Hyaluronidase Treatment, Collagen Fibril Packing ... - Semantic Scholar

Hyaluronidase Treatment, Collagen Fibril Packing, and Normal Transparency in Rabbit Corneas C. J. Connon, MSc; K. M. Meek, PhD; R. H. Newton, PhD; M. C. Kenney, MD, PhD; S. A. Alba, MS; H. Karageozian, MS

ABSTRACT PURPOSE: Hyaluronidase treatment is the initial step of corneaplasty, a treatment under development that induces stromal softening and involves the application of a custom designed forming lens to achieve modification of refractive error. The purpose of this investigation was to examine changes in the arrangement of stromal collagen fibrils after hyaluronidase treatment. METHODS: Rabbit corneas were evaluated by slit-lamp microscopy at 0, 2 and 7 days after treatment and haze was assessed by subjective observation. Molecular and interfibrillar Bragg spacing of corneal collagen were measured from synchrotron x-ray scattering patterns. Transmission electron microscopy and digital image analysis were used to calculate radial distribution functions from the positions of collagen fibrils. The calculated fibril sizes and positions were also used to predict the transmission of visible light through these corneas. RESULTS: Hyaluronidase-treated corneas were shown to have a decreased interfibrillar Bragg spacing of 15% to 21%. Fibril hydration did not

From The Open University, Oxford Research Unit, Oxford, United Kingdom (Connon), the Department of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom (Meek, Newton), CedarsSinai Medical Center, Los Angeles, California (Kenney), and Advanced Corneal Systems, Inc., Irvine, California (Alba, Karageozian) The Open University (CJC), the Medical Research Council (KMM), the Council for the Central Laboratory of the Research Councils (KMM), and the Wellcome Trust (KMM, RN) supported this work. We thank Anthony Gleeson, Sue Slawson, and Liz Towns-Andrews at station 2.1, and James Nicholson, Rob Kehoe, and Pierre Rizkallah at station 7.2 for their encouragement and help at the Daresbury synchrotron. Authors S.A. Alba and H. Karageozian are employees of Advanced Corneal Systems, Inc. The remaining authors have no proprietary interest in the materials presented herein. Correspondence: C. J. Connon, Dept. Optometry and Vision Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, PO Box 905, Cardiff, CF10 3NB, United Kingdom. Tel: 44(0).29.20876317; Fax: 44(0).29.20874859; E-mail: [email protected] Received: June 23, 1999 Accepted: March 5, 2000

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change. Transparency of these corneas remained unaltered. CONCLUSIONS: Hyaluronidase reduced the hydration of the corneal stroma, which led to a more compacted collagen fibril arrangement. This compression was predicted to cause a small reduction in the transmission of visible light through the cornea but not to a point likely to cause visual impairment. [J Refract Surg 2000;16:448-455]

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rganization of collagen fibrils in the corneal stroma is responsible for the shape and transparency of the tissue. The spacing between the fibrils is thought to be governed by the interfibrillar proteoglycans1 which, because of their hydrophilic properties and their association with the fibrils, contribute to corneal rigidity. Proteoglycans are hybrid molecules consisting of a protein to which are attached long chains of highly sulfated repeating disaccharides called glycosaminoglycans. The most common types of glycosaminoglycans found in the adult cornea are keratan sulfate and chondroitin/dermatan sulfate. Advanced Corneal Systems hyaluronidase enzyme (ACS-005, Advanced Corneal Systems, Inc., Irvine, CA) is thought to break the chemical bonds between the sugars in the chondroitin sulfate glycosaminoglycans, softening the cornea and making it easier to reshape. The positions of the Bragg reflections in the low angle, equatorial, x-ray scattering pattern from the corneal stroma can be used to obtain an accurate measure of the average center-to-center interfibrillar spacing of the collagen.2-4 The wide angle x-ray scattering pattern from the corneal stroma also has a strong equatorial Bragg reflection, at about 1.5 nm, which allows the average intermolecular spacing to be computed.3,5 This, in turn, allows the Journal of Refractive Surgery Volume 16 July/August 2000

