- Email: [email protected]
Effect of trypan blue on the elasticity of the human anterior lens capsule
ARTICLE
Effect of trypan blue on the elasticity of the human anterior lens capsule H. Burkhard Dick, MD, Shakhsanam E. Aliyeva, MD, Fritz Hengerer, MD
PURPOSE: To evaluate the effect of trypan blue (VisionBlue) staining on the elastic properties of the human anterior lens capsule. SETTING: Center for Vision Science, Ruhr University Eye Hospital, Bochum, Germany. METHODS: Nineteen anterior capsules were obtained from 19 human eyes at the time of cataract operation. Two same-size strips were prepared from each capsule; 1 was exposed to trypan blue staining for 10 seconds, and the other was a control. The elasticity measurements were performed on both specimens using a modified rheometer. RESULTS: The mean age of the patients was 75.3 years G 13.3 (SD) (range 35 to 89 years). Sixty-three measurements of 19 suitable specimen pairs were performed. In 13 pairs, the elasticity measurements were eligible for statistical analysis. The stress value at the tearing point was statistically significantly lower in the treated group (mean 169 G 55 Pa) than in the control group without staining (mean 252 G 67 Pa) (P Z .023). The difference in stress between the stained and control samples at 3 seconds and 5 seconds was not statistically significant (P Z .093 versus P Z .316). Dynamic frequency sweep measurements showed a statistically significant elevation of the viscosity modulus in the samples stained with trypan blue. CONCLUSIONS: Trypan blue staining affected the biomechanical properties of the human lens capsule and led to a significant reduction in elasticity and an increase in stiffness. These results confirm clinical reports of changes in the capsule by dye and should be taken into consideration in dye-enhanced cataract surgery. J Cataract Refract Surg 2008; 34:1367–1373 Q 2008 ASCRS and ESCRS
First introduced by Gimbel and Neuhann,1 the continuous curvilinear capsulorhexis (CCC) has been the gold standard in cataract surgery for more than 20 years. Compared with other surgical approaches to open the anterior capsule, this technique has a lower complication rate. The main criterion to perform the CCC is the presence of retroillumination from the fundus, which enables viewing of the round edges of the capsulorhexis.
Accepted for publication March 20, 2008. From the Department of Ophthalmology (Dick), Ruhr-University, Bochum, the Department of Ophthalmology (Aliyeva), Johannes Gutenberg-University, Mainz, Germany Center for Vision Science (Hengerer), Bochum, Germany. Neither author has a financial or proprietary interest in any material or method mentioned. Corresponding author: H. Burkhard Dick, MD, Center for Vision Science, Ruhr University Eye Hospital, In der Schornau 23 - 25, D-44892 Bochum, Germany. E-mail: [email protected]. Q 2008 ASCRS and ESCRS Published by Elsevier Inc.
In cases of mature cataract or in other situations in which the red reflex is poor or visualization of the capsule compromised, capsule staining is helpful to visualize the round edges of the capsulorhexis. For this purpose, fluorescein, indocyanine green, and trypan blue are commonly used. The more intense and persistent capsule staining that trypan blue provides is particularly advantageous, making it preferable to other agents for dye-aided cataract surgery. However, it is not known whether the application of trypan blue affects the mechanical characteristics of the lens capsule. The impairment of elastic properties of the lens capsule after application of trypan blue could increase the risk for capsular tears and may result in a higher incidence of intraoperative complications. To ensure the safety of dye-enhanced surgical procedures, quantitative evaluation of capsule elasticity is needed. We performed a prospective study to assess the change in the elastic property of the human lens capsule by application of VisionBlue, a trypan blue dye. 0886-3350/08/$dsee front matter doi:10.1016/j.jcrs.2008.03.041
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PATIENTS AND METHODS
Rheometry
The lens capsules of 19 patients having phacoemulsification were collected. The same surgeon (H.B.D.) performed the capsulorhexis and collected a sampling of specimens using a standard technique. Because various ocular disorders may influence capsule elasticity, patients with the following pathologies were excluded from the study: pseudoexfoliation syndrome, previous intraocular surgery, topical medical treatment within the past 8 weeks, chronic recurrent inflammation, history of ocular trauma, glaucoma, pregnancy, metabolic diseases such as diabetes mellitus, and eyes with mature cataract. The selection of eyes was randomized. During creation of the capsulorhexis, the anterior lens capsule was removed en bloc and placed in a container with balanced salt solution (BSS). All measurements were performed within a maximum of 6 hours after the capsule was removed from the eye. The human lens capsule thickness is at its maximum at the anterior midperiphery, which appears to be located central to the zonular insertion. It increases with age, especially at the anterior pole, while the midperipheral zone stabilizes or slightly decreases after the seventh decade. The thickness of the human lens capsule continuously increases with age, an average of 11.0 to 15.0 mm at the anterior lens pole. Maximum thickness is at the anterior midperiphery, increasing with age from 13.5 to 16.0 mm. The anterior zonular insertion is actually related to a local pre-equatorial thinning, which remains unchanged with age. The thickness of the anterior lens capsules was not measured because this study was an intraindividual comparative analysis. The individuals were not separated by age, sex, or any other potentially discriminating factors. No specimen was intentionally discarded.
Rheometry allows sophisticated and automated measurement of changes in the flow of fluids and deformation of solid materials under various kinds of stress and strain. In this study, the Rheometrics RDS-II Mechanical/Dynamic Spectrometer RMS-800 (Rheometrics Scientific, Inc.) was used (Figure 1). Originally designed to determine the complex flow properties of liquids, this instrument is also equipped to measure the tensile and compressive strength of solid materials. Elastic properties of the lens capsule were assessed as a reaction to application of graduated strength. The Digital Storage Scope HM 208 oscilloscope (Hameg Instruments GmbH) was also used to determine the strength transmitting to the sample. For graphic interpretation, RSI Orchestrator software (version 6.5.8, Rheometric Scientific, Inc.) was used.
