doi: 10.1136/bjo.2009.174409 2010 94: 933-939Br J Ophthalmol

 Rita Mencucci, Iacopo Paladini, Brunilda Brahimi, et al. microscopic studyrecurrent corneal erosion: an electron Alcohol delamination in the treatment of

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Alcohol delamination in the treatment of recurrentcorneal erosion: an electron microscopic study

Rita Mencucci,1 Iacopo Paladini,1 Brunilda Brahimi,1 Ugo Menchini,1

Harminder S Dua,2 Paolo Romagnoli3

ABSTRACTAim To investigate by electron microscopy the plane ofseparation of the epithelial sheet from its substratum inthe procedure of alcohol delamination (ALD) in patientswith recurrent corneal erosion syndrome.Methods Ten cases of recurrent corneal erosions (RCE)secondary to trauma and seven cases related tomapedotefingerprint dystrophy (MDFP) were treatedwith ALD. The epithelial sheets obtained from thesepatients were examined by transmission electronmicroscopy. Similarly sheets obtained from 20 patientsundergoing photorefractive keratectomy (10 bymechanical removal and 10 by ALD) were also examinedas control group. Five further corneal buttons obtained atkeratoplasty were treated with ALD and the epithelialsheet and corresponding stroma were both examined.Results In all specimens, whether removedmechanically or by ALD, the intercellular surfaces did notshow any disruption and desmosomes were preserved.In patients with traumatic RCE and in corneal buttonsobtained at keratoplasty, tissue separation occurredalong the lamina lucida, whereas in patients with MDFPthe whole basal lamina was removed along with theepithelium. Focal areas of basal cell degeneration andepithelial detachment from the basal lamina were alsonoted.Conclusions ALD enables efficient removal of theepithelium with an almost complete preservation of thelamina densa in traumatic RCE. In RCE due to MDFP theepithelium separates from the stroma below the basallamina and may reflect the pathology of the condition.

INTRODUCTIONRecurrent corneal erosion (RCE) is characterised byepisodes of spontaneous breakdown of the cornealepithelium due to defective adhesion to the base-ment membrane.1

It is believed that failure of the epithelial cells toregain and maintain tight adhesive contacts withthe underlying stroma following injury plays a pivotalrole in the pathogenesis of traumatic RCE. Thereare several theories about pathogenesis, includinghemidesmosome weakness, alterations of thecorneal basement membrane, in particular dupli-cation and thickening, and activation of matrixmetalloproteinases as part of an inflammatoryprocess.2 3

The vast majority of patients develop RCEfollowing mechanical trauma involving thebasement membrane, while in some patients itmay be secondary to a primary dystrophy of theepithelial basement membrane also known asmapedotefingerprint dystrophy (MDFP).4 MDFP

is regarded by some authors to be related to anautosomal dominant genetic defect and by othersas a non-hereditary corneal degeneration.4 5 Thedisease is bilateral and frequently asymmetric, mostfrequently asymptomatic but sometimes causingpain. On clinical examination it is characterised byone or more features of map-like subepithelialpatches (maps), epithelial microcysts (dots) andsubepithelial ridges (fingerprints).6 Typical findingsat histology are an abnormal basement membraneprotruding into the epithelium and intraepithelialmicrocysts.7

Slit-lamp examination of RCE reveals punctateepithelial erosions in milder cases (microformerosions) and a frank epithelial defect or a large areaof oedematous non-adherent epithelium in severecases (macroform erosion).8

Mild episodes of RCE can often be successfullymanaged by topical lubrication alone, but thereare cases in which conservative measures fail.A number of treatments have been advocatedfor this group of patients, all of which reporta high degree of success. These include soft, high-water-containing bandage contact lenses (BCL),9

anterior stromal puncture,10 neodymium-doped:yttrium aluminium garnet (ND:YAG) laser punc-ture,11 superficial keratectomy with a diamondburr12 and phototherapeutic keratectomy (PTK).13 14

These treatments have all been used successfully,but are limited by the risk of scarring at the vis-ual axis, refractive changes or by cost andavailability.14

Delamination of the corneal epithelium withdilute alcohol has become a valuable alternative forthe treatment of recurrent corneal erosions. Itseems to be a quick, safe and economical procedurethat can achieve excellent clinical results.15 Treat-ment needs to be undertaken in a controlledmanner because the corneal epithelium is quitesensitive to alcohol damage, which is rapidlyevident when contact between 20% ethanol andthe epithelium is maintained for more than 30 s,as demonstrated in laboratory mammals andhumans.16 17 The effects of alcohol on the epithe-lium being treated, in relation to the underlyingpathology, are poorly defined despite its commonusage in the laser epithelial keratomilieusis (LASEK)procedure and in treating RCE.Espana et al studied normal corneas (without