Hyaluronidase Treatment Results in Compression of Corneal Collagen Fibril Packing/Connon et al

Table Hyaluronidase Treatment in Rabbit Corneas Rabbit/Eye 1/R 1/L 2/R 2/L 3/R 3/L 4/R 4/L 5/L

Treatment

Fixative

Weight After Excision (mg)

1000 IU ACS-005 enzyme* 1000 IU ACS-005 enzyme Untreated Untreated 1000 IU ACS-005 enzyme 1000 IU ACS-005 enzyme Balanced salt solution Untreated Untreated

4% paraformaldehyde 4% paraformaldehyde 4% paraformaldehyde 4% paraformaldehyde Frozen Frozen Frozen Frozen Frozen

Not recorded Not recorded Not recorded Not recorded 27.6 31.8 62.2 60.0 Not recorded

* Advanced Corneal Systems (ACS-005) hyaluronidase enzyme

hydration and hence the refractive index of the collagen fibrils to be determined.6 X-ray scattering is a powerful method for observing changes in the packing of the collagen molecules within the fibrils, and of the fibrils in the corneal stroma. In particular, the measured spacing represents averages from the entire thickness of the cornea through which the x-rays are passed, and spacing from hydrated tissue can be determined. Corneaplasty7 is a technique that is being developed to modify refractive errors in patients. The procedure involves changing the refractive properties of the cornea without cutting or ablating the tissue. This is accomplished by softening the cornea with an injection of ACS-005 hyaluronidase and then placing a custom-designed forming lens, which changes the corneal shape from a baseline configuration to an optimized corneal shape for best visual performance in a matter of 1 to 2 days. At this point, the forming lens is no longer required. The technique has been tested in a rabbit model, and US Food and Drug Administration (FDA) Phase IIa human efficacy studies are underway. The procedure is unique in that it requires only a single injection of enzyme and no other surgical intervention. In contrast, radial keratotomy involves making multiple incisions into the cornea with a scalpel, and laser keratectomy relies on excimer lasers to remove surface tissue from the cornea, both of which can cause postoperative optical problems such as an uneven anterior surface or haze. The exact mechanisms by which corneaplasty might alter refractive error are not clear. There are the mechanical forces exerted by the contact lens, but in addition, the enzyme itself might alter corneal biochemical properties. We used synchrotron x-ray scattering and transmission electron microscopy to measure the packing of the collagen fibrils of rabbit corneas that have Journal of Refractive Surgery Volume 16 July/August 2000

been treated with ACS-005 enzyme. Corneas that underwent this treatment are clinically clear, so it appears that removal of some of the corneal proteoglycans does not change corneal ultrastructure sufficiently to cause a significant increase in light scattering. To investigate this hypothesis, we used a light scattering model to predict how changes in collagen packing would be expected to affect the transmission of visible light through the corneas. MATERIALS AND METHODS Experimental Procedures All experimental procedures were carried out in accordance with the ARVO Resolution on the Use of Animals in Ophthalmic and Vision Research. Five New Zealand White rabbits (males), weighing between 1.5 and 2.0 kg, were included in the study. Two of the rabbits were injected with a 1000 IU of ACS-005 enzyme at four sites into the corneal rim of both eyes, and another rabbit was injected with balanced salt solution into the corneal rim of its left eye, leaving the right eye untouched. The two remaining rabbits had no injection in either eye. The corneas and anterior segments were examined with a slit lamp in a masked fashion by a board-certified ophthalmologist (M.C. Kenney) at days 0, 2, 4, and 7. After 1 week, the animals were sacrificed with intravenous injections of sodium pentobarbital (100mg/kg). All eyes were immediately enucleated and the corneal tissue carefully excised. The corneas were preserved either by freezing then storing at -80° C, or by incubation in 4% paraformaldehyde (Table). They were then shipped from Cedars-Sinai Medical Center, Los Angeles, CA to the Open University Oxford Research Unit, Oxford, UK. The frozen tissue was allowed to thaw before being placed into a x-ray beam or before fixation for transmission electron microscopy; we have

