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From the Department of Research and Development, Instituto Oftalmologico de Alicante and Universidad Miguel Hernandez, Alicante, Spain.
| Abstract |
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METHODS. RI at the anterior stromal surface of bovine corneal buttons was measured after the epithelium was scraped away. Samples were weighed and oven dried to calculate hydration. RI of the stromal bed surface was measured with a modified hand-held Abbé refractometer immediately before and after excimer laser photoablation. Thirty-one untreated persons (group 1: 44 corneas; age range, 1965 years) and eight re-treated patients (group 2: 10 corneas) were examined.
RESULTS. RI of bovine stromal surface was significantly associated with hydration (H) (RI = 1.4067 0.00599H, r = 0.9079, P < 0.001). Photoablation significantly increased the RI of the midstroma (group 1: 1.3721 ± 0.0041 to 1.3839 ± 0.0050; group 2: 1.3717 ± 0.0038 to 1.3819 ± 0.0039). Differences between groups were not significant. In group 1 (n = 31), change in RI (
RI) was significantly related to preoperative RI (
RI = 1.155 0.833RI, r = 0.595, P < 0.001) and RI was significantly related to age (x) (RI = 1.3634 + 0.00026x, r = 0.603, P < 0.001).
CONCLUSIONS. Mammalian corneal stromal RI correlates with hydration. LASIK significantly increases the refractive index of the treated stromal bed, and this equates to an average change in hydration from 4.3 to 2.9. For individual cases, change in RI is associated with the pre-op RI. The lack of any difference between untreated and re-treated corneas suggests that with time hydration returns back to normal levels. The RI in the older corneal stroma is slightly higher relative to the RI in the younger corneal stroma.
Stromal hydration and refractive index can be measured ex vivo, and the information may help us to estimate the change in stromal hydration during LASIK. Techniques for estimating stromal hydration in vitro are available,12 13 14 15 but they are cumbersome and difficult to incorporate in the operating theater where PRK or LASIK procedures are conducted. The refractive index of the corneal surface has been measured in vivo using a modified Abbé refractometer.16
The purposes of this investigation were to (1) evaluate the relationship between stromal hydration and refractive index in vitro; (2) examine the effects of air exposure on the refractive index of the stroma after removing the epithelium in vitro; (3) measure stromal refractive index immediately before and after the application of excimer laser in routine cases of LASIK; and (4) use this information to predict whether there is a significant change in hydration as a direct consequence of excimer laser photoablation.
To the best of our knowledge, this is the first report in which the refractive index of the human corneal stroma has been studied during the LASIK procedure in an experimental manner.
| Methods |
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![]() | (1) |
Because the stromal thickness is directly proportional to stromal hydration,19 20 21 the hydration term in a refractive indexhydration model can be replaced with the thickness. Laing et al.11 developed a model predicting changes in corneal refractive index in relation to changes in thickness as follows:
![]() | (1A) |
For example, using the Fatt and Hedbys21 hydrationthickness relationship and placing the values into model 1A, it can be shown that
![]() | (2) |
Equation 2 can be adjusted to suit any hydrationthickness relationship. Using the Ytteborg and Dohlmann19 model, the Ns H equation changes to
![]() | (3) |
Both models predict that, for a normal H of 3.5, Ns = 1.376.
In summary, for the normal stroma when there is a decrease in hydration and thickness, there is a corresponding increase in refractive index and vice versa.
To test these models, we measured the refractive index of corneal samples over a range of H.
Measurement of Stromal Refractive Index and Hydration In Vitro
The refractive index of bovine corneal samples was measured with a standard Abbé bench model refractometer (Ealing Electro-Optical, Watford, UK). Ten bovine eyes were obtained from a local abattoir. The eyes were removed and transported in a sealed moist chamber with a conjunctival flap to prevent desiccation. The epithelium was removed by gently scraping with a scalpel blade. The cornea was removed from the globe by cutting just within the limbus, and the anterior stromal surface was placed on the measuring prism table of the refractometer. The preliminary adjustments and calibration of the refractometer were performed earlier, in accordance with the suppliers instructions. Using a standard sodium light (sodium D line), we read off the refractive index. The cornea was weighed using a chemical balance and placed in a moist chamber from 1 to 2 hours to allow the sample to dehydrate slowly. Refractive index was again measured, and the sample was weighed and returned to the moist chamber. The cycle was repeated two more times. The sample was dehydrated to calculate H by placing the cornea in a dry oven for 3 days at 80°C to 90°C. All corneal samples were obtained within 30 minutes of death. Bovine samples were used because for economic reasons, they were available fresh and in abundance, without unnecessary killing of laboratory animals.
