Clinical StudyProtective Effects of Trehalose on the Corneal Epithelial Cells
Pasquale Aragona1 Pietro Colosi2 Laura Rania1 Francesca Colosi1 Antonina Pisani3
Domenico Puzzolo3 and Antonio Micali3
1 Department of Experimental Medical-Surgical Sciences Regional Referral Center for the Ocular Surface DiseasesUniversity of Messina Via C Valeria 1 98125 Messina Italy
2 Department of Social and Environmental Health University of Messina Via C Valeria 1 98125 Messina Italy3 Department of Biomedical Sciences and Morphofunctional Imaging Section of Histology and EmbryologyUniversity of Messina Via C Valeria 1 98125 Messina Italy
Correspondence should be addressed to Pasquale Aragona paragonaunimeit
Received 15 February 2014 Revised 15 May 2014 Accepted 27 May 2014 Published 18 June 2014
Academic Editor Winston W-Y Kao
Copyright copy 2014 Pasquale Aragona et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Purpose Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcoholdelamination Methods Twelve patients undergoing laser subepithelial keratomileusis (LASEK) were consecutively included inthe study The right eyes were pretreated with 3 trehalose eye drops whilst left eyes were used as control Epithelial specimenswere processed for cells vitality assessment apoptosis and light and transmission electron microscopy a morphometric analysiswas performed in both groups Results In both trehalose-untreated eyes (TUE) and trehalose-treated eyes (TTE) the percentageof vital cells was similar and no apoptotic cells were observed In TUE the corneal epithelium showed superficial cells withreduced microfolds wing cells with vesicles and dilated intercellular spaces and dark basal cells with vesicles and wide cleftsIn TTE superficial and wing cells were better preserved and basal cells were generally clear with intracytoplasmatic vesicles Themorphometric analysis showed statistically significant differences between the two groups the TTE epithelial height was higherthe basal cells showed larger area and clearer cytoplasm The distribution of desmosomes and hemidesmosomes was significantlydifferent between the groups Conclusions Trehalose administration better preserved morphological and morphometric featuresof alcohol-treated corneal epithelium when compared to controls
1 Introduction
Laser subepithelial keratomileusis (LASEK) is a surgical tech-nique carried out on patients who undergo photorefractivekeratectomy (PRK) for low myopia with thin cornea orwith professions or lifestyles that expose them to traumaso contraindicating the laser in situ keratomileusis (LASIK)[1] The procedure consists of a chemical reduction of theepithelial adhesion to Bowmanrsquos layer by the application ofa dilute solution of ethanol on the corneal surface [1]
Despite the large number of works either in laboratoryanimals [2ndash4] or in humans both in normal [1 5ndash10] and inpathological eyes [11 12] the effects of alcohol on the cornealepithelium are still controversial In fact no significantchanges [1 4 6] orminimal adverse effects [2] to well-evidentdamages [3 5 7ndash9 12] have been described All these changes
were related to the action of ethanol [12] It acts by removingwater and destabilizing either the protein hydrophobic bondsthus unfolding the tertiary protein structure or the hydrogenbonds in hydrophilic areas resulting in protein denaturation[13] Furthermore alcohol penetrates the tissues and substi-tutes inter- and intracellular water consequently shrinkageand hardening of tissues can be observed [14]
In order to prevent the morphological changes inducedby alcohol on the corneal surface the protective action oftrehalose was considered Trehalose is a nonreducing dis-accharide of glucose naturally produced and accumulatedin many living organisms but not in mammals [15] It wasidentified as a key response element needed to protect thecells against a great number of environmental stresses suchas desiccation dehydration cold heat and oxidation [13]Among these functions the protection against desiccation
Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 717835 9 pageshttpdxdoiorg1011552014717835
2 The Scientific World Journal
was widely studied in ophthalmic research as exogenoustrehalose protects corneal epithelial cells from experimentaldrying [16] and was shown to be effective in the treatment ofmoderate to severe human dry eye [17] Furthermore duringdesiccation in vivo it was also demonstrated that trehalosecould effectively suppress apoptotic cell death on the ocularsurface [18]
Aim of the present work was to compare the structureand the ultrastructure of the corneal epithelium in patientsundergoing alcohol delamination with and without trehalosepretreatment
2 Materials and Methods
21 Study Design This is an experimental controlled studyon a model of corneal epithelial alcohol delaminationcurrently used in some refractive surgery procedures Itwas carried at the Regional Referral Center for the Ocu-lar Surface Diseases of the Department of ExperimentalMedical-Surgical Sciences of the University Hospital ofMessina Messina Italy Ethics approval was granted by theInstitutional Review Board of the Department of Experi-mental Medical-Surgical Sciences of the University Hospitalof Messina Messina Italy and the study was conducted inconcordance with the tenets of the Declaration of HelsinkiInformed consentwas obtained fromall the participants afterexplanation of the nature and the possible consequences ofthe study
22 Patients Population The epithelial specimens wereobtained from 24 eyes (12 patients 7 male and 5 femalemean age 263 plusmn 42 years) with a refractive error of minus4 plusmn 28diopters undergoing PRK Inclusion criteria were subjectseligible for refractive surgical procedure with myopia in botheyes below 7 diopters willing to participate to the study andto adhere to the study protocol and who signed the informedconsent Exclusion criteria were systemic or ocular diseasescontraindicating the refractive surgical procedure or subjectswho were not willing to adhere to the study protocol
23 Treatment Protocol Before the surgical procedure theright eyes of the patients were treated as follows a dropof a solution based on 3 trehalose (Thealoz Thea FarmaMilano Italy) followed after 5min by the instillation ofa drop of 04 oxybuprocaine hydrochloride (NovesinaNovartis Farma Origgio VA Italy) This procedure was per-formed every 151015840 for the hour before the surgical procedureso that a total of 5 drops of trehalose were instilled within thehourThe left eyeswere treatedwith only 04oxybuprocainehydrochloride instilled five times for the hour before the sur-gical procedure During the surgical treatment the epithelialspecimens were obtained as follows demarcation of a centralcorneal area with a 90mm diameter cone (J2907 JanachComo Italy) filling of the cone with a 20 solution of ethylalcohol in distilled water for 25 sec emptying of the conewitha Merocel sponge (Medtronics Merocel Mystic CT USA)washing with a solution 1 1 of balanced salt solution anddistilled water and successive drying with a Merocel sponge
The epithelial flaps were obtained by gently detaching thealcoholized epithelium with a blunt spatula and divided inthree specimens for the morphological assessment
24 Epithelial Cells Vitality Evaluation Epithelial cells vitalitywas evaluatedwith the trypan blue dye test [6] One specimenwas incubated for 21015840 in 01 trypan blue in phosphate-buffered saline (PBS) at 37∘C It was then washed severaltimes in warm PBS and incubated in Dulbeccorsquos modifiedEagle culture medium (Sigma-Aldrich Srl Milano Italy)containing 10 of fetal calf serum for 301015840 at 37∘C After afurther wash in warmPBS the specimenwas flatmounted ona slide and photographed with a Zeiss Primo Star LM Vitalcells appeared with unstained nuclei and cytoplasm whiledead cells were blue
25 Apoptosis Assay One specimen was fixed in 10formaldehyde in 02M PBS dehydrated in graded ethanolcleared in xylene and embedded in paraffin (ParaplastSPI Supplies West Chester PA USA) Paraffin blocks wereplaced in a rotarymicrotome (RM2125RT Leica InstrumentsNuszligloch Germany) and 5 120583m sections cut with disposablemetal blades (S35 Feather Safety Razor Co Osaka Japan)were cleared with xylene and rehydrated in graded ethanolAn apoptosis detection kit (Chemicon International Apop-Tag Plus Peroxidase In Situ Apoptosis Detection Kit Temec-ula CA USA) was used [19] The sections were subjected topartial digestion with proteinase K (20120583gmL) and washedin PBS Endogenous peroxidase activity was blocked with 3H2O2in PBS Thereafter the sections were incubated in an
equilibration buffer and then with the TUNEL reaction mix-ture in a humidified chamber at 37∘C for 60min They wereincubated in a stopwash buffer at room temperature andwashedwith PBSThe anti-digoxigenin conjugatewas appliedand incubation in a humidified chamber was performedfor 30min at room temperature The 3-31015840-diaminobenzidine(DAB) was then added for 10min and the slides werecounterstained with Mayerrsquos hematoxylin The sections werewashed in ethanol and xylene mounted in Permount (FisherScientific Fair Lawn NJ USA) and photographed with aZeiss Primo Star LM
26 Light and Transmission Electron Microscopy One spec-imen was immediately fixed in 25 glutaraldehyde 02Min Soslashrensenrsquos phosphate buffer (pH 72) at +4∘C for 4 hwashed with 02M Soslashrensenrsquos phosphate buffer (pH 72)and postfixed in 1 osmium tetroxide (OsO
4) in 02M
Soslashrensenrsquos phosphate buffer (pH 72) at +4∘C for 1 h Thespecimen was dehydrated in graded ethanol and acetone andflatembedded in embedding agent (Durcupan ACM FlukaSigma-Aldrich St Louis MO USA) for a better orientationSemithin sections (1 120583m) were cut with a LKB UltrotomeV ultramicrotome stained with an aqueous solution of 1toluidine blue in 1 borax and 1 pironine and viewed andphotographedwith a Zeiss Primo Star lightmicroscope (LM)From the same specimens used for LM ultrathin sectionsof gold-silver interference color were cut with a diamondknife on a LKB Ultrotome V ultramicrotome collected on