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shown that freezing and thawing does not alter the collagen spacing in the cornea.8 To investigate the effects of any possible drying artifacts during transportation that might have affected the interfibrillar spacing of the collagen fibrils, and to examine changes in corneal swelling potential caused by the enzyme injection treatment, the corneal tissue remaining after a portion had been removed for transmission electron microscopy was equilibrated against a solution of known osmolarity.4 Equilibration was carried out by placing the corneas in 14 kDa cut-off dialysis tubing and leaving them for 5 days at 4°C in a solution of 2% polyethylene glycol (20 kDa) containing 0.15M NaCl. For normal, untreated rabbit corneas, this results in tissue being equilibrated to a level near to physiological hydration with minimal loss of interfibrillar constituents.9 Synchrotron X-ray Scattering The defrosted corneas and the corneas kept in paraformaldehyde were mounted between Mylar windows in clear plastic cells, which were airtight to avoid tissue dehydration. The cells containing the corneas were placed in the x-ray beam so that the x-rays passed through the center of the tissue, along the optical axis. Synchrotron x-ray scattering was carried out at the Central Laboratory of the Research Council (CLRC) synchrotron facility at Daresbury, United Kingdom. Low-angle patterns were collected at Station 2.1 using a fixed camera length of 6 m, an exposure time of 3 minutes, and a 3 x 1 mm beam size. Rat tail tendon was used to calibrate the data. Scattering patterns were collected on a multi-wire gas proportional detector. The interfibrillar Bragg spacing was calculated from the position of the innermost equatorial reflection.3 Wide-angle patterns were collected at Station 7.2, with a camera length of 11 cm, an exposure time of 3 minutes, and a 200-µm diameter collimated beam. The 0.305-nm spacing in calcite was used to calibrate the data. Patterns were collected on a MAR image plate detector. Transmission Electron Microscopy The frozen tissue was thawed and then fixed overnight (12 hours) in 2.5% glutaraldehyde, 0.1 M phosphate buffer (pH 7.2) at 4° C, followed by 1.5 hours in 0.1 M osmium tetroxide at 20° C. The tissue was then dehydrated in ethanol, infiltrated with Spurr’s resin, and polymerized at 70° C for 8 hours. Ultrathin sections (50-nm) were cut and then stained with uranyl acetate and lead citrate.

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The corneal mid-stroma was studied at magnifications of 10 to 15 k on a Jeol 1010 transmission electron microscope. Histomorphometric analysis was done on an image processor. The system is equipped with a CCD camera (Kodak MegaPlus, Model 1.4i) which is able to digitize images in a 512 x 512 pixel raster in 256 gray levels. The analyzer can store 16, 8-bit gray images or binary images. Numerous mathematical morphology functions are available through an image orientated language (Imaging C) derived from Soft-imaging Software, analySIS 2.1. Once collected by the digital camera the images were “cleaned up” through a series of algorithms that reduced background noise while retaining the size and position of each fibril. The images were then binarized, which allowed fast computation of the relative fibril positions and radii within each image. This information could then be used to calculate a radial distribution function, g(r), a mathematical description of the positions of the fibrils with respect to one another and also to predict the transmission of visible light through the cornea. Calculation of the Radial Distribution Function The radial distribution, g(r), is a measure of the average number density of fibril centers at a given distance, r, from any other fibril center relative to the bulk fibril number density. A summary of the method follows but a more detailed description is given by Farrell and Hart.10 Given the complete set of fibril coordinates from a digitized electron microscope image, g(r) was calculated as follows. An arbitrary fibril center was taken as the origin of a set of annuli, radius r and thickness Dr, extending from r = 0 to about 500 nm. The number of fibril centers in each annulus was counted and divided by the area of the annulus, 2prDr. The result was a histogram of the number density of fibril centers at each distance r from the origin. The process was repeated using each fibril in turn as the origin, and the average histogram for all fibrils gave the radial distribution function. The radial distribution function rises from zero (for r = 0) upward until the nearest neighbor distance is reached, at which point there is a distinct peak. As r increases further, the function undulates gently before stabilizing to a constant value, the “correlation distance” rc. The value of g(r) for r > rc represents the bulk number density of fibrils, r, for the whole area covered and is an important parameter in calculating the percentage transmission through the cornea. Radial distribution functions were normalized to allow them to be more easily compared, Journal of Refractive Surgery Volume 16 July/August 2000


