Ultrastructural features of corneas with pellucid marginal degeneration

Ultrastructural Features of Corneas With PellucidMarginal DegenerationSAEED AKHTAR,1* OMAR KIRAT,2 HIND ALKATAN,2 XINHUA SHU,3 AND TURKI ALMUBRAD1

1Cornea Research Chair, Department of Optometry, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia2King Khalid Eye Specialist Hospital, Riyadh, Saudi Arabia3Department of Life Sciences, Glasgow Caledonian University, Glasgow, Scotland

KEY WORDS pellucid marginal degeneration (PMD); degenerated cornea; collagen fibrils;proteoglycans

ABSTRACT PURPOSE: Pellucid marginal degeneration (PMD) of the cornea is a rare ectaticdisorder which typically affects the inferior or superior peripheral cornea in a crescentic fashion.We report histological and ultrastructural features of three PMD corneas. METHODS: The fol-lowing three patients were diagnosed with PMD corneas: (A) one 41-year-old male, (B) one 56-year-old female, and (C) one 31-year-old male. The patients underwent keratoplasty and theexcised corneas were processed for light and electron microscopy to study the ultrastructural fea-tures. RESULTS: Degenerated corneas were observed in the region adjacent to the limbus. Inthe degenerated region of the cornea, the Bowman’s layer had been replaced by collagenous pan-nus and the anterior stroma contained degenerated collagen fibrils (CFs) with very large proteo-glycans (4626420 nm2). The lamellae were fused and keratocytes appeared like fibroblast. Theprelimbal region of the PMD cornea had a degenerated Bowman’s layer and thin undulatinglamellae in the stroma. The CFs of the Bowman’s layer and the stroma were replaced by veryfine microfilaments. The mean of the minimum CF diameter was 1963 nm in London RsinWhite-embedded tissue. CONCLUSIONS: Our observations of the disorganization and degener-ation of CFs suggest that PMD could be related to a disorder in the synthesis of CF. This disorderwas more severe in the cornea adjacent to the limbus compared to the cornea further away fromthe limbus. Microsc. Res. Tech. 76:404–411, 2013. VC 2013 Wiley Periodicals, Inc.

INTRODUCTION

Pellucid marginal degeneration (PMD) is a progres-sive, noninflammatory, corneal, ectatic disorder involv-ing thinning of the inferior cornea (Krachmer, 1978;Sridhar et al., 2004). Characteristically, this thinningoccurs 1–3 mm from the limbus in the 4–8 o’clock posi-tion (Krachmer et al., 1984). This configuration causesthe cornea superior to the ectasia to protrude, causinga “beer belly” configuration, producing a flat verticalmeridian above the thinning with high against-the-rule astigmatism (Krachmer, 1978; Walker et al.,2008). A variant of this condition, superior PMD,causes similar findings in the superior cornea (Leeet al., 2008).

Ultrastructural features of the PMD cornea such asbreaks in Bowman’s layer, disorganization of collagenfibrils (CFs) in the stroma, and degeneration of Desce-met’s membrane have been reported previously (Pouli-quen et al., 1987). Corneal melt was also observed inthe eyes suffering with PMD (Kugler et al., 2011).

Hassell and Birk (2010) reported that the extracellu-lar matrix of corneal stroma consists of collagen withlesser amount of proteoglycans (PGs). The fibril-form-ing types are collagen types I, II, III, V, XI, XXIV, andXXVII. They are rod-like collagens with extensive tri-ple helical domains (ca. 300 nm) containing 990–1020amino acids. These CFs are uniformly distributed inorthogonal lamellae. This uniform distribution of CFs

is maintained by PGs and is responsible for cornealtransparency (Maurice, 1957).

In this study, we report the clinical, histological, andultrastructural features of PMD corneas. We examinedthe corneal region adjacent to the limbus and the pre-limbal region of PMD cornea. The ultrastructuralarchitecture of CFs, PGs, and keratocytes wasinvestigated.

MATERIALS AND METHODS

Tissue procurement and use was in accordance withthe Declaration of Helsinki and local regulations. Ithas been ethically approved by the Local Ethical Com-mittee; King Saud University, Saudi Arabia, and KingKhalid Eye Specialist Hospital, Riyadh, Saudi Arabia.