Preparation of Anterior Capsule Specimen Because the study design implies an intraindividual comparative analysis, paired samples were prepared from each anterior lens capsule. Microscissors were used to divide the capsule into 2 parts shaped like half moons. From each part, 2 same-size strips were cut out under the microscope; 1 strip served as a paired control, and the other was used for the staining. The strain force applied in the rheometer varies depending on the shape and size of the sample; thus, meticulous care was taken to achieve the same size and geometry across all specimens for proper assessment of elastic properties. Samples were immersed in and stained with VisionBlue solution for 10 seconds and then placed in a Petri dish containing BSS. As dehydration might change the biomechanical properties of the tissue, all samples were constantly held in a moisture medium during the entire experiment.
Holders To strain the sample in the rheometer, holders were made from a light, corrosion-resistant material. The purpose of the holders was to provide safe fixation, and their weight had to be as low as possible to minimize its effect on measurement during the swinging process. Therefore, in collaboration with the Max Planck Institute for Polymer Research, Mainz, Germany, a gripping device designed to hold the sample between 2 aluminum clamps by means of a screw was engineered. The self-adhesive cellulose strips were glued to the sharp metallic edges of the clamp to avoid altering the samples and to prevent slippage during measurements. The gripping device was screwed into a bearing base built into the rheometer instead of to a measuring plate for viscous fluids. Fixation of the capsule strips in the aluminum clamps was performed under the light microscope at 64-fold magnification. One end of the sample was placed between the branches of the aluminum clamp. Then, the branches were tightly joined with the help of a forceps and fixed by screwing with moderate pressure. Because the stretching length of the strips had to be identical in all samples (trypan blue and control), the sample was carefully measured on a micrometer scale placed under a Petri dish. Finally, the other end of the sample was fixed in the opposite clamp of the measuring device. To
Tissue Staining Agent VisionBlue contains 0.6 mg dilute ophthalmic solution of trypan blue, 1.9 mg sodium monohydrogen orthophosphate, 0.3 mg sodium dihydrogen orthophosphate, 8.2 mg sodium chloride sodium hydroxide for adjusting the pH to a value between 7.3 and 7.6, and water for injection. This solution has been available in Germany since February 2003; the U.S. Food and Drug Administration approved VisionBlue in December 2004 for the purpose of improving anterior capsule visualization during cataract surgery. It is supplied as a premixed sterile solution in a 0.5 mL glass ampule.
Figure 1. Image depicting the RMS-800 sample staging area and control panels.
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Measurements Dynamic Strain Sweep Before each measurement was taken, equal prestress force (5% for all specimens), controlled by an oscillograph, was applied to each capsule strip. When operating in dynamic mode (dynamic strain sweep), the instrument applies a sinusoidal oscillating strain with a constant low frequency, producing a sinusoidally oscillating stress in the sample. Two measurements were used for each sample analysis, and the range of the lowest measurements for minor movements with a maximum amplitude of 0.5 mm was assessed.
Dynamic Frequency Sweeps The goal of dynamic frequency sweeps is to obtain the frequency dependent dynamic modulus (G) in the linear regime of the material. In this study, the dynamic frequency sweeps were done with a frequency range of 0.1 to 100 radians per second, which corresponds to 0.016 to 15.915 Hz. The step for decade variation was set at 5 points per decade to define the difference in logarithms of the starting and final frequencies in each equally divided segment of exponents. Then, a sinusoidal force with constant amplitude was applied to the sample. Shear Rate Assessment The shear rate was evaluated by applying the tested fluid between 2 metallic plates. One plate was set in rotation with a fixed speed; the second, which was connected to a special device to assess driving torque, was simultaneously passively rotated depending on fluid viscosity. The speed at which the 2 plates moved relative to each other is called shear rate and is expressed in radian per second. In this study, this experiment was performed in a modified version with the purpose of determining the viscoelastic properties of all samples, the ultimate stress value, and a time interval at the rupture point. In strain tests and bending tests, the elasticity modulus is expressed as E0 and the viscosity modulus is expressed as E00 . The complex elasticity modulus E* is a ratio: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 E Z ðE0 Þ þðE00 Þ Using a conversion factor composed from geometry, the width (b) and thickness (d) of the sample, and a constant of device (k Z 895), the elasticity and viscosity modulus were calculated as follows: E0 Z G0 [(b d)/895)]. Because of the potential risk for capsule damage, the samples’ thicknesses were not measured and a value of 10 mm was assigned as a standard for all samples. The elasticity module defines the energy that exerts influence on a material by deformation; it leads to return of the shape after discontinuation of the applied force. The following formula was used to calculate the modulus of elasticity (E0 ):
s E0 Zcosd 3 The modulus of viscosity (E00 ) was calculated as s E00 Zsind 3 where d is the phase angle (phase displacement between stress and deformation vectors), s is stress, and 3 is strain. The tangent of the loss angle was calculated as E00 tandZ 0 E
Statistical Analysis Descriptive statistics were calculated and expressed as the mean value and the standard deviation. Nonparametric analysis was used to assess significant differences between variables. To enable use of SPSS software (version 11, SPSS, Inc.) for statistical analysis, all data were converted from RSI Orchestrator using Microsoft Excel 2003 for MS Windows XP. To present the data and compose histograms, Microsoft Excel and Microsoft Word Version Office (version X) (all Microsoft products, Microsoft Corp.) for Macintosh (Apple, Inc.) were used. Graphic presentation of results was expressed in nonparametric box plots, a univariate dot pattern, and bar graphs. The paired t test was used for linked samples. A P value of 0.05 was considered statistically significant.