by dividing by r. The distinct peak followed by smaller undulations and eventual stability in the radial distribution function reflects the lattice structure of the cornea, with neighboring fibrils being uniformly spaced, but without any long-range order. The position of the primary peak in g(r) was used to determine the average interfibrillar spacing in electron micrographs of cross-sections of collagen fibrils in the corneas examined. The bulk fibril number densities determined from the radial distribution functions of the fibrils were used in the light scattering model, “direct summation of fields.”11 Comparison of the interfibrillar spacing determined from g(r) with the corresponding value determined from synchrotron x-ray scattering allowed any changes during processing for electron microscopy to be taken into account.6 Direct Summation of Fields Model The fraction of light transmitted through the cornea is related to the total scattering cross-section per fibril per unit length, s, by the equation: F(l) = e-rst

(Equation 1)

where t is the thickness of the stroma, r is the bulk number density of fibrils in the stroma, and s the scattering cross-section; a function of the following 1) the size of the fibrils (taken from micrographs), 2) the packing of the fibrils (taken from micrographs), 3) the refractive indices of the hydrated fibrils and hydrated interfibrillar matrix (assumed from x-ray scattering measurements6), and 4) wavelength (l). The model requires a number of assumptions. Estimates are required for the refractive indices of the fibrils (nf) and ground substance (ng). In the normal rabbit cornea, nf = 1.416.6 nf is a function of fibril hydration; if hydration increases, nf will fall. The refractive index ng has the value 1.357 in the normal rabbit cornea6 but may increase if tissue shrinkage causes the density of the interfibrillar proteins, etc. to rise, or be reduced if there is a significant loss of interfibrillar material. The degree to which the latter occurs in enzyme-treated tissue will remain unknown until the proteoglycans in the stroma have been studied in more detail. All parameters used in the calculations are assumed to be uniform throughout the tissue. The details of the calculation are described elsewhere11, so only an outline of the method is given here. Starting with an electron microscope image from the CCD camera of the fibrils in cross section, the first step was to measure the size and position of Journal of Refractive Surgery Volume 16 July/August 2000

each fibril. The area covered by the image was then divided up into a grid and the fibrils were grouped together according to which grid-element they occupied. The field scattered by each fibril depends on its radius and thus varies from one fibril to the next. The scattered fields from all the fibrils in a gridelement were superimposed, taking into account the phase differences introduced by the different fibril locations. The superimposition was used to calculate the differential cross section per fibril, ss(us). This describes the scattering at one given angle, us, for all the fibrils in one grid-element. The process was repeated for each grid-element and the ensemble average over the whole grid represents the mean differential scattering cross section per fibril for the entire stroma. Integration from us = 0 to 2p radians gives the total scattering cross section per fibril, s, from which the transmittance, Ft can then be calculated using Equation 1. Statistics Statistics were used to compare normal distributions of fibril diameters from control and enzymetreated stroma. Using the mean, standard deviation, and sample number of fibril diameters from each population (controls and enzyme-treated), a 95% confidence interval was calculated for each population mean.12 RESULTS Slit-lamp examination of the corneas and anterior segments showed no detectable increase in baseline backscattering. The paraformaldehyde and frozen preserved corneas (rabbits 1 to 4) were analyzed by synchrotron x-rays as described in the Methods section. Figure 1A shows the profiles of the Bragg spacing from the innermost low-angle, equatorial reflections of both the enzyme-treated and control corneas from frozen tissue. Figure 1B shows the low-angle equatorial results from paraformaldehyde-preserved corneas. Figure 1C shows the results from the frozen tissues that were subsequently reequilibrated. These equilibrated tissues had an average hydration (H) of 3.8 for the control tissue and 2.3 for the enzyme-treated tissue, where H = (wet weight – dry weight)/dry weight. The two enzyme-treated corneas in Figure 1A have a reduced Bragg spacing (38.0 ± 1.9 nm and 36.5 ± 1.4 nm) compared to the untreated or balanced salt solution-treated control corneas (47.0 ± 2.3 nm and 43.7 ± 2.2 nm). The small difference between the two control corneas was not significant and therefore a mean control value was calculated by

