Eye (2007) 21, 812–818 & 2007 Nature Publishing Group All rights reserved 0950-222X/07 $30.00 www.nature.com/eye

1 2 1 1 AOAOYSTUDY LABORATORY Short-term effect JY Oh , WR Wee , JH Lee and MK Kim of intracameral triamcinolone acetonide on corneal endothelium using the rabbit model

Abstract triamcinolone acetonide injection when used without filtering and resuspension. Purpose To investigate the effect of Eye (2007) 21, 812–818; doi:10.1038/sj.eye.6702357; intracameral injection of triamcinolone published online 2 June 2006 acetonide on the corneal endothelium in 1Department of Ophthalmology, Seoul rabbit eyes. Keywords: ; effect; endothelium; toxicity; National University College Methods Triamcinolone acetonide (40 mg/ml, triamcinolone of Medicine, Seoul Artificial 0.2 cm3) after filtering and resuspension in Eye Center, Seoul National balanced salt solution (BSS) was injected University Hospital Clinical intracamerally for 3 min into 10 rabbit eyes Research Institute, Seoul, 3 Introduction Korea and irrigated with 5 cm of BSS. Triamcinolone without resuspension and BSS were injected, Triamcinolone acetonide is being used 2Seoul National University, respectively, into five rabbit eyes. Endothelial increasingly to enhance visualization of the Bundang Hospital, toxicity was evaluated and compared by vitreous body in the anterior chamber of the Seongnam, Korea measurements of endothelial cell counts and eye during surgery.1–3 Enhanced central corneal thickness. The endothelial viewing of these tissues can promote a Correspondence: MK Kim, Department of viability was determined using vital staining surgeon’s ability to evaluate clinical structural Ophthalmology, Seoul with alizarin red and trypan blue at 2 h after relationships and may aid in a more effective National University College injection. The scanning electron microscopy anterior with fewer complication. of Medicine, 28 Yongon- (SEM) was performed in one cornea from each In addition, there are reports suggesting that dong, Chongno-gu, Seoul group. intracameral injection of dexamethasone 110-744, Korea Results Endothelial cell counts and central Tel.: þ 82 2 2072 2665; during reduces the 2 Fax: þ 82 2 741 3187. corneal thickness following intracameral postoperative inflammation and subsequently E-mail: kmk9@ injection of triamcinolone acetonide did not the development of postoperative cystoid medimail.co.kr significantly change when compared to macular oedema. However, corneal endothelial controls. The mean percentage of viable toxicity with the intracameral injection of Received: 17 November endothelial cells was 99.50, 99.52, and 99.49% triamcinolone acetonide has not been fully 2005 in the resuspended triamcinolone group, Accepted in revised form: evaluated to date. The preparation of 16 March 2006 triamcinolone without resuspension group, triamcinolone acetonide includes preservatives Published online: 2 June and BSS group, respectively (P ¼ 0.46, such as benzyl alcohol. Therefore, we would 2006 Kruskall–Wallis test). But SEM showed predict that some endothelial damage can result reduced microvilli of endothelial surface in from triamcinolone acetonide or preservatives. None of the authors has a an eye of the triamcinolone without financial or proprietary We investigated the short-term effect of interest in any of the resuspension group. intracameral triamcinolone acetonide on the material or method Conclusions The intracameral injection of corneal endothelium using a rabbit model. mentioned triamcinolone acetonide did not induce a significant visable change of endothelium in Presented in part at the Materials and methods rabbit eyes. However, ultrastructural villi ASCRS Symposium, Washington DC, USA, changes observed suggest a possibility of Animal experiments were performed in April 2005 microstructural damages in endothelium with accordance with the Association for Research Short-term effect of intracameral triamcinolone JY Oh et al 813