The Scientific World Journal 3
uncoated 200ndash300 mesh copper grids and stained withmethanolic uranyl acetate and lead citrate Micrographs weretakenwith a Philips CM-10 transmission electronmicroscope(TEM) at 80 kV
27 Morphometric Analysis A morphometric analysis of thecorneal epithelium in both groups of eyes was carried outon LM micrographs considering the percentage of vitalcells the number of epithelial layers the epithelial thicknessthe area of the basal cells and the optical density of theircytoplasm On TEM images the number of desmosomes(De) of superficial wing andbasal cells andhemidesmosomes(Hd) of the basal cells were evaluated
The percentage of vital cells was counted on 5 micro-graphseye obtained from 5 different nonoverlapping ran-domly chosen microscopic fields For each field a square of150 times 150 120583m was drawn with the Adobe Photoshop 801software and the cells included in the square were countedincluding among the cells touching the square border onlythose from two sides
LM data were obtained from 5 semithin sectionseye(total of 60 sectionsgroup) collecting 1 semithin sectionevery 100 all micrographs were obtained at the same magni-fication of 450x with a Zeiss Primo Star LM and processedwith a Macintosh MacBook using the Adobe Photoshop801 software All images were converted to black and whiteand brought to the final magnification of 1 000x for themorphometric analysis To avoid possible bias from obliquesectioning which would artificially increase the measuredthickness the thinnest portion of the epithelium was mea-sured in all the images On the selected images a straightline perpendicular to the epithelium was tracedThe numberof epithelial layers was counted along the line The overallepithelial thickness was expressed in 120583m and calculatedaccording to the following formula for the conversion frompixels [(length in pixels) times (10 000120583mcm)][(resolution inpixelscm) times (magnification)] As to the basal cells areaand their cytoplasmatic density 10 microscopic fields of100 120583m each were randomly chosen for every group andanalyzed with the public domain ImageJ software (ImageJhttprsbinfonihgovij) For the measurements only basalcells with well evident nuclei were chosen the area wasexpressed in 120583m2 and the cytoplasmatic density was calcu-lated (function analyze gt measure) in optical units (OU)within the levels from 0 (black) to 255 (white)
TEM morphometric analysis was performed on negativefilms (Kodak 4489) obtained from 5 ultrathin sectionseyeat the same voltage exposure time and magnification Thenegative films were acquired (ratio 1 1) with an Epson Per-fection 4180 scanner processed with a Macintosh MacBookusing theAdobe Photoshop 801 software and converted intopositive images at the same final magnification of 18 000xThe absolute number of De was calculated along the interfacebetween adjacent superficial wing and basal cells by countingall De observed in 100 standard fields of 10 120583m each includingthe cellular membranes of two adjacent cells The absolutenumber of Hd was calculated along the interface betweenbasal cells and basement membrane by counting all Hd
observed in 100 standard fields of 10 120583m each including asingle cell
Two experienced histologists masked about the originof the specimens performed all the morphological andmorphometric evaluations All data were expressed in 120583m forlinear values in 120583m2 for cellular area and in OU for opticaldensity between 0 (black) and 255 (white)
28 Statistical Analysis Primary efficacy variables were theepithelial thickness the number of De the area of the basalcells and the optical density of the basal cells cytoplasmSecondary parameters were the number of layers and thenumber of Hd For the analysis of the results the Studentrsquos119905-test for unpaired data was used A 119875 value of le005 wasconsidered statistically significant
3 Results
31 Epithelial Cells Vitality Evaluation and Apoptosis AssayThe trypan blue dye test demonstrated that trehalose-untreated eyes (TUE) (Figure 1(a)) showed a higher amountof not vital cells stained in blue with the dye than trehalose-treated eyes (TTE) (Figure 1(b)) (118plusmn08versus 144plusmn09resp 119875 lt 0001)
The apoptosis assay in both TUE (Figure 1(c)) and TTE(Figure 1(d)) failed to demonstrate positive cells
32 Structural and Ultrastructural Data The corneal epithe-lium obtained from TUE and observed in semithin sectionsat light microscopy showed superficial cells with variableshape and optical density round wing cells with not clearlyevident intercellular borders and basal cells with hyper-chromic nuclei and wide intercellular spaces (Figure 2(a))With TEM corneal superficial cells were flat and showed fewshort irregular microfolds and dilated intercellular spaces(Figures 2(b) and 3(a)) Wing cells showed no apparentchanges whilst intercellular spaces were moderately dilated(Figures 2(b) and 3(b)) Basal cells had elliptical nuclei withevident nucleoli dark and vacuolated cytoplasm and dilatedintercellular spaces (Figures 2(b) and 3(c))Their inferior poleshoweddense cytoplasm fewHd and someblebs surroundedby aggregates of granular material (Figures 2(b) and 3(d))
In the corneal epithelium obtained from TTE observedin semithin sections at light microscopy superficial cellsappeared flat with well-evident intercellular borders wingcells were polygonal with well-evident intercellular bordersand basal cells had polygonal shape and evident intercellularspaces (Figure 2(c)) With TEM corneal superficial cellswere flat and showed regularly arranged microfolds andwell-preserved intercellular borders (Figures 2(d) and 3(e))Wing cells showed well-evident cytoskeleton and regularintercellular borders glued by many De (Figure 2(d)) Occa-sionally some wing cells otherwise of normal morphologyshowed an apparently double nucleus (Figure 3(f)) Basal cellsshowed a clear cytoplasm with only few vesicles a roundeuchromatic nucleus small intercellularwidenings and somedesmosomes (Figures 2(d) and 3(g)) Their inferior poleshowed clear cytoplasm and numerous Hd connected to thelamina lucida of the basement membrane (Figure 3(h))
4 The Scientific World Journal
(a) (b)
(c) (d)
Figure 1 (a b) Light micrographs of trypan blue dye test for epithelial cells vitality In both TUE (a) and TTE (b) positive dead cells (arrow)can be observed Scale bar 80 120583m (c d) Light micrographs of TUNEL reaction of corneal epithelium In both TUE (c) and TTE (d) nopositive cells can be observed Scale bar 30 120583m
Table 1 Morphometric parameters of the corneal epithelium in trehalose-untreated eyes (TUE) and trehalose-treated eyes (TTE)
TTE 68 plusmn 07 577 plusmn 41lowast 271 plusmn 31lowast 244 plusmn 51 94 plusmn 16daggerdagger 63 plusmn 08 = 57 plusmn 1lowast 24 plusmn 21lowast 209 plusmn 24lowastDaggerlowast119875 lt 00001 versus TUEdagger119875 lt 00001 versus the other cell junctions within the TUE groupdaggerdagger119875 lt 00001 versus the other cell junctions in TTE group=119875 = 004 versus darkdark cell junctions in TTE groupDagger119875 lt 00001 versus clear cells of the same group
33 Morphometric Data The results of the morphometricanalysis within each group showed (Table 1) that for basalcells in TUE the number of De was 92 plusmn 11 betweenclearclear cells 57 plusmn 1 between cleardark cells and 42 plusmn1 between darkdark cells (clearclear versus cleardark119875 lt 00001 clearclear versus darkdark 119875 lt 00001and cleardark versus darkdark 119875 lt 00001) For Hda statistically significant difference was observed in theirnumber when clear and dark basal cells were compared(219 plusmn 29 and 181 plusmn 15 resp 119875 lt 00001) InTTE the number of De was 94 plusmn 16 between clearclearcells 63 plusmn 08 between cleardark cells and 57 plusmn 1between darkdark cells (clearclear versus cleardark 119875 lt00001 clearclear versus darkdark 119875 lt 00001 andcleardark versus darkdark 119875 = 004) For Hd a statis-tically significant difference was observed in their number
when clear and dark basal cells were compared (24 plusmn 21 and209 plusmn 24 resp 119875 lt 00001)
Comparing the two groups (Table 1) a statistically sig-nificant higher epithelial thickness in TTE was demonstrated(577 plusmn 41 120583m in TTE and 554 plusmn 35 120583m in TUE resp 119875 lt00001) As the number of De their number was statisticallysignificantly higher in TTE between superficial cells (271 plusmn31 in TTE and 201 plusmn 2 in TUE resp 119875 lt 00001) andbetween dark basal cells (57 plusmn 1 in TTE and 42 plusmn 1 in TUEresp 119875 lt 00001) As to the number of Hd present in theinferior pole of the basal cells it was statistically significantlyhigher in TTE in both the clear (24plusmn21 in TTE and 219plusmn29in TUE resp 119875 lt 00001) and the dark cells (209 plusmn 24 inTTE and 181 plusmn 15 in TUE resp 119875 lt 00001)
No statistically significant differences between bothgroups were found for the number of the cellular layers and
The Scientific World Journal 5
S
W
N
S
W
V
V
VN
B
B
lowast
lowastlowast
lowast
lowast
(a) (c)
(b) (d)
Figure 2 (a) Light micrograph from a semithin section of the corneal epithelium obtained with alcohol delamination in TUE Superficialcells show variable shape and optical density (arrow) basal cells have hyperchromic nuclei and large intercellular spaces (double arrow)Scale bar 25 120583m (b) TEM micrograph of the corneal epithelium obtained with alcohol delamination in TUE Superficial cells (S) show areduced number of apical microfolds (arrow) In the wing cells (W) intracellular vesicles and slightly dilated intercellular spaces (lowast) areevident Basal cells (B) are irregular in their shape and size and show large intercellular spaces (lowast) and a cytoplasm filled with vesicle (V)Many blebs (arrowhead) surrounded by granular material are evident Scale bar 5 120583m (c) Light micrograph from a semithin section of thecorneal epithelium in TTE Both superficial and wing cells show normal shape and well-evident intercellular borders (arrow) basal cells havepolygonal shape andmany small intracytoplasmatic vesicles (double arrow) Scale bar 25120583m (d) TEMmicrograph of the corneal epitheliumin TTE Superficial cells (S) show a normal flattened shape with well-evident apical microfolds (arrow)Wing cells (W) have uniform electrondensity and normal intercellular spaces (arrowhead) Basal cells (B) have variable electron density polygonal shape few cytoplasmatic vesicles(V) round nuclei (N) and small intercellular spaces (lowast) Scale bar 5 120583m