Effects of Exposure on Stromal Refractive Index Ex Vivo
Human eyes were obtained from donors soon after death or from patients undergoing enucleation because of melanoma. In all cases, the first measurement was taken within 1 hour after death or enucleation. The epithelium was removed by gently scraping it away with a scalpel blade. The refractive index of the anterior stromal surface was measured with the bench model refractometer. Refractive index measurements were repeated 5 minutes over the following 0.5 hour. Between measurements, the cornea was exposed to air. The room temperature and relative humidity ranged from 20°C to 23°C and 55% to 60%, respectively. The refractometer calibration was checked according to the manufacturers instructions before any measurements of corneal samples. Three human eyes were investigated.
Refractive Index of Stroma before and after LASIK In Vivo
The refractive index of the corneal surface has been measured in vivo using a refractometer.16 In the present study, the refractive index of the stroma was measured using a modified refractometer (Abbé pocket refractometer; Bellingham & Stanley Ltd., Tunbridge Wells, UK). This particular model is normally used to measure refractive index of sugar solutions. The prism box is opened, and a drop of the liquid is placed on the clean polished surface of the measurement block. The prism box is closed, and the device is pointed toward a light source. The observer views, through the refractometer eyepiece, a circular field with a vertical scale passing through the center. The field of view consists of dark and light regions separated by a horizontal line of demarcation. The position of this boundary line as it crosses the vertical scale is recorded. The scale is fitted as standard and marked off in percentage of sucrose at the eyepiece graticule. A calibration chart, provided by the manufacturer, is used to convert scalar readings to refractive indexes. The scale resolution was 0.0018 U of refractive index. For our purposes, the prism box was removed, the measurement block was placed directly onto the stromal bed, and a small light source was arranged close to the measurement block to provide sufficient illumination so that a refractive index measurement could be made when viewing through the eyepiece. The refractometer was originally calibrated using 10 sucrose solutions of various concentrations (10%40%) at 20°C. The concentrations of the samples were masked, and the codes broken at the end. The testretest reliability of the refractometer was checked on 10 separate occasions by using a soft contact lens (Lunelle ES70; Ocular Sciences, Romsey, UK) mounted on a plastic dome to simulate the corneal stromal bed. Between measurements, the lens was cleaned with saline and returned to the storage vial. All contact surfaces were cleaned and sterilized with a standard surgical grade of alcohol before measurement in subjects.
Subjects
The study was performed in accordance with the tenets of the Declaration of Helsinki (2000). Signed consent was obtained from each subject after they received a full explanation of the procedure and the purpose of the investigation. Each subject was a patient attending for routine LASIK. None of the subjects had any corneal complications or diseases that could adversely affect the expected outcome of LASIK or corneal refractive index.