in Vision and Ophthalmology Statement for Use of and postoperative measurements in corneal thickness Animals in Ophthalmic Vision and Research, and and endothelial cell counts were obtained by subtracting the protocol was approved by the Institutional Review baseline measurements from measurements at 2 h after Board of Seoul National University Hospital, Seoul, injection in each group and the mean change was Korea. compared between the three groups using Twenty New Zealand white rabbits were randomly Kruskal–Wallis test. divided into three groups and designated as Two hours after the injection, the rabbits were killed resuspended triamcinolone group, triamcinolone with an intracardiac injection of potassium chloride. without resuspension group, and BSS group as a Their and a 2.0-mm rim of were removed control. Ten rabbits in the resuspended triamcinolone with a blade and scissors and the diaphragm was group received an intracameral injection of triamcinolone peeled from the cornea. In 17 of a total of 20 corneas (nine acetonide (Tamcetons, Hanall Pharmaceutical, from the resuspended triamcinolone group, four from Seoul, Korea; 40 mg/ml, 0.2 cm3) resuspended in the triamcinolone without resuspension group, and four balanced salt solution (BSS, Santen, Osaka, Japan) to from the BSS group), the endothelial viability was avoid injecting a vehicle present in commercially determined using a live/dead cell assay according to available triamcinolone acetonide; the triamcinolone Taylor and Hunt.5 Corneal buttons were placed acetonide solution was made by the method that a endothelial side up and 0.25% trypan blue was applied triamcinolone aqueous suspension, was left standing dropwise to the endothelium for 90 s. Each cornea was for 30 min and the vehicle was discarded, as rinsed with normal saline to remove excess stain. previously described4 and the remaining triamcinolone Alizarin red S (0.20%; pH 4.2) was then applied to the acetonide suspension was washed with 1 ml of BSS endothelium for 90 s and the buttons were rinsed with three times using a 0.2 mm filter (Millex-FGs, normal saline. After the staining procedure, corneas were Carringtwohill, Co., Cork, Ireland) to thoroughly immersed in the glutaraldehyde fixative solution (2.48%; remove a vehicle possibly remaining in the suspension. osmolality 301 mosm/kg pH 7.2) for 10 min. Each cornea Five rabbits were included in the triamcinolone was prepared by trephining in a centre with a 6 mm of without resuspension group, they received an injection trephine (Storz Ophthalmics, St Louis, MI, USA) to of commercial preparation of triamcinolone reduce biased information about endothelial cell acetonide without filtering or resuspension damages derived from the cutting with scissors during (40 mg/ml, 0.2 cm3). The third group, a control group, the harvest of the cornea. The disc of tissue was had five rabbits, they received an intracameral injection mounted, endothelium uppermost, in saline, under a of BSS. coverslip, on a glass microscope slide and was then Surgery was performed randomly on one eye of a evaluated for endothelial damage with an Olympus BX rabbit. The rabbits were anaesthetized with an 50 light microscope (Olympus Optical Co. Ltd., intramuscular injection of ketamine (30 mg/kg) and Tokyo, Japan). The number of trypan blue-stained xylazine (5 mg/kg). A wire lid speculum was placed to dead cells and unstained live cells was counted in a separate the . An operating microscope was more than 10 medium power field ( Â 200) using a grid in positioned over the eye undergoing surgery. The anterior the microscope to calculate the percentages of dead cells. chamber was entered in the superotemporal quadrant The mean percentage of dead endothelial cells through a long corneal tunnel using a 30-G needle. After was compared between the three groups using entry, 0.2 ml of aqueous humor was withdrawn from the Kruskal–Wallis test. eye and then 0.2 ml of triamcinolone acetonide or BSS The remaining three corneas, one cornea from each was injected intracamerally through a separate 30-G group, were dissected in piece, 3 Â 3 mm from the centre needle. Three minutes later, the anterior chamber was and prepared for scanning electron microscopy (SEM). irrigated with 5 ml of BSS by the bimanual method to Samples were washed in sodium buffer solution wash away the triamcinolone. (PBS) and cut into smaller pieces. For SEM, samples Corneal thickness measurements by ultrasonic were prefixed with 2.5% glutaraldehyde PBS (pH 7.2) pachymetry (Pachymeter echograph, Quantel medical, at 41C overnight. Following several washes in PBS, Clermont-Ferrand, France) and endothelial cell count by samples were kept in 1% osmiumtetraoxide–PBS for specular microscopy (Konan specular microscope SP- the final fixation for 1 h. Samples were then washed 8800, Konan medical Inc., Nishinomiya, Japan) were and dehydrated through serial dilutions of ethanol. made before surgery and every 30 min after surgery. Samples were mounted onto stubs, sputter-coated with Within each group, temporal changes in corneal gold by Polaron SC-500 (VG Microtech, Sussex, UK) and thickness and endothelial cell counts were tested using a finally examined with SEM (JSM 300 JEOL, Tokyo, paired Student’s t-test. The difference between baseline Japan).