Patient’s DetailsPatient A. A 41-year-old male presented with grad-

ual painless decline of visual acuity in both eyes, worsein the left eye. His uncorrected visual acuity (UCVA)

*Correspondence to: Dr. Saeed Akhtar, Cornea Research Chair, Department ofOptometry, College of Applied Medical Sciences, King Saud University, PO Box10219, Riyadh 11433, Saudi Arabia.E-mail: [email protected]; [email protected]

Received 2 November 2012; accepted in revised form 5 January 2013

Contract grant sponsor: National Plan for Science and Technology, King SaudUniversity, Riyadh, Saudi Arabia.

DOI 10.1002/jemt.22180Published online 15 February 2013 in Wiley Online Library (wileyonlinelibrary.com).

VVC 2013 WILEY PERIODICALS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 76:404–411 (2013)

was 20/125 and 8/200 in the right and left eye, respec-tively. His best spectacle corrected visual acuity(BSCVA) was 20/25 and 20/125 in the right and left eye,respectively. The refraction was 11.75–5.00 X85 and12.25–5.75 X90 and the intraocular pressure was 11and 13 mm Hg, in the right and left eye, respectively.

Examination with a slit lamp revealed normaladnexa in both eyes. Both corneas were clear, with aninferior crescentic band of thinning from the 4 to 8o’clock position, with an apparently normal corneaseparating it from the limbus. The central cornea wasvertically flat with steepening over the thin area, thiswas worse in the left eye (Fig. 1A).

The patient underwent insertion of intacs intra-corneal rings in his left eye (Fig. 1B). Postopera-tively, the patient was not satisfied and therefore heunderwent explantation of the intacs and lamellarkeratoplasty.

Patient B. A 56-year-old female who presentedwith a painless gradual decline of visual acuity: morein the right eye than left eye. She had a negative pastocular and medical history.

Her examination revealed UCVA of 5/200 in theright eye and 20/100 in the left eye. Her BSCVA was20/200 in the right eye and 20/40 in the left eye. Hermanifest refraction was 212.00–11.00 X80 in the righteye and 24.00–4.00 X100 in the left eye. The intraocu-

lar pressure was 19 and 17 mm Hg in the right and lefteye, respectively.

Slit lamp examination of the right eye revealednormal lids and conjunctiva. The cornea had an infe-rior cresentic band of thinning about 1–2 mm from thelimbus, running from 4 to 8 o’clock position with over-lying steepening. The remaining cornea between thelimbus and the thin area was apparently normal(Fig. 1C). The central cornea had a mild nebular kera-toconus. Slit lamp examination of the left eye wasunremarkable. Topographic evaluation showed PMDand keratoconus in the right eye and subclinical PMDin the left eye. The patient underwent lamellar kerato-plasty to the right eye.

Patient C. A 31-year-old male who presented withpainless gradual decline of visual acuity in both eyes,worse in the right eye. His past medical and ocular his-tory were unremarkable. His examination revealedUCVA 2/200 and 20/200 in the right eye and left eye,respectively. His BSCVA is 20/200 and 20/30 in theright and left eye, respectively. The intraocular pres-sure was 15 and 11 mm Hg in the right and left eye,respectively.

Slit lamp examination revealed normal adnexae inboth eyes, both corneas had inferior cresentic area ofthinning about 1–2 mm from the limbus running fromthe 4 to 8 o’clock positions, with steepening and mildscarring. The central cornea was clear and vertically

Fig. 1. Clinical pictures of PMD. A: Slit lamp image of the left eyeof patient A, showing inferior corneal thinning (white arrow) withnormal cornea, separating it from the limbus. B: Scheimpflug imageof the left eye of patent A, after Intacs intracorneal ring implantation.The thin arrows show the segment sites and the thick arrow shows

the inferior thinning, with the normal corneal thickness, separatingit from the limbus (PMD). C: Scheimpflug image of the right cornea ofpatient B, showing the inferior thinning (large arrow), with the nor-mal corneal thickness, separating it from the limbus (small arrow),typical of PMD.

ULTRASTRUCTURAL FEATURES OF PELLUCID CORNEAS 405

Microscopy Research and Technique

flat. This situation was worse in the right eye com-pared to the left eye. Rest of the examination was nor-mal in both eyes. The patient underwent penetratingkeratoplasty to the right eye.

Tissue Processing

The tissue was divided into two parts and immedi-ately fixed in different fixatives according to which thefeatures of the cornea were under investigation.