Procedure
LASIK primary procedures and reoperations were performed by experienced surgeons (JLA, JJP-S) who used a technique that has been reported by us.22 For reoperations, the flap was lifted with the LASIK spatula (Alió; Katena, Denville, NJ). After the flap was lifted, the stromal bed was dried with a wet sponge and ablation was performed. An excimer laser (Technolas 217C; Bausch & Lomb, Tampa, FL) was used in all cases (primary and retreatments). No drying of the cornea was performed in any case during the ablation process. In primary cases, a microkeratome (M2; Moria, Antony, France) was used, adjusting the suction ring diameter to the promediated K values of the surgically treated cornea. The flap in all cases was with 9.5 mm with a hinge of 4 mm, tentatively programmed according to the flow chart provided by the company. After the flap was created, it was lifted by the LASIK spatula (Alió; Katena), and the stromal bed was wiped with a wet sponge. After the surface was completely dry, ablation was performed with the same excimer laser. In no case was there any further drying of the cornea during the ablation process. After the experimental measurements were taken, the flap was repositioned both in retreatment and primary LASIK cases, completing the procedure according to our previously reported methods.23 After the flap was created and lifted back, the stromal bed was checked by the surgeon for any irregularities. When the stromal bed was free of any irregularities or complications, the refractometer test block was gently lowered onto the stromal bed. A pen torch light was placed close to the eye to facilitate observation of the dark and light fields. The refractive index was read, the refractometer was gently lifted from the stromal bed, and the LASIK procedure was continued. The refractometer contact surface was wiped with alcohol. Immediately after completing the ablation, the surgeon checked the stromal bed, and the refractive index measurement was repeated. The refractometer was gently removed and the surgical procedure was completed. The stromal surface was not irrigated between refractive index measurements. Data were collected from 44 untreated and 10 re-treated corneas.
| Results |
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Refractive Index of the Stroma before and after Lasik In Vivo
The midstromal refractive index was measured in 31 (44 eyes) untreated patients. This group consisted of 18 men (mean age, 34.3 years; range, 1965; 26 eyes) and 13 women (mean age, 34.1; range, 2554; 18 eyes). The midstromal refractive index was also measured in 10 eyes of eight patients who needed retreatment (six men, two women; mean age, 34.3 years; range, 3043). The refractive index was measured immediately before and after photoablation. The results from individual corneas are in Table 3 . The pre- and postoperative respective mean ± SD refractive indexes were 1.3721 ± 0.0041 and 1.3839 ± 0.0050 for the untreated group and 1.3717 ± 0.0038 and 1.3819 ± 0.0039 for the retreatment group. The difference between the mean refractive index before and after ablation was significant (t-test, P = 5.17 x 1020 for the untreated group and P = 1.44 x 105 for the retreatment group). The difference in refractive index between the two groups just before (group 1) and immediately after (group 2) ablation was not significant (t-test, group 1: P = 0.746; group 2: P = 0.197). Within the untreated group, there was no significant difference in refractive index between men and women (t-test, P = 0.7556). Application of linear regression analysis revealed (1) a significant correlation between refractive index and age in the corneas before ablation but not after ablation (n = 31; before ablation: r = 0.629, P = 0.0002; after ablation: r = 0.221, P > 0.01); (2) the correlation between the individual pairs of refractive indexes immediately before and after ablation was not significant (n = 31; r = 0.163, P > 0.01); and (3), for individual corneas, there was a significant correlation between the actual change in refractive index and the pretreatment refractive index (n = 31; r = 0.595, P = 0.0004).
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| Discussion |
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Change in Refractive Index after Exposure to Air
The refractive index of the stromal surface gradually increased exponentially after exposure to air. This is expected, as mentioned earlier, as a direct consequence of water loss by evaporation. The refractive index increased by +0.006, +0.005, and +0.019 in samples 1, 2, and 3, respectively, after 15 minutes of exposure, with an average change of +0.0027 during the first 5 minutes. Unless there are complications, during the standard LASIK procedure, the bare stroma is not exposed to the air for more than 5 minutes. According to equations 1 2 and 3 , the refractive index would increase by +0.004 to +0.01 for a decline in H from 3.5 to 3.3. This hydration shift is equivalent to a 1.3% change in water content and leads us to conclude that the dehydration of the de-epithelized cornea within the first 5 minutes of exposure is not expected to surpass 1%. Consequently, any change in the stromal refractive index of magnitude greater than +0.0027 during photoablation must be due to other factors. It would be useful to have an indication of how much the refractive index would change under different ambient conditions of temperature and humidity. We would expect the evaporation from the stromal surface to increase further when the ambient temperature is raised above 20°C to 23°C and the relative humidity is lowered below 55% to 60%. However, our intention was to estimate the change in refractive index associated with the water loss expected to occur over the typical time course of excimer laser surgical techniques under normal prevalent conditions.