for the number of De between wing cells clearclear basalcells and cleardark basal cells
When the basal cells area was evaluated a statisticallysignificant difference was found between TUE and TTE(1994 plusmn 421 120583m2 and 2583 plusmn 515 120583m2 resp 119875 lt 00001)(Figure 4)
Considering the optical density of the cytoplasm of thebasal cells a statistically significant difference was foundbetween TUE and TTE (1255 plusmn 24OU and 781 plusmn 214 resp119875 lt 00001) (Figure 5)
4 Discussion
Diluted ethanol is currently used in patients undergoingLASEK to reduce the adhesion of the corneal epitheliumto Bowmanrsquos layer in order to expose the stroma for thesubsequent PRK treatment The theoretical advantage ofLASEK is derived from the repositioning of the epithelialflap over the laser-ablated corneal surface so facilitating thecorneal epithelial healing reducing pain and inflammationand decreasing the stromal haze [4]
6 The Scientific World Journal
N
NN
NN
t
n
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 3 (a) TEM micrograph of superficial cells from the corneal epithelium obtained with alcohol delamination on TUE The cells areflat and show short and irregular microfolds (arrow) and dilated intercellular spaces (lowast) Scale bar 5 120583m (b) TEM micrograph of wing cellsfrom the corneal epithelium obtained with alcohol delamination on TUE The nucleus (N) and the cytoplasm have normal appearanceintercellular spaces are moderately dilated (lowast) Scale bar 2 120583m (c) TEM micrograph of basal cells from the corneal epithelium obtainedwith alcohol delamination on TUEThe cells show elliptical nuclei (N) with evident nucleoli (n) dark and vacuolated cytoplasm and dilatedintercellular spaces (lowast) Scale bar 2 120583m (d) TEM micrograph of the inferior pole of a basal cell from the corneal epithelium obtained withalcohol delamination on TUE The cytoplasm is dense and few hemidesmosomes (arrows) are present Note the presence of cellular blebs(arrowhead) and of an irregular granular material Scale bar 05 120583m (e) TEM micrograph of superficial cells from the corneal epitheliumof TTE The cells are flat show regular microfolds (arrow) and are well-preserved intercellular borders (arrowhead) Scale bar 5120583m (f)TEMmicrograph of wing cells from the corneal epithelium of TTEMany tonofilaments (t) are evident around the apparently double nucleus(N1-N2) intercellular borders are normal with well-evident desmosomes (arrowhead) Scale bar 2 120583m (g) TEM micrograph of basal cells
from corneal epithelium in TTE Basal cells have round euchromatic nucleus (N) clear cytoplasm with only few vesicles small intercellularwidenings (lowast) and some desmosomes (arrow) Scale bar 2120583m (h) TEM micrograph of the inferior pole of a basal cell from the cornealepithelium in TTE The cytoplasm is clear and the hemidesmosomes (arrows) are numerous lowast = lamina lucida of the basement membraneScale bar 05 120583m
The Scientific World Journal 7
350
300
250
200
150
100
50
0 Trehalose-untreated eyes
Trehalose-treated eyes
Basa
l cel
ls ar
ea (e
xpre
ssed
in120583
m2)
1994 plusmn 421 2583 plusmn 515
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Figure 4Morphometric data of the basal cells area in TUE andTTEexpressed in 120583m2 lowast = 119875 lt 00001
255240225210195180165150135120105
907560453015
0 Trehalose-untreated eyes
Trehalose-treated eyes
781 plusmn 214 1255 plusmn 24
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Mea
n op
tical
den
sity
of b
asal
cells
cyto
plas
m(g
ray
scal
e OU
)
Figure 5 Morphometric data of the optical density of the basalcells cytoplasm in TUE and TTE expressed within the levels from0 (black) to 255 (white) optical units (OU) lowast = 119875 lt 00001
However adverse effects of alcohol on the corneal epithe-lial cells have been shown In particular the following changeswere described flattening of apical microvilli [3] and darkcytoplasm in the superficial cells [11] diffuse interruptions ofthe intercellular junctions with enlargement of the intercel-lular spaces [3 8 9] cellular edema [3] blebs of the cellularmembrane [5 8 9] autophagic vacuoles [5] damages of Hdandof the basementmembrane [4 5] and coexistence of clearand dark cytoplasm in the basal cells [5 8 11] Furthermorethe treatmentwith alcohol resulted in an increase of apoptoticbasal cells in both laboratory animals [2 4] and humans [5]
Therefore a treatment able to reduce the structural andultrastructural changes induced by the exposition to dilutedethanol characterized by the absence of side effects canbe considered useful in maintaining a healthier corneal
epithelium thus possibly improving its morphological andfunctional recovery after the surgical procedure
Trehalose a naturally occurring alpha-linked disaccha-ride formed by two molecules of glucose was consideredfor its well-known protective effects in eukaryotic cells [15]Trehalose synthesized by many living organisms but notmammals showed many different functions In anhydro-biosis that is the capability of surviving prolonged periodsof dessication a better resistance to dryness conditionswas obtained by increasing the intracellular trehalose levelsin animal cells [15] in this way proteins and membraneswere protected from denaturation [20] A reduced deathby desiccation of human corneal epithelium in culture wasobtained with the pretreatment of the cells with a 100mMsolution of trehalose [16] Furthermore trehalosemight act asfree radicals scavenger reducing the oxidative damage of thecornea caused byUVB rays and suppressing the expression ofproinflammatory cytokines [21] Finally trehalose based eyedrops were found to be effective in the treatment of moderateto severe human dry eye [17] as it reduced the number ofdead cells on the ocular surface through the suppression ofapoptosis [18]
In the present study trehalose dosage was chosen on thebasis of previous experiences [16] which demonstrated theeffect of the exposure to trehalose for 151015840 on corneal epithelialcells maintained in cultureThis procedure of administrationwas adopted as it fitted appropriately the timing of thesurgical procedures
The positive action of trehalose on the corneal epitheliumcould be demonstrated by the reduction of the morphologicchanges in TTE and by the evaluation of the morphometricdata
As to the morphological aspects structural changes wereevident in TUE In fact a reduced number and length ofthe apical microfolds of the superficial cells a dark andvacuolated cytoplasm of the basal cells a lower number ofHd and a diffuse enlargement of the intercellular spaceswere observed On the contrary in TTE superficial cellsshowed well represented microfolds and basal cells hadclear cytoplasm with few vesicles and showed less evidentintercellular spaces
Basal cells with clear cytoplasm and euchromatic nucleusare considered the normal cells of the corneal epithelium[22] Noxae acting on the cornea were shown to transformthe structural and ultrastructural morphology of the basalcells which become darker with more condensed chromatinand cytoplasm andwider intercellular spacesThesemorpho-logical aspects have been described either in experimentalsuch as induced vitamin A deficiency [23] or in pathologicalconditions such as macular corneal dystrophy [24] andhereditary Thiel-Behnke corneal dystrophy [25] and wererelated to the concomitant deficiencies in Bowmanrsquos layer[24]
It is well known that following ethanol treatment theintracellular water is removed and replaced by alcohol itselfwith consequent changes in the tertiary structure of proteinsand cellular shrinkage [14] On the contrary when the cornealepithelium was pretreated with trehalose this molecule bysubstituting water molecules and forming hydrogen bonds
8 The Scientific World Journal
could stabilize the three-dimensional structure of the pro-teins [26]
The protective role of trehalose on the corneal epitheliumwas also supported by our morphometric results In factin both corneal epithelial groups the number of layers wassuperimposable and the epithelial thickness was within thenormal values [22] However in TTE the epithelium wasstatistically significantly thicker than TUE FurthermoreTTE showed statistically significantly higher values thanTUEfor basal cells area and cytoplasmatic density It was thuspossible to propose that the disaccharide could preserve thephysiological morphology of the epithelial cells in contrastwith the darker vacuolated appearance induced by theethanol alone
The morphometric analysis showed also that the numberof De between adjacent superficial cells was statisticallysignificantly higher in TTE In addition in both groupsDe of adjacent basal cells were statistically significantlyless numerous between dark cells when compared to thejunctions between clear cells or cleardark cells Even if nostatistical data are currently available as far as we know onDe distribution in the intact corneal epithelium these datacould indicate a better adhesion between cellular membranesin TTE and explain the morphological findings of widerintercellular spaces observed between dark cells