Eye Short-term effect of intracameral triamcinolone JY Oh et al 814

Results Endothelial cell counts

Corneal thickness The mean endothelial cell count (7SD) was 29027129 at baseline and 29307141, 29137151, and 29227124 at 0.5, 1, In the resuspended triamcinolone group, the mean and 2 h after injection, respectively, in the suspended central corneal thickness (7SD) was 359732 mmat triamcinolone group. In the triamcinolone without baseline and 335724, 330719, and 327724 mm at 0.5, 1, resuspension group, the mean endothelial cell count (7SD) and 2 h after injection, respectively. In the triamcinolone was 28837109 at baseline and 28937116, 28867122, and without resuspension group, the mean corneal thickness 28737107 at 0.5, 1, and 2 h after injection, respectively. In the (7SD) was 380728 mm at baseline and 360716, 384732, BSS group, the mean endothelial cell count was 29457106 and 390735 mm at 0.5, 1, and 2 h after injection, at baseline and 29217105, 29497110, and 29007109 at 0.5, respectively. In the BSS group, the mean corneal 1, and 2 h after injection, respectively. The mean endothelial thickness (7SD) was 368724 mm at baseline and cell count was not significantly different from baseline when 375725, 370720, and 376719 mm at 0.5, 1, and 2 h compared to 2 h after injection in each group studied after injection, respectively. In each group, central (P ¼ 0.413 in the suspended triamcinolone group, P ¼ 0.395 corneal thickness was not significantly different from in the triamcinolone without resuspension group, and baseline at 2 h after injection (P ¼ 0.241 in suspended P ¼ 0.627 in the BSS group) (Figure 2). The mean change triamcinolone group, P ¼ 0.531 in triamcinolone without between baseline and measurements at postoperative 2 h resuspension group, and P ¼ 0.541 in BSS group) was þ 21.0727.7 in the suspended triamcinolone group, (Figure 1). After comparison of the three groups, the À11.3722.8 in the triamcinolone without resuspension change in central corneal thickness after injection was not group, and À40.0718.1 in the BSS group, respectively significant. The mean change between baseline values ( þ means an increase in endothelial cell count; À means a and at postoperative 2 h was À25.5724.1 mmin decrease in endothelial cell count). There were no significant suspended triamcinolone group, þ 13.5720.0 mmin difference observed between the three groups studied triamcinolone without resuspension group, and (P ¼ 0.502; this P-value encompasses all three groups). þ 11.0717.3 mm in BSS group ( þ means an increase in corneal thickness; À means a decrease in corneal Live/dead cell assay thickness). No one group experienced a change greater than the others (P ¼ 0.418; this P-value encompasses all The vital stain revealed that the mean percentage of dead three groups). endothelial cells (7SD) was 0.5070.23%, 0.4870.20%,

Resuspended triamcinolone group

Triamcinolone without suspension group 500 BSS group

450 m) µ 400

350

300 Mean corneal thickness ( 250

200 Baseline 0.5 1 1.5 2 Time after injection (Hour)

Figure 1 Temporal change, in the mean central corneal thickness, from baseline to the time after anterior chamber injection, in the three groups. The mean central corneal thickness was not significantly different, from baseline and at 2 h after injection, for each group.

Eye Short-term effect of intracameral triamcinolone JY Oh et al 815

Resuspended triamcinolone group

Triamcinolone without suspension group 3200 BSS group

3100 ) 3

3000

2900

2800 Endothelial cell counts (/mm 2700

2600 Baseline 0.5 1 1.5 2 Time after injection (Hour)

Figure 2 Temporal change in the mean endothelial cell count, from baseline to the time after the anterior chamber injection, in the three groups. The mean endothelial cell count was not significantly changed, from baseline and at 2 h after injection, for each group.