RCE) and provided immunohistochemical evidencethat upon alcohol delamination (ALD) the basallamina, as indicated by laminin 5 staining, is almostentirely left on the corneal side while the basal cellmembrane of the epithelium, as indicated byintegrin b 4 staining, is removed almost entirely

1Department of Ophthalmology,University of Florence, Italy2Department of Ophthalmology,Queen’s Medical Centre,University Hospital, Nottingham,UK3Department of Anatomy,Histology and ForensicMedicine, University ofFlorence, Italy

Correspondence toDr Rita Mencucci, Departmentof Oto-Neuro-OphthalmologicalSurgical Sciences e Eye Clinic,Viale GB Morgagni 85, 50134Florence, Italy;rita.mencucci@unifi.it

Accepted 19 February 2010

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with the epithelial flap. Collagen VII, the constituent ofanchoring fibrils, remains on the corneal side.18

Electron microscopic examination of epithelium removedwith ALD from corneas, both affected or unaffected by RCE, hasshown separation of the epithelium from the stroma at the levelof the lamina lucida, with apparently intact hemidesmosomeson the basal epithelial surface.8 19 20 Proteinaceous or cellulardebris underneath the epithelium has also been demonstrated inpatients with RCE. The path followed by alcohol to reach thebasal layer and the relationship between the underlying diseaseand the response to alcohol application are still undetermined.To study these issues, we used electron microscopy to examine:the epithelium following ALD to treat RCE either traumatic orrelated to MDFP, the epithelium removed by mechanicaldebridement and by ALD in cases undergoing PRK and cornealbuttons treated by ALD removed at keratoplasty. The exami-nation focused on the epithelial cells and the plane of cleavage.

PATIENTS AND METHODSSeventeen subjects with RCE who had not responded toconservative measures were treated by ALD (seven men and tenwomen, mean age 3467.9 years). Ten of them had traumaticRCE (five men and five women, mean age 3065 years) and sevenhad RCE related to MPDF (two men, five women, mean age3864.4 years). The control group consisted of 20 patientsundergoing photorefractive keratectomy (PRK) for refractivecorrection (ten men and ten women, mean age 27.964.2 years).In ten of them the epithelium was removed mechanically and inten by ALD. Furthermore, five corneal buttons removed frompatients undergoing keratoplasty for traumatic leucoma (fourmen and one woman, mean age 36.561.5 years) were treated byALD taking care to ensure that the delaminated epithelium wasoutside the area of leucoma. A 4 mm disc of epithelium wasremoved. The technique has been described previously.8

All procedures were carried out in an operating room undertopical anaesthesia with oxibuprocaine cloridrate 0.4% (Novesina0.4%; Novartis Farma, Origgio, Italy) and instillation of 5%betadine into the conjunctival sac. Mechanical debridement wasperformed using a blunt spatula. For ALD, absolute ethyl alcoholwas diluted to 20% with sterile water in a 1 ml syringe. A circularwell sufficient to encompass the area of erosions (optical zonemarker, 4e6 mm in diameter; Bausch and Lomb, Kingston-upon-Thames, UK) was placed onto the cornea and held with firmdownward pressure. A few drops of 20% alcohol were placedinside the well to cover the entire treatment area and left in placefor 30 s. The alcohol was then drained with a surgical sponge(K-Sponge-Katena Products, Denville, New Jersey, USA) and thewell removed from the cornea. The surface of the eye was irri-gated with balanced salt solution to wash away residual alcohol.Using a fresh, dry surgical sponge, the corneal epithelium wasremoved from the treated area as a single sheet. The cornealsurface was gently irrigated again with balanced salt solution andthe BCL was inserted. The patients were treated with netilmicindrops 0.3% (Nettacin 0.3%, preservative free; SIFI, Catania, Italy),four times a day, and topical lubricants (Optive UD, preservativefree; Allergan Italia, Rome, Italy) four times a day. Topicalmedication was discontinued and the BCL removed after about2 weeks. Corneal buttons were also similarly treated with alcohol.