RESULTS The mean age of enrolled patients was 75.3 years G 13.3 (SD) (range 35 to 89 years) (Figure 2). Ten specimens were obtained from women (age: mean 78.3 G 9.3 years, median 81.5 years) and 9 from men (age: mean 68.2 G 14.9 years, median 74 years). Nineteen suitable specimens were collected and mounted for elasticity analysis, and 63 measurements were recorded. As the study design assumed the agerelated change in the elasticity of the human lens capsule, all measurements were numbered from 1 to 19, depending on patient age. 10 9 8
Number of patients
prevent exsiccation, the specimen was continuously moistened with drops of BSS. All manipulations of the capsule were performed under a microscope (Wild M38) using smooth microsurgical instruments in a Petri dish filled with BSS. Before each measurement, the solution was changed and the glass micrometer scale used to evaluate specimen length and width was placed. For the measurement, the whole construction was transferred onto a rubber brick, clamped horizontally between the jaws of the testing device, and then expanded to the full length of sample.
7 6 5 4 3 2 1 0
30-40
40-50
50-60
60-70
70-80
Patient Age (years) Figure 2. Demographic data of patients.
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Figure 4. Intraindividual changes in stress values at the tearing point in each sample of the 13 pairs.
Results from 13 measurements were chosen for statistical analysis. The 6 measurements that were excluded had 1 in 3 assessments that exceeded a doubled standard deviation; each was assumed to be a rupture. These patients were only enrolled for the estimation of dynamic frequency sweep value. In 11 of the accepted 13 pairs, the lower stress value at the tearing point was measured in the group treated with VisionBlue. In 1 pair, the similar stress value was applied in both samples. In the remaining pair, the
stained capsule tore under higher stress than the control sample. The difference between the treatment group and paired control group in mean stress value at the tearing point was statistically significant (P Z .023). The box plot in Figure 3 shows the stress range in both groups (mean 169 G 55 Pa in stained samples versus 252 G 67 Pa in unstained samples). The intraindividual changes in stress value applied at the point of rupture in each of the 13 pairs are shown in Figure 4. The difference in the counterforce value between the stained samples and control samples at 3 seconds (P Z .093) and 5 seconds (P Z .316) of stress is shown in Figures 5 and 6, respectively. It is evident that at the specific moment of measurement, the measuring converter applied smaller counterforce in the stained samples. However, the difference was not statistically significant. The dynamic frequency sweep measurements showed considerable biomechanical changes with or
Figure 5. Differences in counterforce between the stained and control samples after 3 seconds of stretching (P Z .93).
Figure 6. Differences in counterforce between the stained and control samples after 5 seconds of stretching (P Z .316).
Figure 3. The box plot showing a statistically significant higher stress value at the point of rupture in the staining group.
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Figure 7. Intraindividual changes in the modulus of elasticity (E0 ) through 16 measuring frequencies in 19 sample pairs.
without staining between the frequencies of 14 to 15 and 15 to 16. In both trypan blue-stained and control samples, a statistically significant elevation of viscosity modulus was noticed at the frequency intervals between 12 and 13, 13 and 14, and 14 and 15. Figures 7 and 8 show the changes in E0 modulus and E00 modulus, respectively, at all 16 frequencies evaluated in the 19 sample pairs. DISCUSSION Successful completion of CCC is crucial for any phacoemulsification procedure and is associated with a lower complication rate and better surgical outcomes. In eyes with white and mature cataract, poor visibility of the capsule due to a decreased or absent red fundus reflex makes surgery more challenging. Therefore, various staining agents have been brought into routine use in recent years.2,3 Trypan blue was initially applied to examine the endothelial cell layer of donor corneas before corneal transplantation. Melles et al.4 reported the first experience with intraoperative application of trypan blue to stain the anterior capsule and facilitate capsulorhexis for surgery for mature cataract. Several advantages make trypan blue particularly desirable. It creates much darker staining and provides better visualization than other dyes. Also, it lasts longer and usually persists throughout the entire procedure, and it is less expensive because only a small amount need be applied.5 Moreover, recent studies show trypan blue staining can help prevent posterior capsule opacification through a significant decrease in density and viability of lens epithelial cells.6 Numerous studies have confirmed the safety and efficacy of trypan blue staining.7–10 Bhartiya et al.11
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Figure 8. Changes in modulus of viscosity (E00 ).
report successful application of the dye in eyes with corneal opacities involving the visual axis. Except for permanent blue discoloration of a hydrogel intraocular lens12 and the inadvertent staining of the posterior lens capsule and vitreous,13 the reported adverse reactions to VisionBlue use are self-limited and of short duration. Reports of histological analysis14 and electron microscopy findings15 of lens capsules stained with trypan blue have also been published. However, the clinically important aspect, such as the effect of trypan blue on the biomechanical properties of human lens capsule, has not been studied. The present study is the first comparative investigation of the change in the elastic properties of the human anterior capsule after staining with VisionBlue vital dye. Our results show statistically significant lower values at the moment of rupture (P Z .023) in the stained samples than in the control samples. The stained capsules were less elastic and tore under less stretching force. Special attention and care were taken to avoid influencing capsule elasticity by any external factor except staining. Therefore, measurements were taken within 6 hours after sampling, the specimens were kept moist during the entire experiment, and all manipulations of the capsule were performed under the microscope with the help of microsurgical instruments. We used the shortest exposure time (10 seconds) and a standard concentration of dye solution (0.0125%). The probable mechanism of action by which trypan blue alters the stretching capacity of the human lens capsule can only be hypothesized based on previous publications. We suggest that these changes are due to collagen crosslinking induced by a photosensitizing action of the staining agent.6,16 In 2000, Perrone first
