor dynamics. Exp Eye Res 1978;27:135e42. [14] Hayashi M, Yablonski MC, Novack GD. Trabecular outflow facility determined by fluorophotometry in human subjects. Exp Eye Res 1989;48:621e5. [15] Jones RF, Maurice DM. New methods of measuring the rate of aqueous flow in man with fluorescein. Exp Eye Res 1966;5: 208e20. [16] Samuelson DA. A re-evaluation of the comparative anatomy of the Eutherian iridocorneal angle and associated ciliary body musculature. Vet Comp Ophthalmol 1996;6:153e72. [17] Kerrigan LA, Zack D, Quigley HA, Smith SD, Pease ME. TUNELpositive ganglion cells in human primary open-angle glaucoma. Arch Ophthalmol 1997;115:1031e5. [18] Wang ZY, Alm P, Ha ˚kanson R. The contribution of nitric oxide to endotoxin-induced ocular inflammation: interaction with sensory nerve fibres. Br J Pharmacol 1996;118:1537e43. [19] 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 USA 1999;96:9944e8. [20] Er H, Gu ¨ndu ¨z A, Turkoz Y, Cigli A, Is‚ci N. Effects of NG-nitro-Larginine and corticosteroids on aqueous humor levels of nitric
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
ORIGINAL ARTICLE
Nitric oxide levels in the aqueous humor vary in different ocular hypertension experimental models Da-Wen Lu a, Yi-Hao Chen a, Charn-Jung Chang b, Chiao-Hsi Chiang c, Hsin-Yu Yao a,* a
Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan b Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan c School of Pharmacy, National Defense Medical Center, Taipei, Taiwan Received 28 March 2014; accepted 3 August 2014
Available online 25 October 2014
KEYWORDS Experimental glaucoma; Fluorophotometer; Latex microspheres; Nitric oxide
Abstract This study investigated the relationships among intraocular pressure (IOP), nitric oxide (NO) levels, and aqueous flow rates in experimental ocular hypertension models. A total of 75 rabbits were used. One of four different materials [i.e., a-chymotrypsin, latex microspheres (Polybead), red blood cell ghosts, or sodium hyaluronate (Healon GV)] was injected into the eyes of the 15 animals in each experimental group; the remaining 15 rabbits were reserved for a control group. The IOP changes in the five groups were recorded on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120. On postinduction Day 7, the dynamics and NO levels in the aqueous humor were recorded. Significant IOP elevations were induced by a-chymotrypsin (p < 0.01) and Polybead (p < 0.01) on each postinduction day. In the red blood cell ghosts model, significant elevations (p < 0.01) were found on postinduction Days 1e3; Healon GV significantly elevated IOP (p < 0.01) on postinduction Day 1 and Day 2. On postinduction Day 7, the aqueous humor NO levels increased significantly in the models of a-chymotrypsin, Polybead, and red blood cell ghosts (all p < 0.01), while the aqueous flow rates were significantly reduced in the models of a-chymotrypsin and Polybead (p < 0.005). Persistent ocular hypertension models were induced with a-chymotrypsin and Polybead in the rabbits. The Polybead model exhibited the characteristic of an increased
Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. Department of Ophthalmology, Tri-Service General Hospital, Number 325, Section 2, Cheng-Kong Road, Taipei, Taiwan. E-mail addresses: [email protected], [email protected] (H.-Y. Yao). http://dx.doi.org/10.1016/j.kjms.2014.09.004 1607-551X/Copyright ª 2014, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
594
D.-W. Lu et al. aqueous humor NO level, similar to human eyes with acute angle-closure glaucoma and neovascular glaucoma. Copyright ª 2014, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction
Materials and methods
Glaucoma is a major cause of irreversible vision loss worldwide and is considered to be a disease with progressive optic neuropathy. Elevated intraocular pressure (IOP) is a major risk factor for developing glaucomatous optic neuropathy. Thus, lowering IOP is recognized as an effective glaucoma treatment [1]. In the past, various animal models have been used to investigate the pharmacology and pathophysiology of glaucoma, particularly the effects of elevated IOP [2]. The study of animal models for different types of glaucoma has not been highly successful. Most studies have utilized rabbits, or to a lesser extent subhuman primates, such as monkey species [2]. Rabbits exhibit an inherited congenital glaucoma, and