All removed epithelial sheets and the denuded corneal buttonswere immediately fixed with Karnovsky’s mixture in 0.1 mol/lcacodylate buffer, pH7.4, osmicated and embedded in epoxy resin.One to two micrometer thick sections were stained with alkalinetoluidine blue and observed at light microscopy to select areas forelectron microscopic analysis. Thin sections (about 70 nm) were

stainedwith uranyl acetate and lead citrate and observed in a 1010electron microscope (Jeol, Tokyo, Japan) at 80 kV.

RESULTSGeneral features of the corneal epitheliumIn all cases the corneal epithelium appeared as a multilayeredsheet (figure 1A), with flat superficial cells joined by tightjunctions and many desmosomes between cells in all layers(figure 1B). The cells were rich in tonofilaments, which formeda mesh filling most of the cytoplasm without aggregating intofibrils except in the immediate proximity to desmosomes.

Mechanical debridement versus ADL of control specimensUpon mechanical debridement the basal epithelial layer wasvariably preserved. In most areas it appeared intact witha continuous plasma membrane and hemidesmosomes on thebasal cell surface (figure 1C). In very few focal areas there weresmall patches of basal lamina adhering to the basal cell surface.However, proteinaceous material and cell blebs were often seenbeneath the epithelium (figure 1A,C). The basal cell desmosomeswere as well preserved (figure 1B) as in the superficial cell layer(figure 1A).The corneal epithelium in cases that underwent ALD during

photorefractive keratectomy and in corneal buttons appearedsimilar to that seen upon mechanical debridement, with preser-vation of desmosomes (figure 2A). Here, too, the plasmamembrane of the basal cellswas intactwith a few small patches ofadherent basal lamina (figure 2B). Peculiar features were inho-mogeneous cell density and nuclear alterations in some areas. Inparticular, dark cells were seen in the most superficial layers,intermediate electron dense cells deeper in the epithelium and palecells even deeper; all these cells were rich in tonofilaments anddemonstratedwell preserved desmosomes (figure 2A). In the sameor other areas, detachment of chromatin from the nuclear enve-lope was shown by the appearance of a halo of relatively cleargranular material between the nuclear envelope and the mostperipheral chromatin clumps (figure 2C). The stromal bed of thecorneal buttons from whence the epithelium was removed byALD showed the Bowman layer delineated by the lamina densaover most of its surface (figure 2D). Occasional anchoring fibrilswere attached to the deep aspect of the lamina densa.

ADL for traumatic RCEEpithelial specimens obtained by ALD from cases of traumaticRCE appeared similar to those from ALD-treated controls (figure3A), with all cells rich in tonofilaments and well-preserveddesmosomes (figure 3B). The basal lamina did not come off withthe epithelium (figure 3C) except where small patches of basallamina remained attached to the epithelium; it was made up ofa single or a few anastomosed layers of basal lamina. A fewanchoring fibrils could be seen extending from the deep aspect ofthe basal lamina (figure 3D). Well-preserved hemidesmosomeswere visible on thebasal surface of thebasal cell layer (figure 3C,D).Electron-dense material and vesicular debris were presentunderneath the basal cell layer in a few areas. Here too,alcohol-induced artefacts were sometimes seen, that is, inho-mogeneous cytoplasm density among cells and chromatindetachment from the nuclear envelope. Moreover, peculiaralterations in these samples were epithelial atrophy andinterruptions of the basal cell layer in some areas (figure 4A),while in other areas there was lack of hemidesmosomes andan irregular profile of the deep surface of the basal cellsincluding thin projections consistent with the appearance of

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filopodia or lamellipodia, with intact basal cell membrane andno signs of degeneration in the cytoplasm (figure 4B).

ADL for RCE in MDFPUpon ALD for RCE in MDFP a thick ($5 mm) layer was seenattached beneath the basal cell layer (figure 4C,D). This layerwas composed of several, incomplete, anastomosing layers ofbasal lamina attached to many anchoring fibrils. The basallamina close to the epithelium showed several, small interrup-tions and the basal cells showed several hemidesmosomes someof which were separated from the basal lamina (figure 4D).There were foci of intercellular oedema, with disruption ofdesmosomes and vesicular debris (figure 4C).