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described the Argentinean flag sign, a name for the most common complication when performing capsulorhexis in eyes with intumescent cataract stained with trypan blue (D.M. Perrone, MD, ‘‘Argentinean Flag Sign Is Most Common Complication for Intumescent Cataracts,’’ Ocular Surgery News US Edition, Decem ber 15, 2000. Available at: http://www.osnsupersite. com/view.asp?rIDZ14207. Accessed May 6, 2008). When a spontaneous tear in the capsule extends into the periphery, the appearance of the stained capsule beside the white cataract mimics the blue–white–blue arrangement of the Argentinean flag. As shown by Singh et al.,14 the dye substance accumulates primarily in the basement membrane, adjacent to the epithelial layer. The basement membrane capsule consists of multiple lamellae and a 3-dimensional molecular meshwork made primarily of type IV collagen, which is important for the biomechanical properties of the capsule and shows the properties of an elastic structure. The exposure of trypan blue to light may induce a chain of photochemical reactions that results in the production of highly reactive freeoxygen radicals or singlet oxygen. These compounds could interact with the basement membrane and cause collagen crosslinking, leading to a decrease in the elastic properties of the lens capsule.17,18 Wollensak et al.16 evaluated 55 trypan blue–stained porcine anterior lens capsules using various time intervals combined with exposure to white light or with no light exposure. The biomechanical stress–strain measurements were performed using an automated material tester. After treatment with light and trypan blue, at 25% strain, there was a statistically significant increase in stress of up to 70% and in elastic stiffness of 47% and a decrease in the ultimate mechanical strain of up to 13%. Interestingly, there were no biomechanical changes in capsules with trypan blue staining in the absence of light or after a short illumination time of 30 seconds, indicating a light-dependent process. Trypan blue staining of the lens capsule combined with light irradiation for at least 1 minute led to an increase in elastic stiffness at 25% strain and a reduction in the ultimate extensibility. In contrast to our study, porcine cadaver eyes and measurements at a fixed strain were used. Moreover, an intraindividual comparison was not performed. The authors found evidence that their results might have been induced by a photosensitizing action of trypan blue, leading to light-induced collagen crosslinking of the capsule collagen similar to age-related crosslinking. As in our study, the application of trypan blue led to an increase in elastic stiffness. Moreover, we found that anterior capsule elasticity is highly frequency dependent; the elevated frequency of stress led to increased stiffness in both treated and control groups. The samples became less elastic,
more rigid, and more fragile. However, the clinical relevance of this must be investigated in further detail. A recent paper19 reviewed 61 selected articles on dye staining in cataract surgery. The panel members selected 36 articles for the panel methodologist to review and rate according to the strength of the evidence. A level I rating was assigned to properly conducted, well-designed, randomized clinical trials; a level II rating, to well-designed cohort and case-control studies; and a level III rating, to case series and case reports. There was level III evidence that trypan blue and other agents are effective in staining the lens. There was level II evidence that staining the capsule is helpful in completing a capsulorhexis and that it is helpful in patients younger than 5 years and in cases of white cataract. The overall surgical advantage of a completed CCC using dye has not been demonstrated, but this may be related to the outcome measures chosen rather than to a failure to confer advantage. There were substantial data indicating that trypan blue 0.1% is not toxic to the cornea. There were data confirming that dye is safe and effective as an adjunct for capsule visualization in cataract surgery. The authors conclude that it is reasonable to use dye when inadequate capsule visualization may compromise the outcomes in cataract surgery. More studies are needed to confirm a lack of toxicity of trypan blue, particularly in the event of posterior segment or longer duration of exposure. In summary, the present study shows the effect of VisionBlue staining on the elastic properties of the human anterior lens capsule. We believe that ours is the first study of its kind to be performed in human eyes; the results have a great clinical implication and should be taken into consideration in dye-enhanced cataract surgery. Further clinical studies might be designed to compare this effect of trypan blue with that of other currently available staining agents. REFERENCES 1. Gimbel HV, Neuhann T. Continuous curvilinear capsulorhexis [letter]. J Cataract Refract Surg 1991; 17:110–111 2. Pandey SK, Werner L, Escobar-Gomez M, Werner LP, Apple DJ. Dye-enhanced cataract surgery. Part 3: posterior capsule staining to learn posterior continuous curvilinear capsulorhexis. J Cataract Refract Surg 2000; 26:1066–1071 3. Dada VK, Sharma N, Sudan R, Sethi H, Dada T, Pangtey MS. Anterior capsule staining for capsulorhexis in cases of white cataract; comparative clinical study. J Cataract Refract Surg 2004; 30:326–333 4. Melles GRJ, de Waard PWT, Pameyer JH, Beekhuis WH. Trypan blue capsule staining to visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg 1999; 25:7–9 5. Ozturk F, Osher RH. Capsular staining: recent developments. Curr Opin Ophthalmol 2006; 17:42–44 6. Nanavaty MA, Johar K, Sivasankaran MA, Vasavada AR, Praveen MR, Zetterstro¨m C. Effect of trypan blue staining on
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the density and viability of lens epithelial cells in white cataract. J Cataract Refract Surg 2006; 32:1483–1488 Venkatesh R, Das M, Prashanth S, Muralikrishnan R. Manual small incision cataract surgery in eyes with white cataracts. Indian J Ophthalmol 2005; 53:173–176 Chung CF, Liang CC, Lai JSM, Lo ESF, Lam DSC. Safety of trypan blue 1% and indocyanine green 0.5% in assisting visualization of anterior capsule during phacoemulsification in mature cataract. J Cataract Refract Surg 2005; 31:938–942 Wong VWY, Lai TYY, Lee GKY, Lam PTH, Lam DSC. A prospective study on trypan blue capsule staining under air vs under viscoelastic. Eye 2006; 20:820–825 Stu¨rmer J. Das Blaue Wunder in der Kataraktchirurgie. Klin Monatsbl Augenheilkd 2002; 219:191–195 Bhartiya P, Sharma N, Ray M, Sinha R, Vajpayee RB. Trypan blue assisted phacoemulsification in corneal opacities. Br J Ophthalmol 2002; 86:857–859 Werner L, Apple DJ, Crema AS, Izak AM, Pandey SK, Trivedi RH, Ma L. Permanent blue discoloration of a hydrogel intraocular lens by intraoperative trypan blue. J Cataract Refract Surg 2002; 28:1279–1286 Birchall W, Raynor MK, Turner GS. Inadvertent staining of the posterior lens capsule with trypan blue dye during phacoemulsification [photo essay]. Arch Ophthalmol 2001; 119:1082– 1083 Singh AJ, Sarodia UA, Brown L, Jagjivan R, Sampath R. A histological analysis of lens capsules stained with trypan blue for