Refractive Index and Age
The mean refractive index of the midstroma of the 44 normal corneas was 1.372 ± 0.0021, slightly lower than the 1.376 normally quoted in the literature.16 24 30 31 32 We believe this is the first report of human corneal stromal refractive index measurement in vivo, and this value is almost identical with the more recently quoted value for the posterior stromal surfaces in vitro.16 Figure 3 shows the intrastromal refractive index and age for 1 eye from each of the 31 untreated patients. The slight increase in refractive index of the midstroma in the older cornea was a surprise finding that could be accounted for as follows.
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Second, mammalian corneal thickness may increase with age, as a direct consequence of a gradual increase in hydration, and this correlates with a decline in endothelial cell count and function.11
Third, the actual swelling pressure reduces as the state of hydration increases2 ; therefore, in the older stroma, we expect the starting-point hydration to be higher than in the younger stroma. Consequently, a stroma with a relatively higher starting hydration has a relatively lower starting-point swelling pressure. A lower swelling pressure requires a smaller upsurge in IOP before water is effectively expelled from the stroma. The increased IOP may be of short duration, but the time lapse between the increase in IOP and fluid flow from the stroma may be enough to affect the refractive index of the older cornea.
Effect of Excimer Laser Photoablation
The refractive index in the untreated group increased from 1.372 ± 0.0041 to 1.384 ± 0.0050 and in the re-treatment group from 1.372 ± 0.0038 to 1.382 ± 0.0039 (Fig. 4) . The results suggest that the change in intrastromal refractive index affects the overall optical performance of the eye. Using the equation of Fatt and Harris,10 for the untreated patients, this change in refractive index is equivalent to a change in average hydration from 4.30 to 2.86. In percentage terms, the change is equivalent to a loss of water of 7% from a pretreatment average of 81%. The change in refractive index of +0.012 units in the untreated group and +0.010 units in the retreatment group was approximately four times greater than the +0.0027 average change expected from passive evaporation of water from the stromal surface after 5 minutes of exposure to air. Clearly, the photoablative process itself is the cause of the unexpected increases in refractive index. It would be useful to monitor any changes in refractive index of the stromal surface over a time course of up to 1 hour in two groups, before and after photoablation. Such monitoring would have allowed comparison of the time course of refractive index change between the treated midstroma in vivo and the stromal surface ex vivo, but was not attempted because it would have subjected the bare stroma to unnecessary physiological stress and placed the patient in jeopardy.
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After the treated surface is irrigated to remove lose debris and the flap is closed, the pseudomembrane may either remain stable or rehydrate by drawing in water from the surrounding tissue. The observed change in refractive index is unlikely to effect clinically significant levels of refraction. However, the subsequent effects on water flow within the treated cornea may adjust corneal surface topography, and this would have a significant effect on corneal aberrations and the overall optical quality of the retinal image, especially during the immediate postoperative period. Within the retreatment group, the refractive index of the previously ablated stromal bed was no different from that in the untreated eyes suggesting that, the postablation stroma gradually rehydrates during wound healing, reaching a steady state of water conduction.
The results in Figure 5 show that the lower the preoperative refractive index, the greater the expected change immediately after ablation. In turn, this infers that the higher the preoperative hydration, the greater the decrease in postoperative hydration. The post-LASIK regression and excimer laser ablation rate are both associated with stromal hydration.3 38 Kim and Jo38 reported that drying the stroma just before ablation has a more pronounced immediate effect on refraction followed by a more prominent regression. This is linked with the actual amount of tissue removed during the procedure, in which the ablation rate of the stromal dry material increases when stromal hydration decreases.3 A greater mass of collagen and GAGs is removed when less water is present, and vice versa.
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| Footnotes |
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Submitted for publication February 20, 2004; revised April 28, 2004; accepted May 12, 2004.
Disclosure: S. Patel, None; J.L. Alió, None; J.J. Pérez-Santonja, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C.
1734 solely to indicate this fact.
Corresponding author: Sudhir Patel, Vissum/Instituto Oftalmologico de Alicante, Avda de Denia s/n, Edificio Vissum, 03016, Alicante, Spain; sudhir.patel{at}psd.csa.scot.nhs.uk.
| References |
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