DISCUSSIONConventionally, the term ‘basal lamina’ is used to describe theelectron microscopic features of the extracellular matrix onwhich the epithelial layers sit. It is made of an electron lucid

superficial layer, the lamina lucida, composed largely of theglycoprotein laminin and an electron-dense deeper layer, thelamina densa, composed largely of type IV collagen. Both theselayers are secreted by the epithelial cells. Deeper to the laminadensa is a ‘lamina reticularis’, which contains fibronectin andother connective tissue elements. In squamous epithelia, thereticular lamina is secreted by the underlying stromal cells.These separate layers are indistinguishable on light microscopyand the laminae lucida, densa and reticularis are togetherreferred to as the basement membrane.This study has demonstrated that there are some differences

in the morphology of the epithelial sheets removed by ALD inpatients with trauma-related RCE and those with MDFPdystrophy. In traumatic RCE the cleavage plane was primarilybetween the basal cells and the basal lamina. These findingswere similar to those seen in the control samples obtained frompatients who did not have RCE but were being treated ina similar manner with PRK (LASEK). Hemidesmosomes

Figure 1 Transmission electronmicroscopy of corneal epithelium uponmechanical debridement. (A) Fullthickness epithelium with electrondense, presumably proteinaceousmaterial underneath (asterisk). Scalebar, 2.5 mm. (B) Detail of well preservedlateral cell membranes withdesmosomes. Scale bar, 0.25 mm.(C) Detail of basal cell layer, withhemidemosomes, proteinaceous debris(asterisk) and cell blebs (arrowhead)underneath. Scale bar, 0.5 mm.

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remained attached to the epithelial sheet removed. This alsoconcurred with findings seen in corneal buttons removed atkeratoplasty and treated with ALD. In cases with MDFPdystrophy with RCE, the thick, multilayered basal lamina wasremoved entirely with the epithelium.

Alterations in intracellular content affecting both nuclei andcytoplasm together with preservation of intercellular desmo-somal attachments would indicate that alcohol passes through,rather than in between cells to reach the subepithelial stroma.Cells of different electron density were seen in ALD specimen,with the most dense being more superficially located and withuniform density across each single cell. This finding was alike incontrol specimens and in those from RCE, so it cannot beattributed to the disease but should be related to treatment; itwould suggest that alcohol is capable of passing through cellsand that the cell membrane allows permeation of alcohol whilst

retaining its morphological integrity. The patchy nature of thechanges observed would imply that the penetration of alcoholwas not uniform across the area of contact resulting in variableexposure of cells under the area of contact.Examination of the corneal buttons and of the epithelial

sheets removed both from controls and from cases of RCEshowed that the lamina densa was not removed with theepithelium but remained attached to the underlying stroma.This indicates that epithelial delamination took place in generalat the level of the lamina lucida of the basal lamina. Althoughthis was the dominant picture there were areas where some cellsappeared damaged with accumulation of extracellular proteina-ceous material and cell debris underneath. In other areasdelamination occurred beneath the basal lamina, whichremained attached to the epithelium. These inequalities in thelevel of delamination could be related to the effect of alcohol or

Figure 2 Transmission electronmicroscopy of corneal epithelium uponalcohol delamination (ALD). (A) Detail ofwell preserved lateral cell membraneswith intact desmosomes. Scale bar,0.25 mm. (B) Area of basal cell layerwith attached basal lamina down to thelamina reticularis with anchoring fibrils(arrows); the arrowhead points toa hemidesmosome. Scale bar, 0.25 mm.(C) Alcohol-induced artefacts:detachment of chromatin from thenuclear membrane in a nucleus (N), anddark cells (above) alternating with clearcells (below; diamond). Scale bar,0.6 mm. (D) Corresponding stromalsurface of a corneal button from whichthe epithelium was removed by ALD,showing the lamina densa (thin arrows).The star indicates the previous locationof the epithelium. Scale bar, 0.6 mm.

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the mechanical intervention required to physically remove thealcohol treated sheet. There was no correlation observedbetween the level of delamination and the intracellular alter-ations noted such as non-uniform electron density of cells andseparation of chromatin from the nuclear envelope. This wouldindicate that the effects of alcohol on the cells and on the basallamina are independent of each other. These features were seenboth in control specimens and in those from RCE, so they couldnot be attributed to the disease but rather should be ascribed tothe delamination procedure. It was also of interest to note thatmechanical debridement was also associated with changes verysimilar to those seen with ALD, but the epithelium was morefrequently damaged. This would point to a natural cleavageplane at the level of the lamina lucida. A previous study hadshown that mechanical debridement is also associated withdamage to the underlying stroma,19 therefore we suggest thatALD should be preferred to mechanical debridement for thetreatment of RCE, judging from a histological point of view.