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capsulorrhexis in phacoemulsification cataract surgery. Eye 2003; 17:567–570 Rangaraj NR, Ariga M, Thomas J. Comparison of anterior capsule electron microscopy findings with and without trypan blue stain. J Cataract Refract Surg 2004; 30:2241–2242 Wollensak G, Spo¨rl E, Pham D-T. Biomechanical changes in the anterior lens capsule after trypan blue staining. J Cataract Refract Surg 2004; 30:1526–1530 Goosey JD, Zigler JS Jr, Kinoshita JH. Cross-linking of lens crystallins in a photodynamic system: a process mediated by singlet oxygen. Science 1980; 208:1278–1280 Charulatha V, Rajaram A. Influence of different crosslinking treatments on the physical properties of collagen membranes. Biomaterials 2003; 24:759–767 Jacobs DS, Cox TA, Wagoner MD, Ariyasu RG, Karp CL. Capsule staining as an adjunct to cataract surgery; a report from the American Academy of Ophthalmology (Ophthalmic Technology Assessment). Ophthalmology 2006; 113:707–713
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First author: H. Burkhard Dick, MD Department of Ophthalmology, Ruhr-University, Bochum, Germany
Effect of trypan blue on the elasticity of the human anterior lens capsule H. Burkhard Dick, MD, Shakhsanam E. Aliyeva, MD, Fritz Hengerer, MD
PURPOSE: To evaluate the effect of trypan blue (VisionBlue) staining on the elastic properties of the human anterior lens capsule. SETTING: Center for Vision Science, Ruhr University Eye Hospital, Bochum, Germany. METHODS: Nineteen anterior capsules were obtained from 19 human eyes at the time of cataract operation. Two same-size strips were prepared from each capsule; 1 was exposed to trypan blue staining for 10 seconds, and the other was a control. The elasticity measurements were performed on both specimens using a modified rheometer. RESULTS: The mean age of the patients was 75.3 years G 13.3 (SD) (range 35 to 89 years). Sixty-three measurements of 19 suitable specimen pairs were performed. In 13 pairs, the elasticity measurements were eligible for statistical analysis. The stress value at the tearing point was statistically significantly lower in the treated group (mean 169 G 55 Pa) than in the control group without staining (mean 252 G 67 Pa) (P Z .023). The difference in stress between the stained and control samples at 3 seconds and 5 seconds was not statistically significant (P Z .093 versus P Z .316). Dynamic frequency sweep measurements showed a statistically significant elevation of the viscosity modulus in the samples stained with trypan blue. CONCLUSIONS: Trypan blue staining affected the biomechanical properties of the human lens capsule and led to a significant reduction in elasticity and an increase in stiffness. These results confirm clinical reports of changes in the capsule by dye and should be taken into consideration in dye-enhanced cataract surgery. J Cataract Refract Surg 2008; 34:1367–1373 Q 2008 ASCRS and ESCRS
First introduced by Gimbel and Neuhann,1 the continuous curvilinear capsulorhexis (CCC) has been the gold standard in cataract surgery for more than 20 years. Compared with other surgical approaches to open the anterior capsule, this technique has a lower complication rate. The main criterion to perform the CCC is the presence of retroillumination from the fundus, which enables viewing of the round edges of the capsulorhexis.
Accepted for publication March 20, 2008. From the Department of Ophthalmology (Dick), Ruhr-University, Bochum, the Department of Ophthalmology (Aliyeva), Johannes Gutenberg-University, Mainz, Germany Center for Vision Science (Hengerer), Bochum, Germany. Neither author has a financial or proprietary interest in any material or method mentioned. Corresponding author: H. Burkhard Dick, MD, Center for Vision Science, Ruhr University Eye Hospital, In der Schornau 23 - 25, D-44892 Bochum, Germany. E-mail: [email protected]. Q 2008 ASCRS and ESCRS Published by Elsevier Inc.
In cases of mature cataract or in other situations in which the red reflex is poor or visualization of the capsule compromised, capsule staining is helpful to visualize the round edges of the capsulorhexis. For this purpose, fluorescein, indocyanine green, and trypan blue are commonly used. The more intense and persistent capsule staining that trypan blue provides is particularly advantageous, making it preferable to other agents for dye-aided cataract surgery. However, it is not known whether the application of trypan blue affects the mechanical characteristics of the lens capsule. The impairment of elastic properties of the lens capsule after application of trypan blue could increase the risk for capsular tears and may result in a higher incidence of intraoperative complications. To ensure the safety of dye-enhanced surgical procedures, quantitative evaluation of capsule elasticity is needed. We performed a prospective study to assess the change in the elastic property of the human lens capsule by application of VisionBlue, a trypan blue dye. 0886-3350/08/$dsee front matter doi:10.1016/j.jcrs.2008.03.041
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EFFECT OF TRYPAN BLUE ON CAPSULE ELASTICITY
PATIENTS AND METHODS
Rheometry
The lens capsules of 19 patients having phacoemulsification were collected. The same surgeon (H.B.D.) performed the capsulorhexis and collected a sampling of specimens using a standard technique. Because various ocular disorders may influence capsule elasticity, patients with the following pathologies were excluded from the study: pseudoexfoliation syndrome, previous intraocular surgery, topical medical treatment within the past 8 weeks, chronic recurrent inflammation, history of ocular trauma, glaucoma, pregnancy, metabolic diseases such as diabetes mellitus, and eyes with mature cataract. The selection of eyes was randomized. During creation of the capsulorhexis, the anterior lens capsule was removed en bloc and placed in a container with balanced salt solution (BSS). All measurements were performed within a maximum of 6 hours after the capsule was removed from the eye. The human lens capsule thickness is at its maximum at the anterior midperiphery, which appears to be located central to the zonular insertion. It increases with age, especially at the anterior pole, while the midperipheral zone stabilizes or slightly decreases after the seventh decade. The thickness of the human lens capsule continuously increases with age, an average of 11.0 to 15.0 mm at the anterior lens pole. Maximum thickness is at the anterior midperiphery, increasing with age from 13.5 to 16.0 mm. The anterior zonular insertion is actually related to a local pre-equatorial thinning, which remains unchanged with age. The thickness of the anterior lens capsules was not measured because this study was an intraindividual comparative analysis. The individuals were not separated by age, sex, or any other potentially discriminating factors. No specimen was intentionally discarded.