and 0.5170.15% in the suspended triamcinolone group, anterior chamber and that this method helps surgeons to in the triamcinolone without resuspension group, and in remove the vitreous body completely and safely from the the BSS group, respectively. No significant differences anterior chamber. Nonetheless, there are several concerns were noted (P ¼ 0.460; this P-value encompasses all three related to the use of triamcinolone acetonide groups). Therefore, intracameral injection of intraocularly including intraocular pressure elevation,1 triamcinolone acetonide did not cause significant postoperative ,2,6 and corneal endothelial death compared with that in the BSS-injected endothelial toxicity. Regarding corneal toxicity, Chan eyes (Figure 3a–c). et al7 reported that a single 4 mg bolus injection of intravitreal triamcinolone acetonide had no harmful effects on the human corneal endothelium during 6 Electron microscopy months of follow-up, when evaluated with specular SEM revealed that endothelial cells kept hexagonality microscopy. But there has been no prior report evaluating and presented well-defined cell borders in all three endothelial effect of intracameral triamcinolone to our groups regardless of whether they were the knowledge. Therefore, we evaluated the effect on corneal triamcinolone-injected corneas or the controls. However, endothelium of rabbit eyes when exposed to triamcino- microvilli of the endothelial surface were reduced in lone acetonide for 3 min after intracameral injection. number in the triamcinolone without resuspension In our study, 3-min exposure to 8 mg/0.2 ml of group, whereas microvilli were well maintained in the triamcinolone acetonide did not produce significant resuspended triamcinolone group and the BSS group changes in corneal thickness and endothelial cell counts (Figure 4a–c). compared to those before injection and those in control groups. A quantatitive live/dead cell assay showed that endothelial cell viability was not altered after a 3-min Discussion exposure to triamcinolone acetonide. These findings Triamcinolone acetonide is a water-insoluble steroid suggest that short-term injection of triamcinolone with or which inhibits inflammation and has been used as a without resuspension might not affect the viability of the potent anti-inflammatory agent for various intraocular corneal endothelial cells. SEM revealed that microvilli diseases. Recently, Yamakiri et al1 reported that the slightly decreased in number in the triamcinolone intracameral injection of triamcinolone acetonide during without resuspension group, suggesting a possible cataract surgery visualizes the transparent vitreous in the microstructural damage. However, endothelial cells were

Eye Short-term effect of intracameral triamcinolone JY Oh et al 816

Figure 3 Light microscopic view of rabbit corneas stained with trypan blue and alizarin red after 3 min of exposure to a commercial preparation of triamcinolone acetonide without Figure 4 Scanning electron micrographs of the endothelium of resuspension (40 mg/ml) (a), triamcinolone acetonide after rabbit corneas after 3 min of exposure to commercial preparation filtered and resuspended (40 mg/ml) (b) and BSS (c). The of triamcinolone acetonide without resuspension (40 mg/ml) endothelial cells in all three groups had a regular hexagonal (a), triamcinolone acetonide after filtered and resuspended shape with straight, well-demarcated borders (original magni- (b), and BSS (c). The endothelium in triamcinolone without fication  400). resuspension group (a) showed a loss of microvilli whereas the endothelium in the resuspended triamcinolone group (b) and BSS group (c) revealed normal microvilli on the surface (original magnification  1500).