normotensive rabbits and rabbits with different forms of experimental glaucoma have frequently been investigated, particularly when screening drugs to lower IOP [3]. According to Gelatt et al [2], the most common experimental models used to induce glaucoma in rabbits include the following: water loading; injection of red blood cell (RBC) ghosts; injection of a-chymotrypsin; laser-induction; and steroid injections. The water-loading model does not maintain an elevated IOP (> 30 mmHg) or increase the duration of the elevated IOP. In the models of RBC ghost-induced glaucoma, the elevated IOP does not persist for > 3 months. In the a-chymotrypsin model, a long-term period of elevated IOP (> 30 mmHg) was achieved. However, a-chymotrypsin causes intense iridocyclitis, intraocular inflammation, lens dislocation and vitreous precipitates. Laser-induced glaucoma requires additional laser equipment and carries the risk of inducing phthisis bulbi. Steroid-induced glaucoma requires frequent injections and is associated with serious systemic side effects. A novel model of experimental glaucoma or ocular hypertension in rabbits is therefore necessary. In the eyes, nitric oxide (NO) plays a vital role in the pathogenesis of glaucoma and involves regulating the IOP [4], modulating local ocular blood flow [5], and controlling retinal ganglion cell death by apoptosis [6]. The excessive production of NO in glaucomatous eyes is stimulated by the expression of the inducible isoform of NO synthase [7]. It has been demonstrated that excessive NO is produced in the retina and optic disc of rats with experimental glaucoma and in the eyes of glaucoma patients [8e11]. In our previous study, we also found that NO levels in the aqueous humor (AH) differ in patients with different types of glaucoma [10]. In the present study, one of our goals was to compare four different experimental models based on their IOP levels, AH NO levels, and aqueous flow rates. Another goal was to elucidate the relationship between NO and different ocular hypertension models.
Materials for glaucoma induction The following materials were used to set up four different glaucoma models: RBC ghosts, sodium hyaluronate (Healon GV), a-chymotrypsin (Leurquin Co., Neuilly-sur-Marne, France); latex microspheres (Polybead polystyrene, 2.5% Solid-Polybead; PSI, Warrington, PA, USA); 10% sodium fluorescein (NaFl) and tobramycin (Alcon Lab., Fort Worth, TX, USA); and ketamine HCl, xylazine HC1, proparacaine HC1 (Alcaine), and sodium phosphate buffer (pH 7.4; Sigma Chemical Co., St. Louis, MO, USA). The New Zealand (NZW) white rabbits were obtained from the National Taiwan University Hospital Animal Center (Taipei, Taiwan).
Animal anesthesia and preparations for glaucoma induction A total of 75 eyes from 75 NZW white rabbits (weighing 2.5e3 kg, regardless of gender) were used in this in vivo study. Each group was composed of 15 rabbits, which were used for the material induction, and the remaining 15 rabbits were reserved for the control group. Animal care and treatment complied with the guidelines of the Association for Research in Vision and Ophthalmology governing research animals. The NZW white rabbits were anesthetized with an intramuscular injection of ketamine HCl (40 mg/kg) and xylazine HCl (8 mg/kg), and then, two eye drops of 0.5% proparacaine HC1 (Alcaine) were placed in the right eyes of the rabbits. After anesthesia, using two 30-G needles, we injected the four different materials into the right eyes of the rabbits to induce glaucoma; the first injection was into the anterior chamber through the limbus, while the other drained the AH from the anterior chamber. As the surgical wounds healed, infection was prevented by administering topical tobramycin for 4 days [12]. The IOP was initially measured after the inducing materials and was recorded at regular intervals. The IOP changes in the five groups were recorded on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120. On postinduction Day 7, the dynamics and NO levels of the AH in the individuals of each group were recorded.
IOP measurements The IOP changes in the five groups were recorded on the stated postinduction days with a pneumotonometer (Langham, California, USA). Ocular hypertension induction (defined as IOP >30 mmHg) was compared between the induced eyes and the control eyes according to their IOP levels [12].