As to the Hd found in the inferior pole of the basalcells their mean number was calculated in 10 120583m of basalmembrane in order to obtain data from single basal cellsIn fact their ultrastructural aspect was uneven being char-acterized by the coexistence of clear and dark cells In thisway the analysis of a single cell could be performed and thevalues could be referred to as the specific morphological celltype Clear cells in the TTE showed normal values of Hdwhen compared to what reported in a previous work [27]whereas clear cells in TUE and dark cells in both groupsshowed statistically significantly lower values In clear cellsHd maintained their regular connection with the laminalucida of the basement membrane thus confirming that thecleavage plane of ethanol-induced corneal epithelial flaps waslocated between the lamina lucida and the lamina densa ofthe basement membrane [7] On the contrary in dark cellsmembrane-bound blebs of the basal pole and an extracellulargranular-filamentous material similar to that observed inpatients with recurrent corneal erosions [12] were presentthus indicating an irregular cleavage of these cells
5 Conclusions
The evidence reported in previous studies indicating thattrehalose is able to improve the corneal epithelial conditionsin course of diseases such as dry eye together with ourfindings shows that it has a direct therapeutic role on theepithelial cells therefore its use could be advantageous inthe treatment of patients with epithelial damage Howeverfurther studies about the clinical outcome are needed toconfirm the validity of its use
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
Theauthors are indebted toMr Sebastiano Brunetto from theDepartment of Biomedical Sciences and MorphofunctionalImaging for the technical assistance
References
[1] D T Azar R T Ang J-B Lee et al ldquoLaser subepithelialkeratomileusis electronmicroscopy and visual outcomes of flapphotorefractive keratectomyrdquo Current Opinion in Ophthalmol-ogy vol 12 no 4 pp 323ndash328 2001
[2] J B Lee J A Javier J H Chang C C Chen T Kato andD T Azar ldquoConfocal and electron microscopic studies oflaser subepithelial keratomileusis (LASEK) in the white leghornchick eyerdquo Archives of Ophthalmology vol 120 no 12 pp 1700ndash1706 2002
[3] S Y KimW J Sah YW Lim and TWHahn ldquoTwenty percentalcohol toxicity on rabbit corneal epithelial cells electronmicroscopic studyrdquo Cornea vol 21 no 4 pp 388ndash392 2002
[4] I K Song andCK Joo ldquoMorphological and functional changesin the rat cornea with an ethanol-mediated epithelial flaprdquoInvestigative Ophthalmology and Visual Science vol 45 no 2pp 423ndash428 2004
[5] C C Chen J H Chang J B Lee J Javier and D TAzar ldquoHuman corneal epithelial cell viability and morphologyafter dilute alcohol exposurerdquo Investigative Ophthalmology andVisual Science vol 43 no 8 pp 2593ndash2602 2002
[6] B Gabler CWinkler VonMohrenfels A K Dreiss J Marshalland C P Lohmann ldquoVitality of epithelial cells after alcoholexposure during laser-assisted subepithelial keratectomy flappreparationrdquo Journal of Cataract and Refractive Surgery vol 28no 10 pp 1841ndash1846 2002
[7] E M Espana M Grueterich A Mateo et al ldquoCleavage ofcorneal basement membrane components by ethanol exposurein laser-assisted subepithelial keratectomyrdquo Journal of Cataractand Refractive Surgery vol 29 no 6 pp 1192ndash1197 2003
[8] I G Pallikaris I I Naoumidi M I Kalyvianaki and V J Kat-sanevaki ldquoEpi-LASIK comparative histological evaluation ofmechanical and alcohol-assisted epithelial separationrdquo Journalof Cataract and Refractive Surgery vol 29 no 8 pp 1496ndash15012003
[9] A C Browning S Shah H S Dua S V Maharajan T Grayand M A Bragheeth ldquoAlcohol debridement of the cornealepithelium in PRK and LASEK an electron microscopic studyrdquoInvestigative Ophthalmology and Visual Science vol 44 no 2pp 510ndash513 2003
[10] J Y Oh J M Yu and J H Ko ldquoAnalysis of ethanol effectson corneal epitheliumrdquo Investigative Ophthalmology and VisualScience vol 54 no 6 pp 3852ndash3856 2013
[11] H S Dua R Lagnado D Raj et al ldquoAlcohol delamination ofthe corneal epithelium an alternative in the management ofrecurrent corneal erosionsrdquo Ophthalmology vol 113 no 3 pp404ndash411 2006
[12] R Mencucci I Paladini B Brahimi U Menchini H S Duaand P Romagnoli ldquoAlcohol delamination in the treatmentof recurrent corneal erosion an electron microscopic studyrdquo
The Scientific World Journal 9
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
was widely studied in ophthalmic research as exogenoustrehalose protects corneal epithelial cells from experimentaldrying [16] and was shown to be effective in the treatment ofmoderate to severe human dry eye [17] Furthermore duringdesiccation in vivo it was also demonstrated that trehalosecould effectively suppress apoptotic cell death on the ocularsurface [18]
Aim of the present work was to compare the structureand the ultrastructure of the corneal epithelium in patientsundergoing alcohol delamination with and without trehalosepretreatment
2 Materials and Methods
21 Study Design This is an experimental controlled studyon a model of corneal epithelial alcohol delaminationcurrently used in some refractive surgery procedures Itwas carried at the Regional Referral Center for the Ocu-lar Surface Diseases of the Department of ExperimentalMedical-Surgical Sciences of the University Hospital ofMessina Messina Italy Ethics approval was granted by theInstitutional Review Board of the Department of Experi-mental Medical-Surgical Sciences of the University Hospitalof Messina Messina Italy and the study was conducted inconcordance with the tenets of the Declaration of HelsinkiInformed consentwas obtained fromall the participants afterexplanation of the nature and the possible consequences ofthe study
22 Patients Population The epithelial specimens wereobtained from 24 eyes (12 patients 7 male and 5 femalemean age 263 plusmn 42 years) with a refractive error of minus4 plusmn 28diopters undergoing PRK Inclusion criteria were subjectseligible for refractive surgical procedure with myopia in botheyes below 7 diopters willing to participate to the study andto adhere to the study protocol and who signed the informedconsent Exclusion criteria were systemic or ocular diseasescontraindicating the refractive surgical procedure or subjectswho were not willing to adhere to the study protocol
23 Treatment Protocol Before the surgical procedure theright eyes of the patients were treated as follows a dropof a solution based on 3 trehalose (Thealoz Thea FarmaMilano Italy) followed after 5min by the instillation ofa drop of 04 oxybuprocaine hydrochloride (NovesinaNovartis Farma Origgio VA Italy) This procedure was per-formed every 151015840 for the hour before the surgical procedureso that a total of 5 drops of trehalose were instilled within thehourThe left eyeswere treatedwith only 04oxybuprocainehydrochloride instilled five times for the hour before the sur-gical procedure During the surgical treatment the epithelialspecimens were obtained as follows demarcation of a centralcorneal area with a 90mm diameter cone (J2907 JanachComo Italy) filling of the cone with a 20 solution of ethylalcohol in distilled water for 25 sec emptying of the conewitha Merocel sponge (Medtronics Merocel Mystic CT USA)washing with a solution 1 1 of balanced salt solution anddistilled water and successive drying with a Merocel sponge
The epithelial flaps were obtained by gently detaching thealcoholized epithelium with a blunt spatula and divided inthree specimens for the morphological assessment
24 Epithelial Cells Vitality Evaluation Epithelial cells vitalitywas evaluatedwith the trypan blue dye test [6] One specimenwas incubated for 21015840 in 01 trypan blue in phosphate-buffered saline (PBS) at 37∘C It was then washed severaltimes in warm PBS and incubated in Dulbeccorsquos modifiedEagle culture medium (Sigma-Aldrich Srl Milano Italy)containing 10 of fetal calf serum for 301015840 at 37∘C After afurther wash in warmPBS the specimenwas flatmounted ona slide and photographed with a Zeiss Primo Star LM Vitalcells appeared with unstained nuclei and cytoplasm whiledead cells were blue
25 Apoptosis Assay One specimen was fixed in 10formaldehyde in 02M PBS dehydrated in graded ethanolcleared in xylene and embedded in paraffin (ParaplastSPI Supplies West Chester PA USA) Paraffin blocks wereplaced in a rotarymicrotome (RM2125RT Leica InstrumentsNuszligloch Germany) and 5 120583m sections cut with disposablemetal blades (S35 Feather Safety Razor Co Osaka Japan)were cleared with xylene and rehydrated in graded ethanolAn apoptosis detection kit (Chemicon International Apop-Tag Plus Peroxidase In Situ Apoptosis Detection Kit Temec-ula CA USA) was used [19] The sections were subjected topartial digestion with proteinase K (20120583gmL) and washedin PBS Endogenous peroxidase activity was blocked with 3H2O2in PBS Thereafter the sections were incubated in an
equilibration buffer and then with the TUNEL reaction mix-ture in a humidified chamber at 37∘C for 60min They wereincubated in a stopwash buffer at room temperature andwashedwith PBSThe anti-digoxigenin conjugatewas appliedand incubation in a humidified chamber was performedfor 30min at room temperature The 3-31015840-diaminobenzidine(DAB) was then added for 10min and the slides werecounterstained with Mayerrsquos hematoxylin The sections werewashed in ethanol and xylene mounted in Permount (FisherScientific Fair Lawn NJ USA) and photographed with aZeiss Primo Star LM
26 Light and Transmission Electron Microscopy One spec-imen was immediately fixed in 25 glutaraldehyde 02Min Soslashrensenrsquos phosphate buffer (pH 72) at +4∘C for 4 hwashed with 02M Soslashrensenrsquos phosphate buffer (pH 72)and postfixed in 1 osmium tetroxide (OsO
4) in 02M
Soslashrensenrsquos phosphate buffer (pH 72) at +4∘C for 1 h Thespecimen was dehydrated in graded ethanol and acetone andflatembedded in embedding agent (Durcupan ACM FlukaSigma-Aldrich St Louis MO USA) for a better orientationSemithin sections (1 120583m) were cut with a LKB UltrotomeV ultramicrotome stained with an aqueous solution of 1toluidine blue in 1 borax and 1 pironine and viewed andphotographedwith a Zeiss Primo Star lightmicroscope (LM)From the same specimens used for LM ultrathin sectionsof gold-silver interference color were cut with a diamondknife on a LKB Ultrotome V ultramicrotome collected on