1. To study the ultrastructural features and distribu-tion of PG, tissue was fixed in 2.5% glutaraldehydecontaining 0.05% cuprolinic blue (BDH, Dorset,United Kingdom) using a critical electrolyte concen-tration mode within 30 min of removal (Scott andHaigh, 1985). The tissue was embedded in TABB031 resin and polymerized for 24 h at 70�C. Semi-thin sections (0.7 mm) were cut for light microscopyand stained with toluidine blue. Ultrathin cross-sec-tions were cut with a Reichert-Jung UltracutVR micro-tome and collected on 200-mesh copper grids. Thesections were stained with uranyl acetate and leadcitrate for better preservation and to enhance con-trast in the cellular organelles. The sections wereobserved by transmission electron microscopy (JEOL1400 and JEOL 1011; JEOL, Akishima, Japan).

2. To study the CF diameter, corneal buttons were fixedin freshly prepared 4% paraformaldehyde10.5%glutaraldehyde in 0.1 M phosphate for 2 h at 4�Cwithin 30 min of removal. Tissues were embedded inLondon resin (LR) White and polymerized under UVlight for 24 h (Akhtar et al., 2008).

The minimum CF diameter and center-to-centerspacing were measured using the Soft Imaging System(AnalySIS, Soft Imaging System GmbH, M€unster,Germany) analysis program. We chose to measure theminimum diameter of each fibril rather than an aver-age value to avoid errors owing to any obliqueness infibril cross-sections. The area size of the PGs was meas-ured in the anterior and middle stroma of the cornea.

RESULTSLight Microscopic Observation

The epithelium was irregular and thin in some places(Fig. 2A). The Bowman’s layer below the thin epitheliumhad breaks in some places (Fig. 2B). In some places, theBowman’s layer was absent and replaced by collagenouspanus (Fig. 2C). The basal epithelial cells were vacuo-lated and lacked cell organelles (Fig. 2C). In some places,the basal cells were missing (Fig. 2D). Fibroblast-likecells were present in the stroma (Figs. 2A–2D).

Electron Microscopic Observations of the PMDCornea Adjacent to the Limbus

Electron microscopy observations showed cornealdegeneration in the region adjacent to the limbus. Inthe degenerated region, the epithelium was very thinand basal epithelial cells were flat instead of columnar(Fig. 3A). Below the epithelium, hemidesmosomeswere degenerated and the Bowman’s layer wasreplaced by disorganized CFs (Fig. 3A). In the anteriorstroma, lamellae were thin and fused to one anotherand contained degenerated CFs. There were large

Fig. 2. Light micrograph of PMD cornea. A: Part of the corneashowing degenerated epithelium, Bowman’s layer, stroma, and kera-tocytes. B: Part of the cornea showing thin epithelium and break inthe Bowman’s layer (arrowhead). C: Part of the cornea showing large

empty basal epithelial cells without cell organelles. Bowman’s layerwas replaced by fibrous pannus. D: Part of the cornea showing thatpannus is pushing epithelium. BE, basal epithelial cell; BW, Bow-man’s layer; E, epithelium; KR, keratocyte; P, pannus; S, stroma.

406 S. AKHTAR ET AL.


lucent spaces in the stroma (Fig. 3B). The CFs did nothave any shape in these regions and were associatedwith very large PGs of area size 482 nm2 (Fig. 3B). Thelamellae in the middle stroma were also very thin andcontained numerous vacuoles (Fig. 3C). The CFs inthese regions were twisted, irregularly distributed,and running in random directions (Figs. 3D and 4A).Throughout the stroma, there were very thin microfi-brils present between the lamellae junction and withinthe lamellae around the CFs (Fig. 4B). In some parts ofthe middle stroma, lamellae were undulating and CFsin these regions were sparsely distributed (Figs. 4Cand 4D). The appearance of the keratocytes was verysimilar to the fibroblast (Fig. 4C).

Electron Microscopic Observation of thePrelimbal Region of the PMD Cornea

Most of the basal epithelial cells were degeneratedand vacuolated (Fig. 5A). The basement membrane at

the subepithelial region was very thick and containedthick, electron-dense filaments (Fig. 5A). The hemides-mosomes were degenerated and the CFs of the Bow-man’s layer were replaced by patches of microfilaments(Fig. 5B). In some places, the normal Bowman’s layerwas present. Throughout the stroma, lamellae werevery thin and undulating (Fig. 5C). Between theselamellae, very large keratocytes similar to fibroblastswere embedded (Fig. 5C). These keratocytes containedthe normal cell organelles. The CFs in the lamellaewere replaced by patches of microfilaments, as seen inthe Bowman’s layer (Fig. 5D). In some places in thestroma, CFs were running in random directions(Fig. 5E). Some of the lamellae contained normal longi-tudinally running CFs decorated with PGs (Fig. 5F).

