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The Influence of Lamellar Orientation on CornealMaterial Behavior: Biomechanical and StructuralChanges in an Avian Corneal Disorder

Craig Boote,1 Ahmed Elsheikh,2 Wael Kassem,2 Christina S. Kamma-Lorger,1

Paul M. Hocking,3 Nick White,4 Chris F. Inglehearn,5 Manir Ali,5 and Keith M. Meek1

PURPOSE. Retinopathy, globe enlarged (RGE) is an inheritedgenetic disease of chickens with a corneal phenotype charac-terized by loss of tissue curvature and changes in peripheralcollagen fibril alignment. This study aimed to characterize thematerial behavior of normal and RGE chicken corneas underinflation and compare this with new spatial- and depth-re-solved microstructural information to investigate how stromalfibril architecture determines corneal behavior under intraoc-ular pressure (IOP).

METHODS. Six RGE chicken corneas and six age-matched nor-mal controls were tested using trephinate inflation and theirstress-strain behavior determined as a function of posteriorpressure. Second harmonic generation mulitphoton micros-copy was used to compare the in-plane appearance and degreeof through-plane interlacing of collagen lamellae between nor-mal and mutant corneas.

RESULTS. RGE corneas displayed a 30130% increase in materialstiffness [Etangent(RGE) 0.94 0.18 MPa to 3.09 0.66 MPa;Etangent(normals) 0.72 0.13 MPa to 1.34 0.35 MPa] (P 0.05). The normal in-plane disposition of anterior collagen inthe peripheral cornea was altered in RGE but through-planelamellar interlacing was unaffected.

CONCLUSIONS. This article demonstrates changes in corneal ma-terial behavior in RGE that are qualitatively consistent withmicrostructural collagen alterations identified both herein andpreviously. This study indicates that, in general, changes instromal fibril orientation may significantly affect corneal mate-rial behavior and thereby its response to IOP. (Invest Ophthal-mol Vis Sci. 2011;52:12431251) DOI:10.1167/iovs.10-5962

The cornea is the transparent component of the eyes outerlayer. It provides a tough, protective envelope for theocular contents and, in terrestrial vertebrates, also accounts foraround two-thirds of the eyes refractive power.1 The cornealstroma is formed by the superposition of several hundredindividual lamellae that align roughly with the corneal surface,each comprising parallel, uniform-diameter collagen fibrils in ahydrated matrix.2 However, the number, thickness, and prefer-ential alignment of lamellae vary considerably between stromaldepths and regions.3 In addition, these same parameters showmarked species-dependent differences, presumably driven by theneed to balance diverse biomechanical prerequisites imposed byenvironment and behavior with the universal requirement foroptical transparency.4

Corneal mechanical behavior is heavily influenced by stro-mal collagen architecture. Since collagen fibrils are strongestaxially, lamellar anisotropy may be expected to confer direc-tion dependency on the corneal elastic modulus,5 as shownexperimentally.610 Allied to this, the degree of interactionbetween layers,11 for example, via anterior-posterior lamellarinterlacing,1214 may also impinge on the stromas ability tosupport corneal tension.15 From a clinical point of view, thestress-bearing ability of the cornea is likely to be important indetermining the topography of the anterior corneal surface andits response to disease, refractive surgery, and changes inintraocular pressure (IOP).1618 However, to what degree, ifany, collagen fibril organization influences the precise shapethat the cornea adopts in response to a given IOP remains anopen question.

Animal vision diseases in which normal eye development iscompromised can provide valuable opportunities for investi-gating the relationship between corneal structure, mechanicalbehavior, and function. Retinopathy, globe enlarged (RGE) isan autosomal recessive disease of chickens,19,20 arising from anin-frame 3-bp deletion in the cone -transducin gene GNB3,21

and is characterized clinically by progressive retinal degenera-tion and blindness by 1 month posthatch.22,23 Loss of func-tional vision triggers secondary globe enlargement and associ-ated severe flattening of the cornea.22,23 Previously we haveshown using x-ray scattering that the development of mis-shapen corneas in RGE is accompanied by in-plane alterationsin dominant peripheral collagen orientation.24,25 We also useda numerical finite-element approach to demonstrate that thesemicrostructural alterations are biomechanically consistent withchanges in tissue stress predicted from the reshaping of theanterior globe.25 However, a definitive link between micro-structural, biomechanical, and morphometric corneal changesin RGE has not yet been established, in large part because thematerial properties of the RGE cornea have not been charac-terized. The present study was designed to provide this missinginformation by comparing the behavior of normal and RGEchicken corneas under varying simulated IOP, using trephinate

From the 1Structural Biophysics Group and 4Vision Science Bio-imaging, School of Optometry and Vision Sciences, Cardiff University,Cardiff, United Kingdom; 2Ocular Biomechanics Group, School ofEngineering, University of Liverpool, Liverpool, United Kingdom; 3Ro-slin Institute and Royal (Dick) School of Veterinary Studies, Universityof Edinburgh, Roslin BioCentre, Midlothian, United Kingdom; and5Leeds Institute of Molecular Medicine, University of Leeds, St. JamessHospital, Leeds, United Kingdom.