Rheometry allows sophisticated and automated measurement of changes in the flow of fluids and deformation of solid materials under various kinds of stress and strain. In this study, the Rheometrics RDS-II Mechanical/Dynamic Spectrometer RMS-800 (Rheometrics Scientific, Inc.) was used (Figure 1). Originally designed to determine the complex flow properties of liquids, this instrument is also equipped to measure the tensile and compressive strength of solid materials. Elastic properties of the lens capsule were assessed as a reaction to application of graduated strength. The Digital Storage Scope HM 208 oscilloscope (Hameg Instruments GmbH) was also used to determine the strength transmitting to the sample. For graphic interpretation, RSI Orchestrator software (version 6.5.8, Rheometric Scientific, Inc.) was used.
Preparation of Anterior Capsule Specimen Because the study design implies an intraindividual comparative analysis, paired samples were prepared from each anterior lens capsule. Microscissors were used to divide the capsule into 2 parts shaped like half moons. From each part, 2 same-size strips were cut out under the microscope; 1 strip served as a paired control, and the other was used for the staining. The strain force applied in the rheometer varies depending on the shape and size of the sample; thus, meticulous care was taken to achieve the same size and geometry across all specimens for proper assessment of elastic properties. Samples were immersed in and stained with VisionBlue solution for 10 seconds and then placed in a Petri dish containing BSS. As dehydration might change the biomechanical properties of the tissue, all samples were constantly held in a moisture medium during the entire experiment.
Holders To strain the sample in the rheometer, holders were made from a light, corrosion-resistant material. The purpose of the holders was to provide safe fixation, and their weight had to be as low as possible to minimize its effect on measurement during the swinging process. Therefore, in collaboration with the Max Planck Institute for Polymer Research, Mainz, Germany, a gripping device designed to hold the sample between 2 aluminum clamps by means of a screw was engineered. The self-adhesive cellulose strips were glued to the sharp metallic edges of the clamp to avoid altering the samples and to prevent slippage during measurements. The gripping device was screwed into a bearing base built into the rheometer instead of to a measuring plate for viscous fluids. Fixation of the capsule strips in the aluminum clamps was performed under the light microscope at 64-fold magnification. One end of the sample was placed between the branches of the aluminum clamp. Then, the branches were tightly joined with the help of a forceps and fixed by screwing with moderate pressure. Because the stretching length of the strips had to be identical in all samples (trypan blue and control), the sample was carefully measured on a micrometer scale placed under a Petri dish. Finally, the other end of the sample was fixed in the opposite clamp of the measuring device. To
Tissue Staining Agent VisionBlue contains 0.6 mg dilute ophthalmic solution of trypan blue, 1.9 mg sodium monohydrogen orthophosphate, 0.3 mg sodium dihydrogen orthophosphate, 8.2 mg sodium chloride sodium hydroxide for adjusting the pH to a value between 7.3 and 7.6, and water for injection. This solution has been available in Germany since February 2003; the U.S. Food and Drug Administration approved VisionBlue in December 2004 for the purpose of improving anterior capsule visualization during cataract surgery. It is supplied as a premixed sterile solution in a 0.5 mL glass ampule.
Figure 1. Image depicting the RMS-800 sample staging area and control panels.
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Measurements Dynamic Strain Sweep Before each measurement was taken, equal prestress force (5% for all specimens), controlled by an oscillograph, was applied to each capsule strip. When operating in dynamic mode (dynamic strain sweep), the instrument applies a sinusoidal oscillating strain with a constant low frequency, producing a sinusoidally oscillating stress in the sample. Two measurements were used for each sample analysis, and the range of the lowest measurements for minor movements with a maximum amplitude of 0.5 mm was assessed.
Dynamic Frequency Sweeps The goal of dynamic frequency sweeps is to obtain the frequency dependent dynamic modulus (G) in the linear regime of the material. In this study, the dynamic frequency sweeps were done with a frequency range of 0.1 to 100 radians per second, which corresponds to 0.016 to 15.915 Hz. The step for decade variation was set at 5 points per decade to define the difference in logarithms of the starting and final frequencies in each equally divided segment of exponents. Then, a sinusoidal force with constant amplitude was applied to the sample. Shear Rate Assessment The shear rate was evaluated by applying the tested fluid between 2 metallic plates. One plate was set in rotation with a fixed speed; the second, which was connected to a special device to assess driving torque, was simultaneously passively rotated depending on fluid viscosity. The speed at which the 2 plates moved relative to each other is called shear rate and is expressed in radian per second. In this study, this experiment was performed in a modified version with the purpose of determining the viscoelastic properties of all samples, the ultimate stress value, and a time interval at the rupture point. In strain tests and bending tests, the elasticity modulus is expressed as E0 and the viscosity modulus is expressed as E00 . The complex elasticity modulus E* is a ratio: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 E Z ðE0 Þ þðE00 Þ Using a conversion factor composed from geometry, the width (b) and thickness (d) of the sample, and a constant of device (k Z 895), the elasticity and viscosity modulus were calculated as follows: E0 Z G0 [(b d)/895)]. Because of the potential risk for capsule damage, the samples’ thicknesses were not measured and a value of 10 mm was assigned as a standard for all samples. The elasticity module defines the energy that exerts influence on a material by deformation; it leads to return of the shape after discontinuation of the applied force. The following formula was used to calculate the modulus of elasticity (E0 ):
s E0 Zcosd 3 The modulus of viscosity (E00 ) was calculated as s E00 Zsind 3 where d is the phase angle (phase displacement between stress and deformation vectors), s is stress, and 3 is strain. The tangent of the loss angle was calculated as E00 tandZ 0 E
Statistical Analysis Descriptive statistics were calculated and expressed as the mean value and the standard deviation. Nonparametric analysis was used to assess significant differences between variables. To enable use of SPSS software (version 11, SPSS, Inc.) for statistical analysis, all data were converted from RSI Orchestrator using Microsoft Excel 2003 for MS Windows XP. To present the data and compose histograms, Microsoft Excel and Microsoft Word Version Office (version X) (all Microsoft products, Microsoft Corp.) for Macintosh (Apple, Inc.) were used. Graphic presentation of results was expressed in nonparametric box plots, a univariate dot pattern, and bar graphs. The paired t test was used for linked samples. A P value of 0.05 was considered statistically significant.