NO in ocular hypertension experimental models
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Fluorophotometric method for determining aqueous flow rates On postinduction Day 7, we placed four drops of the NaFl tracer into the center of the cornea and scanned the pupil using fluorophotometry at regular intervals to observe the AH dynamics. According to the method devised by Yablonski [13], the measurement of the permeability (flow of the fluorescent dye from the cornea to the anterior chamber) was based upon fluorescent dynamics after topical application of the fluorescent trace [14]. Mathematically, the cornea and anterior chamber of the eye can be regarded as two compartments that are separated by a semipermeable membrane; if fluorescein is introduced into the smaller compartment (the cornea), it will quickly diffuse through the semipermeable membrane into the larger compartment. Eventually, the concentrations between the two compartments will reach equilibrium, with a constant ratio between the two compartments. The following equations describe this process. The change in the concentration of the cornea: dCc/dt Z Ka.ca (Ca e Cc)
(1)
and the change in the concentration of the anterior chamber: dCa/dt Z Ko*Ca þ Ka.ca (Cc Ca)
(2)
where Cc and Ca are the fluorescein concentrations in the cornea and the anterior chamber, respectively; Ko is the transfer coefficient for anterior chamber loss; and Kc.ca and Ka.ca are the transfer coefficients of fluorescein between the cornea and anterior chamber, and vice versa. Where Vc and Vac refer to the volume of the cornea and the volume of the anterior chamber, respectively, we have: Vac Ka.ca Z Vc Kc.ca
(3)
Measurement of aqueous flow To reach the final concentration of 0.025% NaFl, we diluted 0.25 mL of the stock solution (10% w/v) with 9.75 mL of 25 mM sodium phosphate buffer (pH 7.4). The fluorescence intensity was measured on a scanning fluorophotometer (Fluorotron Master, OcuMetrics, Mountain View, CA, USA). Fluorescence intensity was linearly related to a NaFl concentration between 1 nmol/L and 10,000 nmol/L. The NaFl concentration was calculated from the fluorescence using a standard calibration curve [15].
cold absolute alcohol, mixing thoroughly, incubating for 30 minutes at 4 C, and then centrifuging for 6 minutes at 13,000 rpm (Denver Instrument, Arizona, USA). Finally, the NO analyzer (Sievers 280 NOA, Sievers Inc., Boulder, CO, USA) was used for the chemiluminescence method of assaying the supernatants [10].
Statistical analysis The t test for paired samples was used to determine the level of statistical significance. A p value < 0.05 was considered statistically significant.
Results Among the four materials used to induce the experimental models of ocular hypertension, the a-chymotrypsin-induced eyes (15 eyes of 15 rabbits) exhibited more serious inflammatory reactions than the other groups. On postinduction Day 7, all 15 eyes in the a-chymotrypsin-induced rabbit group exhibited serious intraocular inflammation. On postinduction Day 120, six eyes in the a-chymotrypsininduced rabbit group exhibited lens dislocation. The data showed that significant IOP elevations in the eyes were induced with a-chymotrypsin (p < 0.01) and Polybead (p < 0.01) on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120 (Fig. 1 and Table 1). Successful induction of ocular hypertension was achieved in the eyes induced with a-chymotrypsin on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120 and in the eyes induced with Polybead on postinduction Days 1e3, Day 7, Day 14, Day 30, and Day 60. Significant IOP elevations were only found on postinduction Days 1e3 in the eyes induced with RBC ghosts (p < 0.01) and on Days 1 and 2 with Healon GV (p < 0.01). After postinduction Day 7, the rabbits induced with RBC ghosts and Healon GV failed to achieve significant IOP elevations (Table 1). On postinduction Day 7, the AH NO levels increased significantly in the models induced with a-chymotrypsin (p < 0.005), Polybead (p < 0.005), and RBC ghosts (p < 0.01). No significant change was observed in the Healon GV-induced models (Table 1). The aqueous flow
Detection of NO levels in the AH On postinduction Day 7, the change in NO levels was measured in the experimental and control eyes by obtaining ocular humor via clear corneal paracentesis. To avoid contact with blood vessels or damage to the iris or other intraocular structures, 1 mL syringes with 30-gauge needles were used. All samples were kept on ice and frozen at 70 C until the protein extraction. Extraction was performed in a 2-mL test tube by adding a double volume of
Figure 1. Time distribution of the average intraocular pressure values (mean SD, n Z 15) of the New Zealand white rabbits. The intraocular pressure changes in the five groups were recorded on postinduction Days 1e3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120. * p < 0.05.