Our electron microscopic findings, as far as controls areconcerned, match well with the immunohistochemical findingsreported by Espana et al.18 These authors did not analyse cases ofRCE. On the other hand our findings do not correlate with those

reported by Chen et al who examined, by immunohistochem-istry, epithelial sheets removed mechanically from corneas withRCE.21 The found laminin 5 (lamina lucida and densa) andcollagen VII (lamina reticularis) in the epithelial sheet suggestingthat their mechanical effort was more forceful and took them toa deeper plane. Retention of the lamina densa on the denudedstroma, as is normally the case with ALD, would leave behinda smooth surface facilitating the migration and adhesion ofregenerating epithelial cells.The occasional finding in RCE, but not in control specimens,

of cells with intact basal cell membrane but devoid of hemi-desmosomes and with an irregular profile including thinprojections of filopodia or lamellipodia (the distinction betweenthe two being impossible at electron microscopy) may beinterpreted as an expression of a repair processes consequent toan erosion, with cells still able to move and not yet firmlyattached to the basal lamina. In particular, the lack of hemi-desmosomes in the presence of an intact basal cell membraneand in the absence of alterations in the cytoplasm is compatiblewith cell migration and reshaping but not with cell pathology.The limited areas of epithelial atrophy should be indicative ofincomplete repair of previous erosions.

Figure 3 Transmission electronmicroscopy of corneal epitheliumremoved by alcohol delamination (ALD)in cases of traumatic recurrent cornealerosion (RCE). (A) Full thicknessepithelial sheet. Scale bar, 6 mm.(B) Detail of well preserved lateral cellmembranes with desmosomes. Scalebar, 0.125 mm. (C) Detail of basal celllayer, with hemidesmosomes(arrowheads). Scale bar, 0.125 mm.(D) Area of basal cell layer with a thin,attached basal lamina down to thelamina reticularis with anchoring fibrils(arrows). The arrowheads point tohemidesmosomes. Scale bar,0.125 mm.

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The difference observed with MDFP dystrophy where thethickened basal lamina was removed completely with theepithelium is novel. Despite signs of degeneration of the basalcell layer with intercellular oedema, occasional cell death andhemidesmosome detachment, the basal lamina was moreadherent to the epithelial sheet than to the underlying stroma.These findings also suggest that in this type of dystrophya derangement in the molecular and ultrastructural organisationof the anterior corneal stroma may contribute to the occurrenceof erosions.

Competing interests None.

Patient consent Obtained.

Ethics approval This study was conducted with the approval of the University ofFlorence. Tissue procurement and use was carried out in accordance with thedeclaration of Helsinki and local ethics regulations.

Provenance and peer review Not commissioned; externally peer reviewed.

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16. Kim SY, Sah WJ, Lim YW, et al. Twenty percent alcohol toxicity on rabbit cornealepithelial cells. Electron microscopic study. Cornea 2002;21:388e92.

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Figure 4 Transmission electronmicroscopy of corneal epithelium fromcases of recurrent corneal erosion(RCE). (A) Traumatic RCE; area ofepithelial atrophy and interruption(double arrowhead) with proteinaceousmaterial (asterisk) and cell blebsunderneath. Scale bar, 3 mm.(B) Traumatic RCE: area of basal celllayer with intact membrane but lackinghemidesmosomes and expandinginto thin cell projections with theaspect of filopodia or lamellipodia.Scale bar, 0.3 mm. (C) Case ofmapedotefingerprint dystrophy(MDFD): the basal epithelial layershows oedema (diamonds) and singlecell degeneration (star) and hasattached a thick basement membranewith multiple layers of basal lamina(asterisk). Scale bar, 3 mm. (D) Case ofMDFD: detail of the multilayered basallamina (asterisk) with many anchoringfibrils (arrows); some hemidesmosomesare not attached to the basal lamina(arrowheads). Scale bar, 0.6 mm.

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18. Espana EM, Grueterich M, Mateo A, et al. Cleavage of corneal basement membranecomponents by ethanol exposure in laser-assisted subepithelial keratectomy.J Cataract Refract Surg 2003;29:1192e7.

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20. Pallikaris IG, Naoumidi II, Kalyvianaki MI, et al. Epi-LASIK: comparative histologicalevaluation of mechanical and alcohol-assisted epithelial separation. J CataractRefract Surg 2003;29:1496e501.

21. Chen YT, Huang FC, Tseng SY, et al. The cleavage plane of corneal epithelialadhesion complex in traumatic recurrent corneal erosion. Mol Vis 2006;12:196e204.

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