RESULTS The mean age of enrolled patients was 75.3 years G 13.3 (SD) (range 35 to 89 years) (Figure 2). Ten specimens were obtained from women (age: mean 78.3 G 9.3 years, median 81.5 years) and 9 from men (age: mean 68.2 G 14.9 years, median 74 years). Nineteen suitable specimens were collected and mounted for elasticity analysis, and 63 measurements were recorded. As the study design assumed the agerelated change in the elasticity of the human lens capsule, all measurements were numbered from 1 to 19, depending on patient age. 10 9 8
Number of patients
prevent exsiccation, the specimen was continuously moistened with drops of BSS. All manipulations of the capsule were performed under a microscope (Wild M38) using smooth microsurgical instruments in a Petri dish filled with BSS. Before each measurement, the solution was changed and the glass micrometer scale used to evaluate specimen length and width was placed. For the measurement, the whole construction was transferred onto a rubber brick, clamped horizontally between the jaws of the testing device, and then expanded to the full length of sample.
7 6 5 4 3 2 1 0
30-40
40-50
50-60
60-70
70-80
Patient Age (years) Figure 2. Demographic data of patients.
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Figure 4. Intraindividual changes in stress values at the tearing point in each sample of the 13 pairs.
Results from 13 measurements were chosen for statistical analysis. The 6 measurements that were excluded had 1 in 3 assessments that exceeded a doubled standard deviation; each was assumed to be a rupture. These patients were only enrolled for the estimation of dynamic frequency sweep value. In 11 of the accepted 13 pairs, the lower stress value at the tearing point was measured in the group treated with VisionBlue. In 1 pair, the similar stress value was applied in both samples. In the remaining pair, the
stained capsule tore under higher stress than the control sample. The difference between the treatment group and paired control group in mean stress value at the tearing point was statistically significant (P Z .023). The box plot in Figure 3 shows the stress range in both groups (mean 169 G 55 Pa in stained samples versus 252 G 67 Pa in unstained samples). The intraindividual changes in stress value applied at the point of rupture in each of the 13 pairs are shown in Figure 4. The difference in the counterforce value between the stained samples and control samples at 3 seconds (P Z .093) and 5 seconds (P Z .316) of stress is shown in Figures 5 and 6, respectively. It is evident that at the specific moment of measurement, the measuring converter applied smaller counterforce in the stained samples. However, the difference was not statistically significant. The dynamic frequency sweep measurements showed considerable biomechanical changes with or
Figure 5. Differences in counterforce between the stained and control samples after 3 seconds of stretching (P Z .93).
Figure 6. Differences in counterforce between the stained and control samples after 5 seconds of stretching (P Z .316).
Figure 3. The box plot showing a statistically significant higher stress value at the point of rupture in the staining group.
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Figure 7. Intraindividual changes in the modulus of elasticity (E0 ) through 16 measuring frequencies in 19 sample pairs.
without staining between the frequencies of 14 to 15 and 15 to 16. In both trypan blue-stained and control samples, a statistically significant elevation of viscosity modulus was noticed at the frequency intervals between 12 and 13, 13 and 14, and 14 and 15. Figures 7 and 8 show the changes in E0 modulus and E00 modulus, respectively, at all 16 frequencies evaluated in the 19 sample pairs. DISCUSSION Successful completion of CCC is crucial for any phacoemulsification procedure and is associated with a lower complication rate and better surgical outcomes. In eyes with white and mature cataract, poor visibility of the capsule due to a decreased or absent red fundus reflex makes surgery more challenging. Therefore, various staining agents have been brought into routine use in recent years.2,3 Trypan blue was initially applied to examine the endothelial cell layer of donor corneas before corneal transplantation. Melles et al.4 reported the first experience with intraoperative application of trypan blue to stain the anterior capsule and facilitate capsulorhexis for surgery for mature cataract. Several advantages make trypan blue particularly desirable. It creates much darker staining and provides better visualization than other dyes. Also, it lasts longer and usually persists throughout the entire procedure, and it is less expensive because only a small amount need be applied.5 Moreover, recent studies show trypan blue staining can help prevent posterior capsule opacification through a significant decrease in density and viability of lens epithelial cells.6 Numerous studies have confirmed the safety and efficacy of trypan blue staining.7–10 Bhartiya et al.11
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Figure 8. Changes in modulus of viscosity (E00 ).