Digital Imaging

The digital image analysis of CFs showed that theminimum mean diameter was 1963 nm and

Fig. 3. Electron micrograph of periphery of PMD cornea adjacent tolimbus. A: Epithelium, basement membrane, and hemidesmosomeswere degenerated. Bowman’s layer was replaced by degenerated fi-brous pannus. B: Part of anterior stroma containing degenerated col-lagen fibrils, large PGs, and lucent spaces. C: Part of middle stroma

showing thin lamellae containing numerous vacuoles. D: Part of mid-dle stroma showing undulating collagen fibrils and a large lucentspace. CF, collagen fibrils; E, epithelium; H, hemidesmosomes; L,lamella; LU, lucent spaces; P, pannus; PG, Proteoglycans; S, stroma;V, vacuoles; arrowhead, degenerate hemidesmosome.



interfibrillar spacing 4868 nm, in all parts of thecorneal stroma (Figs. 6A and 6B).

The digital image analysis showed that PGs werevery large in size in the degenerated region of the cor-nea adjacent to the limbus compared to the PGs size inthe prelimbal part of the cornea (Figs. 6B and 6D). Themean area size of the PGs in the region adjacent to thelimbus was 4626420 nm2 compared to the PGs meanarea in the prelimbal part of the cornea, which was82661 nm2.

DISCUSSION

PMD is a rare ectatic disorder which typicallyaffects the inferior peripheral cornea in a crescenticfashion. The condition is most commonly found inmales, usually appearing between the second and thefifth decades of life, and affects all ethnicities (Jinabhaiet al., 2011). Our clinical observations showed thatpatients had a degenerated cornea at the prelimbal

region. Kugler et al. (2011) described corneal melt afterintrastromal corneal ring segments (ICRSs) wereinserted in four cases. The degeneration of the corneaof our two patients was similar to the marginal degen-eration of the cornea of the patients described byKugler et al. (2011). The patients described by Kugleret al. (2011) had degenerated corneas after ICRS im-plantation, whereas our patients had degenerated cor-neas without insertion of an ICRS implant.

The ultrastructural features of the cornea adjacentto the limbus and prelimbal area of the cornea wereremarkably different, as described in the RESULTSsection. The prelimbal part of the cornea had a degen-erated stroma which contained disorganized CFs andvery large PGs. The keratoconus cornea contained athick basement membrane with large filaments,breaks in the Bowman’s layer, and undulating lamel-lae (Akhtar et al., 2008). Similar to the keratoconuscornea, the corneal stroma of the prelimbal area of the

Fig. 4. Electron micrograph of middle stroma of PMD cornea adjacentto limbus. A: Randomly running collagen fibrils and a large lucent space.B: Numerous microfilaments replacing collagen fibrils. C: Undulating

thin lamellae containing sparsely arranged collagen fibrils. D: Cross-sec-tion of collagen fibrils showing large spacing between collagen fibrils.CF, collagen fibrils; L, lamella; LU, lucent spaces; MF, microfilament.



Fig. 5. Electron micrograph of prelimbal region of the PMDcornea. A: Part of epithelium showing degenerated hemidesmo-somes, thick basement membrane containing electron-densefilaments, and Bowman’s layer. B: Part of Bowman’s layer contain-ing microfilaments. C: Part of anterior stroma showing thin undulat-ing lamellae and large keratocytes. D: Part of anterior stromallamellae containing microfilaments which replaced collagen fibrils.

E: Randomly running collagen fibrils in the middle stroma. F: Longi-tudinally running collagen fibrils decorated with proteoglycans inmiddle stroma. BM, basement membrane; BW, Bowman’s layer; CF,collagen fibrils; E, epithelium; F, filaments; H, hemidesmosomes;KR, keratocyte; L, lamella; MF, microfilament; PG, proteoglycans; S,stroma.



PMD cornea had also a thick basement membranewith large filaments, breaks in the Bowman’s layer,and undulating lamellae. These features of the PMDcornea were different from the normal cornea whichhad a thin basement membrane, normal Bowman’slayer, and parallel running lamellae. Numerous lucentspaces were observed in the PMD stroma. The degen-eration of CFs and PGs causes deregulation of cornealhydration and excess water is trapped in the stroma toform lakes. These lakes appeared as lucent spaces inthe stroma under the electron microscope. The lucentspaces were not artefacts. The lucent spaces wereobserved in the stroma of bullous keratopathy andFuch’s dystrophy.