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Figure 1. The profiles of the interfibrillar Bragg spacings showing a decrease in the center-to-center fibril spacing between the mean control and enzyme-treated corneas. A) frozen then thawed enzyme-treated and control corneas. —●— control (4/L); —◆— control (4/R); —❍— enzyme-treated (3/L); —✧— enzymetreated (3/R). B) paraformaldehyde-preserved enzyme-treated and control corneas. —●— control (2/L); —◆— control (2/R); —❍— enzyme-treated (1/L); —✧— enzyme-treated (1/R). C) re-equilibrated enzyme-treated and control corneas. —●— control (4/L); —◆— control (4/R); —❍— enzyme-treated (3/L); —✧— enzyme-treated (3/R). For information on the individual corneas refer to the Table.

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averaging the Bragg spacings. The mean Bragg spacing of the enzyme-treated corneas was 18% lower than the mean Bragg spacing from the controls. Similar differences of 15% and 21% respectively were found between the enzyme-treated and untreated corneas following paraformaldehyde preservation (Figure 1B) and equilibration (Figure 1C). A similar decrease in mean interfibrillar spacing (18%) was observed from the radial distribution data obtained from the electron-optical images of frozen cornea, via image analysis (Figs 2A and 2B). With the aforementioned assumptions, we have made a first approximation as to what occurs after removal of hyaluronidase-sensitive proteoglycans from the cornea by applying the summation of fields method to predict the effect of this treatment on light scattering. Figure 3A shows the predicted transmission in the visible light range for the enzyme-treated corneas compared to a normal rabbit cornea with no injection. There is only a small reduction in expected transmission following removal of hyaluronidase-sensitive proteoglycans that may not be experimentally significant. Even if it is, with 83% of the original transmission maintained at 400 nm (specimen 3/L), no appreciative loss of vision would be expected. In theory any increase in collagen fibril diameters and/or intermolecular spacing following removal of hyaluronidase-sensitive proteoglycans would be expected to change the refractive indices, nf and ng, used in the summation of fields model above. To examine this, mean fibril diameters were measured from over 3500 fibril cross-sections in electron micrographs. The control (4R and 5L) diameters had a mean value of 37.93 nm and the enzyme-treated had a mean value of 40.93 nm. The 95% confidence interval for the control diameters was ±2.33 nm and for the enzyme-treated diameters was ±2.35 nm. These results inferred a small difference between the control and the enzyme-treated fibril diameters, but the difference was not significant. The packing of the collagen molecules within the fibrils was determined from the wide-angle x-ray pattern. The Bragg spacing was found to be 0.157 nm in all the tissues and the reflection had a similar profile in each case. This suggests that the mean

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Figure 2. A) Digitized electron-optical images of control (4/L) and enzyme-treated (3/L) cornea mid-stroma in cross-section visibly indicates a compression of the collagen fibril packing in the enzyme-treated cornea (Scale bars = 400 nm).