The Scientific World Journal 3
uncoated 200ndash300 mesh copper grids and stained withmethanolic uranyl acetate and lead citrate Micrographs weretakenwith a Philips CM-10 transmission electronmicroscope(TEM) at 80 kV
27 Morphometric Analysis A morphometric analysis of thecorneal epithelium in both groups of eyes was carried outon LM micrographs considering the percentage of vitalcells the number of epithelial layers the epithelial thicknessthe area of the basal cells and the optical density of theircytoplasm On TEM images the number of desmosomes(De) of superficial wing andbasal cells andhemidesmosomes(Hd) of the basal cells were evaluated
The percentage of vital cells was counted on 5 micro-graphseye obtained from 5 different nonoverlapping ran-domly chosen microscopic fields For each field a square of150 times 150 120583m was drawn with the Adobe Photoshop 801software and the cells included in the square were countedincluding among the cells touching the square border onlythose from two sides
LM data were obtained from 5 semithin sectionseye(total of 60 sectionsgroup) collecting 1 semithin sectionevery 100 all micrographs were obtained at the same magni-fication of 450x with a Zeiss Primo Star LM and processedwith a Macintosh MacBook using the Adobe Photoshop801 software All images were converted to black and whiteand brought to the final magnification of 1 000x for themorphometric analysis To avoid possible bias from obliquesectioning which would artificially increase the measuredthickness the thinnest portion of the epithelium was mea-sured in all the images On the selected images a straightline perpendicular to the epithelium was tracedThe numberof epithelial layers was counted along the line The overallepithelial thickness was expressed in 120583m and calculatedaccording to the following formula for the conversion frompixels [(length in pixels) times (10 000120583mcm)][(resolution inpixelscm) times (magnification)] As to the basal cells areaand their cytoplasmatic density 10 microscopic fields of100 120583m each were randomly chosen for every group andanalyzed with the public domain ImageJ software (ImageJhttprsbinfonihgovij) For the measurements only basalcells with well evident nuclei were chosen the area wasexpressed in 120583m2 and the cytoplasmatic density was calcu-lated (function analyze gt measure) in optical units (OU)within the levels from 0 (black) to 255 (white)
TEM morphometric analysis was performed on negativefilms (Kodak 4489) obtained from 5 ultrathin sectionseyeat the same voltage exposure time and magnification Thenegative films were acquired (ratio 1 1) with an Epson Per-fection 4180 scanner processed with a Macintosh MacBookusing theAdobe Photoshop 801 software and converted intopositive images at the same final magnification of 18 000xThe absolute number of De was calculated along the interfacebetween adjacent superficial wing and basal cells by countingall De observed in 100 standard fields of 10 120583m each includingthe cellular membranes of two adjacent cells The absolutenumber of Hd was calculated along the interface betweenbasal cells and basement membrane by counting all Hd
observed in 100 standard fields of 10 120583m each including asingle cell
Two experienced histologists masked about the originof the specimens performed all the morphological andmorphometric evaluations All data were expressed in 120583m forlinear values in 120583m2 for cellular area and in OU for opticaldensity between 0 (black) and 255 (white)
28 Statistical Analysis Primary efficacy variables were theepithelial thickness the number of De the area of the basalcells and the optical density of the basal cells cytoplasmSecondary parameters were the number of layers and thenumber of Hd For the analysis of the results the Studentrsquos119905-test for unpaired data was used A 119875 value of le005 wasconsidered statistically significant
3 Results
31 Epithelial Cells Vitality Evaluation and Apoptosis AssayThe trypan blue dye test demonstrated that trehalose-untreated eyes (TUE) (Figure 1(a)) showed a higher amountof not vital cells stained in blue with the dye than trehalose-treated eyes (TTE) (Figure 1(b)) (118plusmn08versus 144plusmn09resp 119875 lt 0001)
The apoptosis assay in both TUE (Figure 1(c)) and TTE(Figure 1(d)) failed to demonstrate positive cells
32 Structural and Ultrastructural Data The corneal epithe-lium obtained from TUE and observed in semithin sectionsat light microscopy showed superficial cells with variableshape and optical density round wing cells with not clearlyevident intercellular borders and basal cells with hyper-chromic nuclei and wide intercellular spaces (Figure 2(a))With TEM corneal superficial cells were flat and showed fewshort irregular microfolds and dilated intercellular spaces(Figures 2(b) and 3(a)) Wing cells showed no apparentchanges whilst intercellular spaces were moderately dilated(Figures 2(b) and 3(b)) Basal cells had elliptical nuclei withevident nucleoli dark and vacuolated cytoplasm and dilatedintercellular spaces (Figures 2(b) and 3(c))Their inferior poleshoweddense cytoplasm fewHd and someblebs surroundedby aggregates of granular material (Figures 2(b) and 3(d))
In the corneal epithelium obtained from TTE observedin semithin sections at light microscopy superficial cellsappeared flat with well-evident intercellular borders wingcells were polygonal with well-evident intercellular bordersand basal cells had polygonal shape and evident intercellularspaces (Figure 2(c)) With TEM corneal superficial cellswere flat and showed regularly arranged microfolds andwell-preserved intercellular borders (Figures 2(d) and 3(e))Wing cells showed well-evident cytoskeleton and regularintercellular borders glued by many De (Figure 2(d)) Occa-sionally some wing cells otherwise of normal morphologyshowed an apparently double nucleus (Figure 3(f)) Basal cellsshowed a clear cytoplasm with only few vesicles a roundeuchromatic nucleus small intercellularwidenings and somedesmosomes (Figures 2(d) and 3(g)) Their inferior poleshowed clear cytoplasm and numerous Hd connected to thelamina lucida of the basement membrane (Figure 3(h))
4 The Scientific World Journal
(a) (b)
(c) (d)
Figure 1 (a b) Light micrographs of trypan blue dye test for epithelial cells vitality In both TUE (a) and TTE (b) positive dead cells (arrow)can be observed Scale bar 80 120583m (c d) Light micrographs of TUNEL reaction of corneal epithelium In both TUE (c) and TTE (d) nopositive cells can be observed Scale bar 30 120583m
Table 1 Morphometric parameters of the corneal epithelium in trehalose-untreated eyes (TUE) and trehalose-treated eyes (TTE)
TTE 68 plusmn 07 577 plusmn 41lowast 271 plusmn 31lowast 244 plusmn 51 94 plusmn 16daggerdagger 63 plusmn 08 = 57 plusmn 1lowast 24 plusmn 21lowast 209 plusmn 24lowastDaggerlowast119875 lt 00001 versus TUEdagger119875 lt 00001 versus the other cell junctions within the TUE groupdaggerdagger119875 lt 00001 versus the other cell junctions in TTE group=119875 = 004 versus darkdark cell junctions in TTE groupDagger119875 lt 00001 versus clear cells of the same group
33 Morphometric Data The results of the morphometricanalysis within each group showed (Table 1) that for basalcells in TUE the number of De was 92 plusmn 11 betweenclearclear cells 57 plusmn 1 between cleardark cells and 42 plusmn1 between darkdark cells (clearclear versus cleardark119875 lt 00001 clearclear versus darkdark 119875 lt 00001and cleardark versus darkdark 119875 lt 00001) For Hda statistically significant difference was observed in theirnumber when clear and dark basal cells were compared(219 plusmn 29 and 181 plusmn 15 resp 119875 lt 00001) InTTE the number of De was 94 plusmn 16 between clearclearcells 63 plusmn 08 between cleardark cells and 57 plusmn 1between darkdark cells (clearclear versus cleardark 119875 lt00001 clearclear versus darkdark 119875 lt 00001 andcleardark versus darkdark 119875 = 004) For Hd a statis-tically significant difference was observed in their number
when clear and dark basal cells were compared (24 plusmn 21 and209 plusmn 24 resp 119875 lt 00001)
Comparing the two groups (Table 1) a statistically sig-nificant higher epithelial thickness in TTE was demonstrated(577 plusmn 41 120583m in TTE and 554 plusmn 35 120583m in TUE resp 119875 lt00001) As the number of De their number was statisticallysignificantly higher in TTE between superficial cells (271 plusmn31 in TTE and 201 plusmn 2 in TUE resp 119875 lt 00001) andbetween dark basal cells (57 plusmn 1 in TTE and 42 plusmn 1 in TUEresp 119875 lt 00001) As to the number of Hd present in theinferior pole of the basal cells it was statistically significantlyhigher in TTE in both the clear (24plusmn21 in TTE and 219plusmn29in TUE resp 119875 lt 00001) and the dark cells (209 plusmn 24 inTTE and 181 plusmn 15 in TUE resp 119875 lt 00001)
No statistically significant differences between bothgroups were found for the number of the cellular layers and
The Scientific World Journal 5
S
W
N
S
W
V
V
VN
B
B
lowast
lowastlowast
lowast
lowast
(a) (c)
(b) (d)