Supported by MRC Grants No. G0600755 and G0501050, TheWellcome Trust Grant No. 074165, and Biotechnology and BiologicalSciences Research Council (BBSRC). KM is a Royal Society-WolfsonResearch Merit Award holder.

Submitted for publication May 27, 2010; revised September 9,2010; accepted October 2, 2010.

Disclosure: C. Boote, None; A. Elsheikh, None; W. Kassem,None; C.S. Kamma-Lorger, None; P.M. Hocking, None; N. White,None; C.F. Inglehearn, None; M. Ali, None; K.M. Meek, None

Corresponding author: Craig Boote, Structural Biophysics Group,School of Optometry and Vision Sciences, Cardiff University, MaindyRoad, Cardiff CF24 4LU, UK; bootec@cf.ac.uk.

Cornea

Investigative Ophthalmology & Visual Science, March 2011, Vol. 52, No. 3Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 1243

corneal inflation. In addition, second harmonic generation(SHG) multiphoton microscopy was used to compare the in-plane appearance of stromal lamellae, and their degree ofthrough-plane interlacing, between normal and mutant tissue.Our overall aim was to gain qualitative experimental insightinto the influence of collagen lamellar orientation on cornealbehavior under IOP.

METHODS

Animal Details

Layer type White Leghorn chickens are maintained at the Roslin Insti-tute (Edinburgh, UK). All husbandry and experimental techniques areperformed under a Home Office project license in accordance with theARVO statement for the Use of Animals in Ophthalmic and VisionResearch. Birds are floor-reared with a daily photoperiod of 14 hourslight (intensity, 2030 lux) and 10 hours darkness, and transferred toindividual cages at 16 weeks of age for pedigree breeding. The deri-vation and maintenance of the RGE line at Roslin has been described indetail by Inglehearn et al.22

Tissue Harvesting

Six homozygous (RGE/RGE) blind White Leghorn chickens and sixnormally sighted wild-type birds were euthanatized with an overdose ofsodium pentabarbitone at 9 months old, an age at which the globeenlargement and corneal restructuring caused by the disease have pro-ceeded to completion.25 Immediately after death the eyes were enucle-ated and the posterior sclera punctured several times with a needle toaid perfusion of the preservation medium and minimize corneal swell-ing before the experiments. The right globes were then placed insilicon oil for subsequent inflation testing, and the left eyes wereremoved to 4% paraformaldehyde and stored at 4C for microscopicassessment. The latter group also received a scleral suture to mark thesuperior corneal position.

Corneal Inflation Testing

Immediately before testing central (CCT) and peripheral (PCT) cornealthickness was measured via ultrasound pachymetry (DGH Pachmate55; DGH Technologies, Exton, PA). The average values of CCT (SD)were 334 34 m (normals) and 309 15 m (RGE). The corneaswere then excised such that approximately 3 mm of the attachedscleral rim was retained. Specimens were tested on a custom-devel-oped corneal inflation rig designed to assess corneal deformationunder simulated IOP (Fig. 1). A detailed description of this equipmentmay be found elsewhere.26 In brief, it enables uninterrupted cycles ofpreselected pressure to be applied to the posterior cornea, while thedeformation of the anterior surface is simultaneously measured. Spec-imens were connected along their rings of scleral tissue to the rigspressure chamber using only mechanical clamps and without the needfor adhesive, while allowing the cornea and limbus complete freedomto deform.

The pressure chamber was filled with a saline solution and con-nected to a reservoir whose height was computer controlled to set theapplied pressure. Chamber pressure was recorded using an FDW-seriespressure transducer (RDP Electronics, Wolverhampton, UK). Through-out the tests the saline solution in the pressure chamber was regulatedat 37C using a temperature controller and water bath (Fig. 1). Thespecimens anterior surface was wetted regularly with a viscous tearfilm supplement (Visco-tears; Novartis, Sydney, Australia) to preventdrying. The wetting took place just before the start of each newpressure cycle and involved the release of three drops onto the clamp-ing area above the cornea so that the tear film supplement would flowslowly over the speci

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