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Table 1 The IOP, aqueous nitric oxide levels, and aqueous dynamics in the glaucomatous eyes of New Zealand white rabbits on postinduction Day 7 after inducing the different materials (n Z 15). Groups Aqueous humor level (mM) IOP (mmHg) Aqueous flow Flow rate (ul/min) [AC]slope [Cora]slope D (aver.) Ko (min1) K(a.ca) min1 K(c.ca) min1
a-chymotrypsin
Latex microspheres (Polybead)
135.5 14.5**
RBC ghost
Healon GV
Control eyes
149.8 15.7**
87.6 6.1*
29.1 3.7
28.1 3.7
31.3 3.9**
28.5 3.1**
15.9 2.5
15.1 2.1
15.1 2.3
0.9 0.2**
1.4 0.3**
2.5 0.5
2.5 0.6
2.6 0.5
0.0023 0.0012 5.9 0.005 0.007 0.002
0.001* 0.0002** 1.8 0.002 0.002** 0.0008*
0.003 0.003 6.9 0.008 0.009 0.003
0.001* 0.001* 1.8 0.002 0.004** 0.001*
0.0047 0.0048 6.6 0.014 0.031 0.01
0.001 0.001 1.5 0.001 0.019 0.005
0.005 0.005 4.5 0.017 0.017 0.006
0.0003 0.0001 1.1* 0.003 0.007* 0.002
0.0044 0.0044 6.6 0.015 0.031 0.01
0.001 0.001 1.5 0.003 0.019 0.005
Data are presented as mean SD. * p < 0.01. ** p < 0.005. AC Z anterior chamber; Cora Z cornea; D Z the flow equivalent to loss of fluorescein by diffusion; IOP Z intraocular pressure; Ko Z the transfer coefficient for anterior chamber loss; K(c.ca) and K(a.ca) Z the transfer coefficients of fluorescein between the cornea and anterior chamber, respectively; RBC Z red blood cell.
rates on this day were significantly reduced in those eyes induced with a-chymotrypsin (p < 0.005) and Polybead (p < 0.005). No significant change was recorded in the RBC ghost- and Healon GV-induced models (Table 1).
Discussion In the present study, we used four different materials to induce an experimental model of ocular hypertension. Significant IOP elevation was achieved in the rabbit models induced with a-chymotrypsin and Polybead from postinduction Day 1 to postinduction Day 120. Both models caused ocular hypertension for at least 60 days. Neither ghost RBCs nor Healon GV achieved significant IOP elevation after postinduction Day 7. On postinduction Day 7, the AH NO level increased significantly in the models induced with a-chymotrypsin, Polybead, and RBC ghosts. Healon GV did not cause an increased NO level in AH. The aqueous flow rates were significantly decreased with a-chymotrypsin and Polybead. Neither ghost RBCs or Healon GV resulted in a significantly decreased aqueous rate. The a-chymotrypsininduced model also had more intraocular inflammation than the other groups. AH is released from the ciliary process and returned to the venous system after passing through the trabecular meshwork [16]. In our previous study, we found that the AH NO level was higher in glaucomatous eyes than in nonglaucomatous eyes [10]. The main reason for the higher AH NO level in the glaucomatous eyes was proposed to be the inflammatory reaction in response to the elevated IOP [17]. Alternatively, Wang et al [18] believe that glaucoma is a chronic disease and any increases in AH NO levels arise to compensate for the impaired aqueous outflow that results from pathogenic conditions. In rabbit models, it has been demonstrated that NO synthase 2 is present in the ocular
tissue and that glutamate is elevated in the vitreous humor. Excessive NO may be caused by elevated IOP [19e21]. In our study, we found increased AH NO levels in a-chymotrypsin-, Polybead-, and RBC ghost-induced models. There was no significant change in the AH NO level in the Healon GV-induced model. In our previous study [10], we found that neovascular glaucoma (NVG) and acute angle-closure glaucoma (AACG) patients had the highest NO levels in the AH. Recent studies have demonstrated that NVG patients also have higher levels of vascular endothelial growth factor (VEGF) in their AH [22]. As VEGF is known to increase the release of NO from human tissues [23], it is possible that the elevation of VEGF leads to the higher levels of NO in the AH of NVG patients. In addition, these patients also suffer from breakdown of the bloodeaqueous barrier and chronic inflammation in the anterior chamber, both of which might also contribute to elevation of NO levels. The excessively high NO levels in the AHs of NVG patients may indicate that the pathology of NVG is usually complicated in diabetic retinopathy, which is related to the development of neovascularization in the iris and in the anterior chamber angle of the eyes [24]. In AACG patients, inflammatory reactions are usually present in the anterior chamber, which might partially explain the elevated NO levels in the AH. Chiou et al [11] have demonstrated significantly higher NO levels in the AHs of AACG patients than in cataract patients. An acute attack of glaucoma supposedly exacerbates the inflammation, thereby inducing a higher NO level in the AH. Thus, in NVG and AACG patients, we hypothesized that the increased NO levels in the AH may be a manifestation of the inflammatory status of the anterior chamber [10]. In our study, both a-chymotrypsin and Polybead caused significant IOP elevations and increased AH NO levels. The reason for the increased AH NO levels in these models may be that both materials caused various degrees of intraocular