Figure 2 (a) Light micrograph from a semithin section of the corneal epithelium obtained with alcohol delamination in TUE Superficialcells show variable shape and optical density (arrow) basal cells have hyperchromic nuclei and large intercellular spaces (double arrow)Scale bar 25 120583m (b) TEM micrograph of the corneal epithelium obtained with alcohol delamination in TUE Superficial cells (S) show areduced number of apical microfolds (arrow) In the wing cells (W) intracellular vesicles and slightly dilated intercellular spaces (lowast) areevident Basal cells (B) are irregular in their shape and size and show large intercellular spaces (lowast) and a cytoplasm filled with vesicle (V)Many blebs (arrowhead) surrounded by granular material are evident Scale bar 5 120583m (c) Light micrograph from a semithin section of thecorneal epithelium in TTE Both superficial and wing cells show normal shape and well-evident intercellular borders (arrow) basal cells havepolygonal shape andmany small intracytoplasmatic vesicles (double arrow) Scale bar 25120583m (d) TEMmicrograph of the corneal epitheliumin TTE Superficial cells (S) show a normal flattened shape with well-evident apical microfolds (arrow)Wing cells (W) have uniform electrondensity and normal intercellular spaces (arrowhead) Basal cells (B) have variable electron density polygonal shape few cytoplasmatic vesicles(V) round nuclei (N) and small intercellular spaces (lowast) Scale bar 5 120583m
for the number of De between wing cells clearclear basalcells and cleardark basal cells
When the basal cells area was evaluated a statisticallysignificant difference was found between TUE and TTE(1994 plusmn 421 120583m2 and 2583 plusmn 515 120583m2 resp 119875 lt 00001)(Figure 4)
Considering the optical density of the cytoplasm of thebasal cells a statistically significant difference was foundbetween TUE and TTE (1255 plusmn 24OU and 781 plusmn 214 resp119875 lt 00001) (Figure 5)
4 Discussion
Diluted ethanol is currently used in patients undergoingLASEK to reduce the adhesion of the corneal epitheliumto Bowmanrsquos layer in order to expose the stroma for thesubsequent PRK treatment The theoretical advantage ofLASEK is derived from the repositioning of the epithelialflap over the laser-ablated corneal surface so facilitating thecorneal epithelial healing reducing pain and inflammationand decreasing the stromal haze [4]
6 The Scientific World Journal
N
NN
NN
t
n
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 3 (a) TEM micrograph of superficial cells from the corneal epithelium obtained with alcohol delamination on TUE The cells areflat and show short and irregular microfolds (arrow) and dilated intercellular spaces (lowast) Scale bar 5 120583m (b) TEM micrograph of wing cellsfrom the corneal epithelium obtained with alcohol delamination on TUE The nucleus (N) and the cytoplasm have normal appearanceintercellular spaces are moderately dilated (lowast) Scale bar 2 120583m (c) TEM micrograph of basal cells from the corneal epithelium obtainedwith alcohol delamination on TUEThe cells show elliptical nuclei (N) with evident nucleoli (n) dark and vacuolated cytoplasm and dilatedintercellular spaces (lowast) Scale bar 2 120583m (d) TEM micrograph of the inferior pole of a basal cell from the corneal epithelium obtained withalcohol delamination on TUE The cytoplasm is dense and few hemidesmosomes (arrows) are present Note the presence of cellular blebs(arrowhead) and of an irregular granular material Scale bar 05 120583m (e) TEM micrograph of superficial cells from the corneal epitheliumof TTE The cells are flat show regular microfolds (arrow) and are well-preserved intercellular borders (arrowhead) Scale bar 5120583m (f)TEMmicrograph of wing cells from the corneal epithelium of TTEMany tonofilaments (t) are evident around the apparently double nucleus(N1-N2) intercellular borders are normal with well-evident desmosomes (arrowhead) Scale bar 2 120583m (g) TEM micrograph of basal cells
from corneal epithelium in TTE Basal cells have round euchromatic nucleus (N) clear cytoplasm with only few vesicles small intercellularwidenings (lowast) and some desmosomes (arrow) Scale bar 2120583m (h) TEM micrograph of the inferior pole of a basal cell from the cornealepithelium in TTE The cytoplasm is clear and the hemidesmosomes (arrows) are numerous lowast = lamina lucida of the basement membraneScale bar 05 120583m
The Scientific World Journal 7
350
300
250
200
150
100
50
0 Trehalose-untreated eyes
Trehalose-treated eyes
Basa
l cel
ls ar
ea (e
xpre
ssed
in120583
m2)
1994 plusmn 421 2583 plusmn 515
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Figure 4Morphometric data of the basal cells area in TUE andTTEexpressed in 120583m2 lowast = 119875 lt 00001
255240225210195180165150135120105
907560453015
0 Trehalose-untreated eyes
Trehalose-treated eyes
781 plusmn 214 1255 plusmn 24
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Mea
n op
tical
den
sity
of b
asal
cells
cyto
plas
m(g
ray
scal
e OU
)
Figure 5 Morphometric data of the optical density of the basalcells cytoplasm in TUE and TTE expressed within the levels from0 (black) to 255 (white) optical units (OU) lowast = 119875 lt 00001
However adverse effects of alcohol on the corneal epithe-lial cells have been shown In particular the following changeswere described flattening of apical microvilli [3] and darkcytoplasm in the superficial cells [11] diffuse interruptions ofthe intercellular junctions with enlargement of the intercel-lular spaces [3 8 9] cellular edema [3] blebs of the cellularmembrane [5 8 9] autophagic vacuoles [5] damages of Hdandof the basementmembrane [4 5] and coexistence of clearand dark cytoplasm in the basal cells [5 8 11] Furthermorethe treatmentwith alcohol resulted in an increase of apoptoticbasal cells in both laboratory animals [2 4] and humans [5]
Therefore a treatment able to reduce the structural andultrastructural changes induced by the exposition to dilutedethanol characterized by the absence of side effects canbe considered useful in maintaining a healthier corneal
epithelium thus possibly improving its morphological andfunctional recovery after the surgical procedure
Trehalose a naturally occurring alpha-linked disaccha-ride formed by two molecules of glucose was consideredfor its well-known protective effects in eukaryotic cells [15]Trehalose synthesized by many living organisms but notmammals showed many different functions In anhydro-biosis that is the capability of surviving prolonged periodsof dessication a better resistance to dryness conditionswas obtained by increasing the intracellular trehalose levelsin animal cells [15] in this way proteins and membraneswere protected from denaturation [20] A reduced deathby desiccation of human corneal epithelium in culture wasobtained with the pretreatment of the cells with a 100mMsolution of trehalose [16] Furthermore trehalosemight act asfree radicals scavenger reducing the oxidative damage of thecornea caused byUVB rays and suppressing the expression ofproinflammatory cytokines [21] Finally trehalose based eyedrops were found to be effective in the treatment of moderateto severe human dry eye [17] as it reduced the number ofdead cells on the ocular surface through the suppression ofapoptosis [18]
In the present study trehalose dosage was chosen on thebasis of previous experiences [16] which demonstrated theeffect of the exposure to trehalose for 151015840 on corneal epithelialcells maintained in cultureThis procedure of administrationwas adopted as it fitted appropriately the timing of thesurgical procedures
The positive action of trehalose on the corneal epitheliumcould be demonstrated by the reduction of the morphologicchanges in TTE and by the evaluation of the morphometricdata
As to the morphological aspects structural changes wereevident in TUE In fact a reduced number and length ofthe apical microfolds of the superficial cells a dark andvacuolated cytoplasm of the basal cells a lower number ofHd and a diffuse enlargement of the intercellular spaceswere observed On the contrary in TTE superficial cellsshowed well represented microfolds and basal cells hadclear cytoplasm with few vesicles and showed less evidentintercellular spaces
Basal cells with clear cytoplasm and euchromatic nucleusare considered the normal cells of the corneal epithelium[22] Noxae acting on the cornea were shown to transformthe structural and ultrastructural morphology of the basalcells which become darker with more condensed chromatinand cytoplasm andwider intercellular spacesThesemorpho-logical aspects have been described either in experimentalsuch as induced vitamin A deficiency [23] or in pathologicalconditions such as macular corneal dystrophy [24] andhereditary Thiel-Behnke corneal dystrophy [25] and wererelated to the concomitant deficiencies in Bowmanrsquos layer[24]
It is well known that following ethanol treatment theintracellular water is removed and replaced by alcohol itselfwith consequent changes in the tertiary structure of proteinsand cellular shrinkage [14] On the contrary when the cornealepithelium was pretreated with trehalose this molecule bysubstituting water molecules and forming hydrogen bonds
8 The Scientific World Journal
could stabilize the three-dimensional structure of the pro-teins [26]
The protective role of trehalose on the corneal epitheliumwas also supported by our morphometric results In factin both corneal epithelial groups the number of layers wassuperimposable and the epithelial thickness was within thenormal values [22] However in TTE the epithelium wasstatistically significantly thicker than TUE FurthermoreTTE showed statistically significantly higher values thanTUEfor basal cells area and cytoplasmatic density It was thuspossible to propose that the disaccharide could preserve thephysiological morphology of the epithelial cells in contrastwith the darker vacuolated appearance induced by theethanol alone
The morphometric analysis showed also that the numberof De between adjacent superficial cells was statisticallysignificantly higher in TTE In addition in both groupsDe of adjacent basal cells were statistically significantlyless numerous between dark cells when compared to thejunctions between clear cells or cleardark cells Even if nostatistical data are currently available as far as we know onDe distribution in the intact corneal epithelium these datacould indicate a better adhesion between cellular membranesin TTE and explain the morphological findings of widerintercellular spaces observed between dark cells