A

B Figure 2. B) The normalized radial distribution functions from enzyme-treated and control corneas allows an objective measurement of the observed compression. — enzyme-treated (3/R); ---- enzyme-treated (3/L); - - - control (4/L); ........ control (4/R). For information on the individual corneas refer to the Table.

A

intermolecular spacing, and hence the fibril refractive index, nf, was normal in the enzyme-treated corneas. DISCUSSION Statistical analyses were not performed because beam time restrictions at the synchrotron x-ray source limited the number of specimens that could be examined. However, x-ray scattering yields average values generated from thousands of individual interfibrillar spacings throughout each cornea and can therefore be considered with some significance. The use of ACS-005 hyaluronidase to soften the cornea for re-shaping, by breaking up the sugars in the glycosaminoglycan chain of the proteoglycans, has the effect of decreasing the interfibrillar Bragg spacing by about 18%. This was shown by


B Figure 3. A) Calculated light transmission spectra for enzymetreated and control corneas. B) Calculated light transmission spectrum for a control cornea shows the predicted effects of reducing uniformly the interfibrillar spacing by 15% and 20%, while keeping all other parameters constant.

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synchrotron x-ray scattering and by radial distribution functions from micrographs of enzyme-treated corneas and was found in all samples, regardless of the method of preservation used. It can be inferred from the lower Bragg spacings that the enzymetreated corneas reached a lower final hydration than the controls. This is consistent with the fact that the wet weights of the corneas measured immediately after excision were significantly lower in the enzyme-treated corneas (Table). It is accepted that proteoglycans present in the stroma are essential for swelling to take place to any extent13 and that this swelling is essentially uniform. Collagen fibrils in the cornea do not swell appreciably above physiological hydration.3 The swelling forces between the fibrils, however, are due to fixed charges (Donnan potentials) and the fixed charge concentration in the stroma is due, in part, to the glycosaminoglycans on the proteoglycan molecules and to transient mobile ion binding to proteins.13 Glycosaminoglycans such as chondroitin/ dermatan sulfate and keratan sulfate contribute to the fixed charge by virtue of their sulfonic acid groups (and the carboxyl groups in chondroitin/ dermatan sulfate). The reduced tissue hydration suggests that the enzyme-treated corneas have a lower overall fixed charge than the control corneas. This is supported by the observation that in the equilibrated corneas, identical osmotic conditions produced a lower final hydration in the enzymetreated cornea. Since the enzyme hyaluronidase breaks up glycosaminoglycans, the loss of fixed charge could be attributed to the action of hyaluronidase on the stromal glycosaminoglycans. A reduced swelling potential caused by this loss of fixed charge would be expected to lead to a decrease in the interfibrillar spacing, as we have observed. There are a number of possible consequences of a decrease in the interfibrillar spacing. Clearly it will cause an increase in the bulk fibril number density and affect the interference between light photons scattered by different fibrils. These changes are taken into account in the direct summation of fields model. However, the other assumptions mentioned earlier also need to be reviewed. 1) The reduction in fibril spacing might not occur uniformly throughout the depth of the stroma, which could lead to a refractive index gradient within the tissue. Corneal refractive index is known to vary slightly with depth in the normal cornea14 but