NO in ocular hypertension experimental models inflammation. However, Polybead, a type of latex microsphere, induced the experimental model with less intraocular inflammation and tissue destruction than achymotrypsin. In the current study, the Polybead model exhibited the characteristics of high AH NO levels in the AH and fewer complications; therefore, it may be an acceptable experimental ocular hypertension model for the further investigation of NVG and AACG. In the past, many materials have shown the blockage of the trabecular meshwork and elevated IOP in the eyes of rabbit models, mimicking human glaucoma. However, disadvantages have existed in each of the various glaucoma and ocular hypertension models, with major disadvantages including a shorter period of IOP elevation and a lesser degree of IOP elevation (< 30 mmHg). In our study, a-chymotrypsin and Polybead resulted in significant IOP elevations from postinduction Day 1 to Day 120, whereas RBC ghosts and Healon GV did not achieve significant IOP elevations after postinduction Day 7. Bead microspheres were used to induce experimental glaucoma by obstructing aqueous outflow, and acute glaucoma was induced after the intracameral or intravitreous injection of polystyrene beads in the various animal models [25]. Weber et al [26] have proposed that IOP elevation most likely results from the bead obstruction of aqueous outflow, bead-induced swelling within the trabecular meshwork, or infiltration of the drainage channels by inflammatory cells. We found that the Polybead-induced experimental model had significant IOP elevations lasting for 120 days and IOP > 30 mmHg lasting for 60 days. a-Chymotrypsin is a commonly used material for inducing experimental glaucoma. However, induction with a-chymotrypsin destroys the fibrin of the zonular fibers in the anterior chamber, causing lens dislocation and serious intraocular inflammation [21]. Such effects complicate the intraocular condition and interfere with the investigation of the pathology of glaucoma or drug mechanisms in experimental models. Although the a-chymotrypsin-induced model caused a longer period of IOP elevation than the Polybead model, it also caused more severe intraocular inflammation. Healon GV, a common material for inducing IOP elevation in ocular surgery, was not a good experimental model, according to our results. After injecting Healon GV into the eyes, the IOP was elevated but slowly declined to normal within 3 days. On Day 7 after the Healon GV induction, the experimental model showed no significant IOP change. Compared to the other models, Healon GV resulted in a poor effect on experimental ocular hypertension. The RBC ghost model has been addressed previously, and it confirmed the possibility of secondary glaucoma due to trabecular obstruction [27,28]. According to the previous study, the model easily produced IOP elevation for 7e36 days and was not associated with intraocular inflammation. However, our study revealed that the IOP elevation induced with the RBC ghost did not last for 7 days. This fact most likely contributed to the variable results of the RBC ghostinduced models. Interestingly, the AH NO levels were significantly elevated in the RBC ghost-induced eyes despite the lack of significant changes in IOP or aqueous flow rate. We believe that RBC ghosts can still induce transient IOP elevation in experimental eyes, while the effect of transient IOP elevation caused the subsequent responses observed as elevated AH NO levels on postinduction Day 7. In the present
597 study, the RBC ghost initially caused IOP elevation in the experimental eyes, but the effect of IOP elevation did not persist after 7 days. Hence, a longer period of higher IOP is difficult for the RBC ghosts to achieve. We believe that the RBC ghost may not impede the trabecular meshwork entirely, and a longer period of elevated IOP may not be easily achieved. In the present study, Healon GV- and RBCinduced rabbit models initially caused elevated IOPs, but the period of elevated IOP was shorter, leading us to suppose that neither material caused a sufficient period of elevated IOP or subsequent pathological changes. In conclusion, we provided comparable results for the IOP and AH NO levels and the aqueous flow rate for each of the four different ocular hypertension models. According to the present results, RBC ghost- and Healon GV- induced models initially caused IOP elevation, but the change was not significant after 7 days postinduction. a-Chymotrypsin and Polybead were the best experimental models. In addition, the Polybead-induced model exhibited fewer complications than the a-chymotrypsin induced model. Based on previous studies, Polybead exhibited similar features (increased AH NO levels) in both NVG and AACG patients, and it may constitute a suitable model for investigating these types of glaucoma.
Acknowledgments This research work was partially supported by National Science Council grants (89-2314-B-016-011 and 89-2314-B016-070) and was utilized to fulfill the dissertation requirement for C.J.C.
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