As to the Hd found in the inferior pole of the basalcells their mean number was calculated in 10 120583m of basalmembrane in order to obtain data from single basal cellsIn fact their ultrastructural aspect was uneven being char-acterized by the coexistence of clear and dark cells In thisway the analysis of a single cell could be performed and thevalues could be referred to as the specific morphological celltype Clear cells in the TTE showed normal values of Hdwhen compared to what reported in a previous work [27]whereas clear cells in TUE and dark cells in both groupsshowed statistically significantly lower values In clear cellsHd maintained their regular connection with the laminalucida of the basement membrane thus confirming that thecleavage plane of ethanol-induced corneal epithelial flaps waslocated between the lamina lucida and the lamina densa ofthe basement membrane [7] On the contrary in dark cellsmembrane-bound blebs of the basal pole and an extracellulargranular-filamentous material similar to that observed inpatients with recurrent corneal erosions [12] were presentthus indicating an irregular cleavage of these cells
5 Conclusions
The evidence reported in previous studies indicating thattrehalose is able to improve the corneal epithelial conditionsin course of diseases such as dry eye together with ourfindings shows that it has a direct therapeutic role on theepithelial cells therefore its use could be advantageous inthe treatment of patients with epithelial damage Howeverfurther studies about the clinical outcome are needed toconfirm the validity of its use
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
Theauthors are indebted toMr Sebastiano Brunetto from theDepartment of Biomedical Sciences and MorphofunctionalImaging for the technical assistance
References
[1] D T Azar R T Ang J-B Lee et al ldquoLaser subepithelialkeratomileusis electronmicroscopy and visual outcomes of flapphotorefractive keratectomyrdquo Current Opinion in Ophthalmol-ogy vol 12 no 4 pp 323ndash328 2001
[2] J B Lee J A Javier J H Chang C C Chen T Kato andD T Azar ldquoConfocal and electron microscopic studies oflaser subepithelial keratomileusis (LASEK) in the white leghornchick eyerdquo Archives of Ophthalmology vol 120 no 12 pp 1700ndash1706 2002
[3] S Y KimW J Sah YW Lim and TWHahn ldquoTwenty percentalcohol toxicity on rabbit corneal epithelial cells electronmicroscopic studyrdquo Cornea vol 21 no 4 pp 388ndash392 2002
[4] I K Song andCK Joo ldquoMorphological and functional changesin the rat cornea with an ethanol-mediated epithelial flaprdquoInvestigative Ophthalmology and Visual Science vol 45 no 2pp 423ndash428 2004
[5] C C Chen J H Chang J B Lee J Javier and D TAzar ldquoHuman corneal epithelial cell viability and morphologyafter dilute alcohol exposurerdquo Investigative Ophthalmology andVisual Science vol 43 no 8 pp 2593ndash2602 2002
[6] B Gabler CWinkler VonMohrenfels A K Dreiss J Marshalland C P Lohmann ldquoVitality of epithelial cells after alcoholexposure during laser-assisted subepithelial keratectomy flappreparationrdquo Journal of Cataract and Refractive Surgery vol 28no 10 pp 1841ndash1846 2002
[7] E M Espana M Grueterich A Mateo et al ldquoCleavage ofcorneal basement membrane components by ethanol exposurein laser-assisted subepithelial keratectomyrdquo Journal of Cataractand Refractive Surgery vol 29 no 6 pp 1192ndash1197 2003
[8] I G Pallikaris I I Naoumidi M I Kalyvianaki and V J Kat-sanevaki ldquoEpi-LASIK comparative histological evaluation ofmechanical and alcohol-assisted epithelial separationrdquo Journalof Cataract and Refractive Surgery vol 29 no 8 pp 1496ndash15012003
[9] A C Browning S Shah H S Dua S V Maharajan T Grayand M A Bragheeth ldquoAlcohol debridement of the cornealepithelium in PRK and LASEK an electron microscopic studyrdquoInvestigative Ophthalmology and Visual Science vol 44 no 2pp 510ndash513 2003
[10] J Y Oh J M Yu and J H Ko ldquoAnalysis of ethanol effectson corneal epitheliumrdquo Investigative Ophthalmology and VisualScience vol 54 no 6 pp 3852ndash3856 2013
[11] H S Dua R Lagnado D Raj et al ldquoAlcohol delamination ofthe corneal epithelium an alternative in the management ofrecurrent corneal erosionsrdquo Ophthalmology vol 113 no 3 pp404ndash411 2006
[12] R Mencucci I Paladini B Brahimi U Menchini H S Duaand P Romagnoli ldquoAlcohol delamination in the treatmentof recurrent corneal erosion an electron microscopic studyrdquo
The Scientific World Journal 9
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
uncoated 200ndash300 mesh copper grids and stained withmethanolic uranyl acetate and lead citrate Micrographs weretakenwith a Philips CM-10 transmission electronmicroscope(TEM) at 80 kV
27 Morphometric Analysis A morphometric analysis of thecorneal epithelium in both groups of eyes was carried outon LM micrographs considering the percentage of vitalcells the number of epithelial layers the epithelial thicknessthe area of the basal cells and the optical density of theircytoplasm On TEM images the number of desmosomes(De) of superficial wing andbasal cells andhemidesmosomes(Hd) of the basal cells were evaluated
The percentage of vital cells was counted on 5 micro-graphseye obtained from 5 different nonoverlapping ran-domly chosen microscopic fields For each field a square of150 times 150 120583m was drawn with the Adobe Photoshop 801software and the cells included in the square were countedincluding among the cells touching the square border onlythose from two sides
LM data were obtained from 5 semithin sectionseye(total of 60 sectionsgroup) collecting 1 semithin sectionevery 100 all micrographs were obtained at the same magni-fication of 450x with a Zeiss Primo Star LM and processedwith a Macintosh MacBook using the Adobe Photoshop801 software All images were converted to black and whiteand brought to the final magnification of 1 000x for themorphometric analysis To avoid possible bias from obliquesectioning which would artificially increase the measuredthickness the thinnest portion of the epithelium was mea-sured in all the images On the selected images a straightline perpendicular to the epithelium was tracedThe numberof epithelial layers was counted along the line The overallepithelial thickness was expressed in 120583m and calculatedaccording to the following formula for the conversion frompixels [(length in pixels) times (10 000120583mcm)][(resolution inpixelscm) times (magnification)] As to the basal cells areaand their cytoplasmatic density 10 microscopic fields of100 120583m each were randomly chosen for every group andanalyzed with the public domain ImageJ software (ImageJhttprsbinfonihgovij) For the measurements only basalcells with well evident nuclei were chosen the area wasexpressed in 120583m2 and the cytoplasmatic density was calcu-lated (function analyze gt measure) in optical units (OU)within the levels from 0 (black) to 255 (white)
TEM morphometric analysis was performed on negativefilms (Kodak 4489) obtained from 5 ultrathin sectionseyeat the same voltage exposure time and magnification Thenegative films were acquired (ratio 1 1) with an Epson Per-fection 4180 scanner processed with a Macintosh MacBookusing theAdobe Photoshop 801 software and converted intopositive images at the same final magnification of 18 000xThe absolute number of De was calculated along the interfacebetween adjacent superficial wing and basal cells by countingall De observed in 100 standard fields of 10 120583m each includingthe cellular membranes of two adjacent cells The absolutenumber of Hd was calculated along the interface betweenbasal cells and basement membrane by counting all Hd
observed in 100 standard fields of 10 120583m each including asingle cell
Two experienced histologists masked about the originof the specimens performed all the morphological andmorphometric evaluations All data were expressed in 120583m forlinear values in 120583m2 for cellular area and in OU for opticaldensity between 0 (black) and 255 (white)
28 Statistical Analysis Primary efficacy variables were theepithelial thickness the number of De the area of the basalcells and the optical density of the basal cells cytoplasmSecondary parameters were the number of layers and thenumber of Hd For the analysis of the results the Studentrsquos119905-test for unpaired data was used A 119875 value of le005 wasconsidered statistically significant
3 Results
31 Epithelial Cells Vitality Evaluation and Apoptosis AssayThe trypan blue dye test demonstrated that trehalose-untreated eyes (TUE) (Figure 1(a)) showed a higher amountof not vital cells stained in blue with the dye than trehalose-treated eyes (TTE) (Figure 1(b)) (118plusmn08versus 144plusmn09resp 119875 lt 0001)
The apoptosis assay in both TUE (Figure 1(c)) and TTE(Figure 1(d)) failed to demonstrate positive cells
32 Structural and Ultrastructural Data The corneal epithe-lium obtained from TUE and observed in semithin sectionsat light microscopy showed superficial cells with variableshape and optical density round wing cells with not clearlyevident intercellular borders and basal cells with hyper-chromic nuclei and wide intercellular spaces (Figure 2(a))With TEM corneal superficial cells were flat and showed fewshort irregular microfolds and dilated intercellular spaces(Figures 2(b) and 3(a)) Wing cells showed no apparentchanges whilst intercellular spaces were moderately dilated(Figures 2(b) and 3(b)) Basal cells had elliptical nuclei withevident nucleoli dark and vacuolated cytoplasm and dilatedintercellular spaces (Figures 2(b) and 3(c))Their inferior poleshoweddense cytoplasm fewHd and someblebs surroundedby aggregates of granular material (Figures 2(b) and 3(d))