The CF diameters of PMD cornea (1963 nm in LRWhite resin) were smaller compared to the normal cor-neal diameters (2462nm in LR White resin) describedby Akhtar et al. (2008). The PG mean area (4626420nm2) was larger compared to the normal corneal PGsmean area (92 nm2) described by Akhtar et al. (2008).The ultrastructural disorganization of CFs in the PMDcornea could be owing to the deregulation of PGs.

The PGs in the corneal stroma are members of thesmall leucine-rich proteoglycan (Linsenmayer et al.,1990) gene family and are thought to regulate collage-nous matrix assembly in connective tissue because oftheir bifunctional character: the protein moiety thatbinds CFs and the highly charged hydrophilic glycos-aminoglycans that regulate interfibrillar spacing (Has-sell et al., 1983; Iozzo, 1997; Linsenmayer et al., 1990).In addition to interactions with CFs, corneal stromalPGs also play a role in corneal hydration owing to thehighly negative charge of their sulfated carbohydratemoieties and the glycosaminoglycan chains (Liu et al.,2003).

Keratocan, lumican, and mimecan/osteoglycin arethe major keratan sulfate-containing proteoglycans(KSPG) in vertebrate corneal stroma. KSPGs areuniquely abundant in the cornea and have long beenthought to be essential for corneal transparency. Lumi-can constitutes only about half of the corneal KSPGs(Carlson et al., 2005). It has been reported that micelacking lumican revealed an age-dependent cornealopacity and a high proportion of abnormally thick CFs

Fig. 6. Electron micrograph of PMD cornea. A: Electron micrographof collagen fibrils of PMD corneal stroma. B: Digital image obtainedafter processing the image shown in (A), indicating the presence ofthe collagen fibrils of different diameters. C: Electron micrograph ofthe proteoglycans of anterior stroma of PMD cornea. D: Digital imageobtained after processing image shown in (C), implying that PGs are

large in size. CF, collagen fibrils; PG, proteoglycans. Collagen fibril di-ameter: Red, 10–20 nm; Green, 20–30 nm. Proteoglycans area size:Red, 50–350 nm2; Green, 350–650 nm2. Blue, 650–950 nm2; Yellow,950–1,250 nm2; Aqua, 1,250–1,550 nm2; Pink, 1,550–1,850 nm2;Brown, 1,850–2,150 nm2; and Dark green, 2,150–1,450 nm2.



in the corneal stroma (Chakravarti et al., 1998, 2000;Saika et al., 2000). It has been suggested that a criticallevel of small PGs, that is, lumican and keratocan, isessential for CFs organization but that overabundanceis not detrimental to extracellular matrix morphogene-sis (Carlson et al., 2005). The disorganization of CFs inthe PMD cornea could be related to the disorder oflumican and keratocan, which are essential for CForganization.

Funderburg et al. (1990) investigated the presenceof keratan sulfate by immunohistochemistry. Theyreported that the intensity of keratan sulfate wassignificantly reduced in PMD corneas. The KSPGs,including keratocan, play a unique role in maintainingthe appropriate corneal shape to ensure normal vision(Liu et al., 2003). Khan et al. (2005) carried out studieson keratocan gene (KERA) mutation on a PMD patientand suggested that mutation disrupts the leucine-richrepeats function.

This study also showed the presence of microfila-ments which replaced the CFs in the stroma andaggregated in the interlamellar junction. These micro-filaments had been observed in bullous keratopathyand were strongly labeled with antibody against thebig-h3 protein (keratoepithelin) (Akhtar et al., 2001).The big-h3 protein is closely associated with collagenVI (Hirano et al., 1996). It could be possible that thesemight cause disruption in the formation of collagen VIin the pellucid cornea.

The major fibril-forming collagens of the adult cor-neal stromal ECM are types I and V (Birk et al., 1986).The collagen fibrils of the corneal stroma are hetero-typic fibrils: both of these two collagen types are pres-ent in each collagen fibril. Type V is involved ininitiating fibril assembly (Wenstrup et al., 2004, 2006)and regulating fibril diameter (Birk et al., 1990). Itcould be possible that synthesis and organization ofcollagen types 1 and V was severely disturbed andcaused degeneration of CFs in pellucid corneal stroma.

Our ultrastructural studies of PMD corneas suggestthat degeneration of the stroma could be owing to adisorder of the KSPGs lumican and keratocan. Thederegulation of the KSPGs might disturb the uniformarrangement of the CFs and that disturbance mightlead to the degeneration of the cornea.

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Ultrastructural features of corneas with pellucid marginal degeneration