Eye Short-term effect of intracameral triamcinolone JY Oh et al 817

found to keep hexagonality and well-defined cell borders difference of viability till 12 h. Cell death like apoptosis is in all the three groups. known to appear high 2 h after exposure of certain The preservatives in the preparation of triamcinolone damage.13 Moreover, rabbit endothelial cells can be (40 mg/1 ml) include: benzyl alcohol 0.045% and other regenerated after damage. Therefore, we chose 2 h after components such as sodium carboxymethyl cellulose injection as an examination time to decrease bias related 2 mg, polysorbate 2 mg, sodium chloride 9 mg and to endothelial proliferation although possibility that cell sodium hydroxide. Reduction of microvilli might be damage took longer time than 2 h might not be excluded. owing to corneal cytotoxicity of benzyl alcohol or other In conclusion, our study showed that a 3-min exposure additives like carboxymethyl cellulose because microvilli to triamcinolone acetonide (40 mg/ml) had no toxic effect were maintained in eyes injected with triamcinolone on the endothelial function and viability in rabbit cornea. acetonide after being washed and resuspended in BSS. However, changes observed in ultrastructural villi Also, there is a report demonstrating that toxic effects of suggest a possible microstructural damage to triamcinolone on the could be caused by endothelium after exposure to commercial out of the preservatives alone or by the osmolarity of the vehicles, bottle triamcinolone acetonide without resuspension. of various commercially available corticosteroids, when Therefore, cautious use of the intracameral injection of injected intravitreally in rabbit eyes.8 triamcinolone, after washing and resuspension in BSS, is The cytotoxicity of triamcinolone has been reported in recommended. in vitro cultures.9 In a report of a 5-day exposure of triamcinolone acetonide including benzyl alcohol 0.025%, the numbers of ARPE19 cells were reduced References significantly. The threshold of resistance against the triamcinolone preparation may be different between 1 Yamakiri K, Uchino E, Kimura K, Azad RV. Intracameral retinal pigment epithelium cells and corneal endothelial triamcinolone helps to visualize and remove the vitreous cells. The exposure time is an essential factor when cell body in anterior chamber in cataract surgery. Am J damage is considered. Therefore, in vitro outcome with Ophthalmol 2004; 138: 650–652. long-term exposure in the above study, might be 2 Tan DT, Chee SP, Lim L, Theng J, Van Ede M. Randomized clinical trial of Surodex steroid drug delivery system for expected to have different results when compared to our cataract surgery anterior versus posterior placement of two in vivo data that show no changes in viability except a Surodex in the eye. Ophthalmology 2001; 108: 2172–2181. slight microstructural damage after short-term exposure. 3 Burk SE, Da Mata AP, Snyder ME, Schneider S, Osher RH, We believe our model has more relevance to clinical Cionni RJ. Visualizing vitreous using Kenalog suspension. cataract surgery because anterior chamber was irrigated J Cataract Refract Surg 2003; 29: 645–651. 4 Peyman GA, Cheema R, Conway MD, Fang T. with BSS soon after injection of triamcinolone in cataract Triamcinolone acetonide as an aid to visualization of the surgery. vitreous and the posterior hyaloid during pars plana The present study has several limitations. First, unlike vitrectomy. Retina 2000; 20: 554–555. the human counterpart, rabbit corneal endothelium 5 Taylor MJ, Hunt CJ. Dual staining of corneal endothelium maintains mitotic activity and regenerates after thermal with trypan blue and alizarin red S: importance of 10,11 pH for the dye–lake reaction. Br J Ophthalmol 1981; 65: and chemical insult. There are better models 815–819. 10 12 including the cat and monkey eye where mitotic 6 Sakamoto T, Enaida H, Kubota T, Nakahara M, Yamakiri K, activity is rare. We used the rabbit model for pragmatic Yamashita T et al. Incidence of acute endophthalmitis after reasons including the lower cost associated with triamcinolone-assisted pars plana vitrectomy. Am J maintaining rabbits, the ease of obtaining pachymetry Ophthalmol 2004; 138: 137–138. 7 Chan CK, Fan DS, Chan WM, Lai WW, Lee VY, Lam DS. measurements and corneal photographs, the Ocular-hypertensive response and corneal endothelial morphological similarities between rabbit and human changes after intravitreal triamcinolone injections in corneas, and the general familiarity of the medical Chinese subjects: a 6-month follow-up study. Eye 2005; 19: community with the rabbit model. To reduce the bias 625–630. from the proliferative potential in the rabbit, we 8 Hida T, Chandler D, Arena JE, Machemer R. Experimental and clinical observations of the intraocular toxicity of examined the cells 2 h after exposure, for which corneal commercial corticosteroid preparations. Am J Ophthalmol 13–16 endothelial cells could not regenerate. Second, we 1986; 101: 190–195. did not evaluate the endothelial change when the cornea 9 Yeung CK, Chan KP, Chiang SW, Pang CP, Lam DS. The was exposed to triamcinolone acetonide for more than toxic and stress responses of cultured human retinal 3 min. A long-term exposure study is needed to evaluate pigment epithelium (ARPE19) and human glial cells (SVG) in the presence of triamcinolone. Invest Ophthalmol Vis Sci cytotoxicity during surgery. Third, the cell viability was 2003; 44: 5293–5300. examined 2 h after injection. Actually, we preliminarily 10 Van Horn DL, Sendele DD, Seideman S, Buco PJ. checked the viability up to 12 h, and we did not find any Regenerative capacity of the corneal endothelium

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in rabbit and cat. Invest Ophthalmol Vis Sci 1977l; 16: 14 Judge AJ, Najafi K, Lee DA, Miller KM. Corneal endothelial 597–613. toxicity of topical anesthesia. Ophthalmology 1997; 104: 11 Olsen EG, Davanger M. The healing of rabbit corneal 1373–1379. endothelium. Acta Ophthalmol 1984; 62: 796–807. 15 Li Y, Feng G, Yi Y, Zhong X, Zheng H. Observation of the 12 Van Horn DL, Hyndiuk RA. Endothelial wound repair in endothelial healing in rabbit corneal alkali wounds by primate cornea. Exp Eye Res 1975; 21: 113–124. alizarin red S-trypan blue staining method. Yan Ke Xue Bao 13 Holly GP, Alam A, Kiri A, Edelhauser HF. Effect of 1999; 15: 218–220. indocyanine green intraocular stain on human and rabbit 16 Medin W, Davanger M. Toxic effects of ouabain on the corneal endothelial structure and viability. An in vitro study. rabbit corneal endothelium. Acta Ophthalmol (Copenh) 1993; J Cataract Refract Surg 2002; 28: 1027–1033. 71: 365–370.

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