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the structural implications of this, and in particular any association with changes in the fibril packing, have not yet been investigated. 2) Our wide-angle data implies that the refractive index of the collagen fibrils themselves does not change. 3) The reduction in the interfibrillar spacing implies that the tissue thins following corneaplasty. From Equation 1, this would have the effect of reducing light scattering. A recent study has found a measurable decrease in total corneal thickness following corneaplasty.15 4) The reduction in the interfibrillar spacing might lead to an increase in the refractive index of the interfibrillar matrix. Theoretically, this would also have the effect of reducing light scattering but, in this case, the refractive index of the cornea as a whole would increase. Unfortunately, attempts to measure the refractive index of treated corneas have not provided reproducible results. The experimental results and the theoretical considerations above suggest that the important factor with respect to possible increased light scattering following corneaplasty is the change in the arrangement and spacing (bulk fibril number density) of the collagen fibrils. To see the effect of uniformly compressing the collagen fibrils, assuming that everything except the bulk fibril number density remains constant, a transmission of visible light through the enzyme-treated corneas was predicted. This was accomplished by artificially reducing the measured interfibrillar spacing from the micrographs of a normal cornea (specimen 5/L) by 15% and 20% (Fig 3B). This resulted in two curves, which gave a similar reduction in transmission to the enzyme-treated tissues in Figure 3A. This similarity indicates that the predicted small reduction in transmission following corneaplasty may be a real effect related directly to the decrease in interfibrillar spacing. To date, FDA Phase I safety studies have been completed in the United States and offshore. In addition, Phase IIa safety and efficacy studies are being conducted offshore in several dozen patients with improvement of visual acuity in all eyes. The lack of postoperative haze appears to be due to the fact that the stoma can tolerate a small compression of its constituent collagen fibrils; without this ability it would lead to a perceptible increase in light scattering. The combination of ACS-005 hyaluronidase treatment and specially designed



contact lenses, therefore, seem to offer an alternative procedure to surgery for improvement of visual acuity. REFERENCES 1. Borcherding MS, Blackik LJ, Sittig RA, Bizzell JU, Breen M, Weinstein HG. Proteoglycans and collagen fiber organization in human corneoscleral tissue. Exp Eye Res 1975;21: 59-70. 2. Worthington CR, Inouye H. X-ray diffraction study of the cornea. Int J Biol Macromol 1985;7:2-8. 3. Meek KM, Fullwood NJ, Cooke PH, Elliott GF, Maurice DM, Quantock AJ, Wall RS, Worthington CR. Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration. Biophys J 1991;60:467-474. 4. Meek KM, Leonard DW. Ultrastructure of the corneal stroma: a comparative study. Biophys J 1993;64:273-280. 5. Fratzl P, Fratzl-Zelman N, Klaushofer K. Collagen packing and mineralization. An x-ray scattering investigation of turkey leg tendon. Biophys J 1993;64:260-265. 6. Leonard DW, Meek KM. Refractive indices of the collagen fibrils and extrafibrillar material of the corneal stroma. Biophys J 1997;72:1382-1387. 7. Karageozian HL, Baker P, Kenney MC, May D, Harris A, Nesburn, A, Karageozian, VH. Intrastromal application of


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ACS-005 enzyme for reshaping of human corneas. Invest Ophthalmol Vis Sci 1996;37(suppl):S68. Fullwood NJ, Meek KM. An ultrastructural, time-resolved study of freezing in the corneal stroma. J Mol Biol 1994;236:749-758. Fullwood NJ. Ultrastructural studies of cornea and sclera. Ph.D. Thesis. The Open University, Milton Keynes, U.K.: The Open University;1992:39-40. Farrell RA, Hart RW. On the theory of the spatial organisation of macromolecules in connective tissue. Bul Math Biophys 1969;31:727-760. Freund DE, McCally RL, Farrell RA, Cristol SM, L'Hernault NL, Edelhauser HF. Ultrastructure in anterior and posterior stroma of perfused human and rabbit corneas: relation to transparency. Invest Ophthalmol Vis Sci 1995;36:15081523. Campbell RC. Statistics for Biologists. 2nd ed. Cambridge, UK: Cambridge University Press; 1974:142-144. Hodson SA. Corneal stromal swelling. Prog Ret and Eye Res 1997;16:99-116. Patel S, Marshall J, Fitzke FW. Refractive index of the human corneal epithelium and stroma. J Refract Surg 1995;11:100-105. Karageozian HL, Sebag J, Ansari RR, Shibuya R, Alba SA. Dynamic light scattering detects structural changes induced by intravitreal and intracorneal hyaluronidase injection. Invest Ophthalmol Vis Sci 1998;39(suppl):S803.

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