report successful application of the dye in eyes with corneal opacities involving the visual axis. Except for permanent blue discoloration of a hydrogel intraocular lens12 and the inadvertent staining of the posterior lens capsule and vitreous,13 the reported adverse reactions to VisionBlue use are self-limited and of short duration. Reports of histological analysis14 and electron microscopy findings15 of lens capsules stained with trypan blue have also been published. However, the clinically important aspect, such as the effect of trypan blue on the biomechanical properties of human lens capsule, has not been studied. The present study is the first comparative investigation of the change in the elastic properties of the human anterior capsule after staining with VisionBlue vital dye. Our results show statistically significant lower values at the moment of rupture (P Z .023) in the stained samples than in the control samples. The stained capsules were less elastic and tore under less stretching force. Special attention and care were taken to avoid influencing capsule elasticity by any external factor except staining. Therefore, measurements were taken within 6 hours after sampling, the specimens were kept moist during the entire experiment, and all manipulations of the capsule were performed under the microscope with the help of microsurgical instruments. We used the shortest exposure time (10 seconds) and a standard concentration of dye solution (0.0125%). The probable mechanism of action by which trypan blue alters the stretching capacity of the human lens capsule can only be hypothesized based on previous publications. We suggest that these changes are due to collagen crosslinking induced by a photosensitizing action of the staining agent.6,16 In 2000, Perrone first
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described the Argentinean flag sign, a name for the most common complication when performing capsulorhexis in eyes with intumescent cataract stained with trypan blue (D.M. Perrone, MD, ‘‘Argentinean Flag Sign Is Most Common Complication for Intumescent Cataracts,’’ Ocular Surgery News US Edition, Decem ber 15, 2000. Available at: http://www.osnsupersite. com/view.asp?rIDZ14207. Accessed May 6, 2008). When a spontaneous tear in the capsule extends into the periphery, the appearance of the stained capsule beside the white cataract mimics the blue–white–blue arrangement of the Argentinean flag. As shown by Singh et al.,14 the dye substance accumulates primarily in the basement membrane, adjacent to the epithelial layer. The basement membrane capsule consists of multiple lamellae and a 3-dimensional molecular meshwork made primarily of type IV collagen, which is important for the biomechanical properties of the capsule and shows the properties of an elastic structure. The exposure of trypan blue to light may induce a chain of photochemical reactions that results in the production of highly reactive freeoxygen radicals or singlet oxygen. These compounds could interact with the basement membrane and cause collagen crosslinking, leading to a decrease in the elastic properties of the lens capsule.17,18 Wollensak et al.16 evaluated 55 trypan blue–stained porcine anterior lens capsules using various time intervals combined with exposure to white light or with no light exposure. The biomechanical stress–strain measurements were performed using an automated material tester. After treatment with light and trypan blue, at 25% strain, there was a statistically significant increase in stress of up to 70% and in elastic stiffness of 47% and a decrease in the ultimate mechanical strain of up to 13%. Interestingly, there were no biomechanical changes in capsules with trypan blue staining in the absence of light or after a short illumination time of 30 seconds, indicating a light-dependent process. Trypan blue staining of the lens capsule combined with light irradiation for at least 1 minute led to an increase in elastic stiffness at 25% strain and a reduction in the ultimate extensibility. In contrast to our study, porcine cadaver eyes and measurements at a fixed strain were used. Moreover, an intraindividual comparison was not performed. The authors found evidence that their results might have been induced by a photosensitizing action of trypan blue, leading to light-induced collagen crosslinking of the capsule collagen similar to age-related crosslinking. As in our study, the application of trypan blue led to an increase in elastic stiffness. Moreover, we found that anterior capsule elasticity is highly frequency dependent; the elevated frequency of stress led to increased stiffness in both treated and control groups. The samples became less elastic,
more rigid, and more fragile. However, the clinical relevance of this must be investigated in further detail. A recent paper19 reviewed 61 selected articles on dye staining in cataract surgery. The panel members selected 36 articles for the panel methodologist to review and rate according to the strength of the evidence. A level I rating was assigned to properly conducted, well-designed, randomized clinical trials; a level II rating, to well-designed cohort and case-control studies; and a level III rating, to case series and case reports. There was level III evidence that trypan blue and other agents are effective in staining the lens. There was level II evidence that staining the capsule is helpful in completing a capsulorhexis and that it is helpful in patients younger than 5 years and in cases of white cataract. The overall surgical advantage of a completed CCC using dye has not been demonstrated, but this may be related to the outcome measures chosen rather than to a failure to confer advantage. There were substantial data indicating that trypan blue 0.1% is not toxic to the cornea. There were data confirming that dye is safe and effective as an adjunct for capsule visualization in cataract surgery. The authors conclude that it is reasonable to use dye when inadequate capsule visualization may compromise the outcomes in cataract surgery. More studies are needed to confirm a lack of toxicity of trypan blue, particularly in the event of posterior segment or longer duration of exposure. In summary, the present study shows the effect of VisionBlue staining on the elastic properties of the human anterior lens capsule. We believe that ours is the first study of its kind to be performed in human eyes; the results have a great clinical implication and should be taken into consideration in dye-enhanced cataract surgery. Further clinical studies might be designed to compare this effect of trypan blue with that of other currently available staining agents. REFERENCES 1. Gimbel HV, Neuhann T. Continuous curvilinear capsulorhexis [letter]. J Cataract Refract Surg 1991; 17:110–111 2. Pandey SK, Werner L, Escobar-Gomez M, Werner LP, Apple DJ. Dye-enhanced cataract surgery. Part 3: posterior capsule staining to learn posterior continuous curvilinear capsulorhexis. J Cataract Refract Surg 2000; 26:1066–1071 3. Dada VK, Sharma N, Sudan R, Sethi H, Dada T, Pangtey MS. Anterior capsule staining for capsulorhexis in cases of white cataract; comparative clinical study. J Cataract Refract Surg 2004; 30:326–333 4. Melles GRJ, de Waard PWT, Pameyer JH, Beekhuis WH. Trypan blue capsule staining to visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg 1999; 25:7–9 5. Ozturk F, Osher RH. Capsular staining: recent developments. Curr Opin Ophthalmol 2006; 17:42–44 6. Nanavaty MA, Johar K, Sivasankaran MA, Vasavada AR, Praveen MR, Zetterstro¨m C. Effect of trypan blue staining on
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First author: H. Burkhard Dick, MD Department of Ophthalmology, Ruhr-University, Bochum, Germany