In the corneal epithelium obtained from TTE observedin semithin sections at light microscopy superficial cellsappeared flat with well-evident intercellular borders wingcells were polygonal with well-evident intercellular bordersand basal cells had polygonal shape and evident intercellularspaces (Figure 2(c)) With TEM corneal superficial cellswere flat and showed regularly arranged microfolds andwell-preserved intercellular borders (Figures 2(d) and 3(e))Wing cells showed well-evident cytoskeleton and regularintercellular borders glued by many De (Figure 2(d)) Occa-sionally some wing cells otherwise of normal morphologyshowed an apparently double nucleus (Figure 3(f)) Basal cellsshowed a clear cytoplasm with only few vesicles a roundeuchromatic nucleus small intercellularwidenings and somedesmosomes (Figures 2(d) and 3(g)) Their inferior poleshowed clear cytoplasm and numerous Hd connected to thelamina lucida of the basement membrane (Figure 3(h))
4 The Scientific World Journal
(a) (b)
(c) (d)
Figure 1 (a b) Light micrographs of trypan blue dye test for epithelial cells vitality In both TUE (a) and TTE (b) positive dead cells (arrow)can be observed Scale bar 80 120583m (c d) Light micrographs of TUNEL reaction of corneal epithelium In both TUE (c) and TTE (d) nopositive cells can be observed Scale bar 30 120583m
Table 1 Morphometric parameters of the corneal epithelium in trehalose-untreated eyes (TUE) and trehalose-treated eyes (TTE)
TTE 68 plusmn 07 577 plusmn 41lowast 271 plusmn 31lowast 244 plusmn 51 94 plusmn 16daggerdagger 63 plusmn 08 = 57 plusmn 1lowast 24 plusmn 21lowast 209 plusmn 24lowastDaggerlowast119875 lt 00001 versus TUEdagger119875 lt 00001 versus the other cell junctions within the TUE groupdaggerdagger119875 lt 00001 versus the other cell junctions in TTE group=119875 = 004 versus darkdark cell junctions in TTE groupDagger119875 lt 00001 versus clear cells of the same group
33 Morphometric Data The results of the morphometricanalysis within each group showed (Table 1) that for basalcells in TUE the number of De was 92 plusmn 11 betweenclearclear cells 57 plusmn 1 between cleardark cells and 42 plusmn1 between darkdark cells (clearclear versus cleardark119875 lt 00001 clearclear versus darkdark 119875 lt 00001and cleardark versus darkdark 119875 lt 00001) For Hda statistically significant difference was observed in theirnumber when clear and dark basal cells were compared(219 plusmn 29 and 181 plusmn 15 resp 119875 lt 00001) InTTE the number of De was 94 plusmn 16 between clearclearcells 63 plusmn 08 between cleardark cells and 57 plusmn 1between darkdark cells (clearclear versus cleardark 119875 lt00001 clearclear versus darkdark 119875 lt 00001 andcleardark versus darkdark 119875 = 004) For Hd a statis-tically significant difference was observed in their number
when clear and dark basal cells were compared (24 plusmn 21 and209 plusmn 24 resp 119875 lt 00001)
Comparing the two groups (Table 1) a statistically sig-nificant higher epithelial thickness in TTE was demonstrated(577 plusmn 41 120583m in TTE and 554 plusmn 35 120583m in TUE resp 119875 lt00001) As the number of De their number was statisticallysignificantly higher in TTE between superficial cells (271 plusmn31 in TTE and 201 plusmn 2 in TUE resp 119875 lt 00001) andbetween dark basal cells (57 plusmn 1 in TTE and 42 plusmn 1 in TUEresp 119875 lt 00001) As to the number of Hd present in theinferior pole of the basal cells it was statistically significantlyhigher in TTE in both the clear (24plusmn21 in TTE and 219plusmn29in TUE resp 119875 lt 00001) and the dark cells (209 plusmn 24 inTTE and 181 plusmn 15 in TUE resp 119875 lt 00001)
No statistically significant differences between bothgroups were found for the number of the cellular layers and
The Scientific World Journal 5
S
W
N
S
W
V
V
VN
B
B
lowast
lowastlowast
lowast
lowast
(a) (c)
(b) (d)
Figure 2 (a) Light micrograph from a semithin section of the corneal epithelium obtained with alcohol delamination in TUE Superficialcells show variable shape and optical density (arrow) basal cells have hyperchromic nuclei and large intercellular spaces (double arrow)Scale bar 25 120583m (b) TEM micrograph of the corneal epithelium obtained with alcohol delamination in TUE Superficial cells (S) show areduced number of apical microfolds (arrow) In the wing cells (W) intracellular vesicles and slightly dilated intercellular spaces (lowast) areevident Basal cells (B) are irregular in their shape and size and show large intercellular spaces (lowast) and a cytoplasm filled with vesicle (V)Many blebs (arrowhead) surrounded by granular material are evident Scale bar 5 120583m (c) Light micrograph from a semithin section of thecorneal epithelium in TTE Both superficial and wing cells show normal shape and well-evident intercellular borders (arrow) basal cells havepolygonal shape andmany small intracytoplasmatic vesicles (double arrow) Scale bar 25120583m (d) TEMmicrograph of the corneal epitheliumin TTE Superficial cells (S) show a normal flattened shape with well-evident apical microfolds (arrow)Wing cells (W) have uniform electrondensity and normal intercellular spaces (arrowhead) Basal cells (B) have variable electron density polygonal shape few cytoplasmatic vesicles(V) round nuclei (N) and small intercellular spaces (lowast) Scale bar 5 120583m
for the number of De between wing cells clearclear basalcells and cleardark basal cells
When the basal cells area was evaluated a statisticallysignificant difference was found between TUE and TTE(1994 plusmn 421 120583m2 and 2583 plusmn 515 120583m2 resp 119875 lt 00001)(Figure 4)
Considering the optical density of the cytoplasm of thebasal cells a statistically significant difference was foundbetween TUE and TTE (1255 plusmn 24OU and 781 plusmn 214 resp119875 lt 00001) (Figure 5)
4 Discussion
Diluted ethanol is currently used in patients undergoingLASEK to reduce the adhesion of the corneal epitheliumto Bowmanrsquos layer in order to expose the stroma for thesubsequent PRK treatment The theoretical advantage ofLASEK is derived from the repositioning of the epithelialflap over the laser-ablated corneal surface so facilitating thecorneal epithelial healing reducing pain and inflammationand decreasing the stromal haze [4]
6 The Scientific World Journal
N
NN
NN
t
n
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 3 (a) TEM micrograph of superficial cells from the corneal epithelium obtained with alcohol delamination on TUE The cells areflat and show short and irregular microfolds (arrow) and dilated intercellular spaces (lowast) Scale bar 5 120583m (b) TEM micrograph of wing cellsfrom the corneal epithelium obtained with alcohol delamination on TUE The nucleus (N) and the cytoplasm have normal appearanceintercellular spaces are moderately dilated (lowast) Scale bar 2 120583m (c) TEM micrograph of basal cells from the corneal epithelium obtainedwith alcohol delamination on TUEThe cells show elliptical nuclei (N) with evident nucleoli (n) dark and vacuolated cytoplasm and dilatedintercellular spaces (lowast) Scale bar 2 120583m (d) TEM micrograph of the inferior pole of a basal cell from the corneal epithelium obtained withalcohol delamination on TUE The cytoplasm is dense and few hemidesmosomes (arrows) are present Note the presence of cellular blebs(arrowhead) and of an irregular granular material Scale bar 05 120583m (e) TEM micrograph of superficial cells from the corneal epitheliumof TTE The cells are flat show regular microfolds (arrow) and are well-preserved intercellular borders (arrowhead) Scale bar 5120583m (f)TEMmicrograph of wing cells from the corneal epithelium of TTEMany tonofilaments (t) are evident around the apparently double nucleus(N1-N2) intercellular borders are normal with well-evident desmosomes (arrowhead) Scale bar 2 120583m (g) TEM micrograph of basal cells
from corneal epithelium in TTE Basal cells have round euchromatic nucleus (N) clear cytoplasm with only few vesicles small intercellularwidenings (lowast) and some desmosomes (arrow) Scale bar 2120583m (h) TEM micrograph of the inferior pole of a basal cell from the cornealepithelium in TTE The cytoplasm is clear and the hemidesmosomes (arrows) are numerous lowast = lamina lucida of the basement membraneScale bar 05 120583m
The Scientific World Journal 7
350
300
250
200
150
100
50
0 Trehalose-untreated eyes
Trehalose-treated eyes
Basa
l cel
ls ar
ea (e
xpre
ssed
in120583
m2)
1994 plusmn 421 2583 plusmn 515
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Figure 4Morphometric data of the basal cells area in TUE andTTEexpressed in 120583m2 lowast = 119875 lt 00001
255240225210195180165150135120105
907560453015
0 Trehalose-untreated eyes
Trehalose-treated eyes
781 plusmn 214 1255 plusmn 24
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Mea
n op
tical
den
sity
of b
asal
cells
cyto
plas
m(g
ray
scal
e OU
)
Figure 5 Morphometric data of the optical density of the basalcells cytoplasm in TUE and TTE expressed within the levels from0 (black) to 255 (white) optical units (OU) lowast = 119875 lt 00001
However adverse effects of alcohol on the corneal epithe-lial cells have been shown In particular the following changeswere described flattening of apical microvilli [3] and darkcytoplasm in the superficial cells [11] diffuse interruptions ofthe intercellular junctions with enlargement of the intercel-lular spaces [3 8 9] cellular edema [3] blebs of the cellularmembrane [5 8 9] autophagic vacuoles [5] damages of Hdandof the basementmembrane [4 5] and coexistence of clearand dark cytoplasm in the basal cells [5 8 11] Furthermorethe treatmentwith alcohol resulted in an increase of apoptoticbasal cells in both laboratory animals [2 4] and humans [5]
Therefore a treatment able to reduce the structural andultrastructural changes induced by the exposition to dilutedethanol characterized by the absence of side effects canbe considered useful in maintaining a healthier corneal
epithelium thus possibly improving its morphological andfunctional recovery after the surgical procedure
Trehalose a naturally occurring alpha-linked disaccha-ride formed by two molecules of glucose was consideredfor its well-known protective effects in eukaryotic cells [15]Trehalose synthesized by many living organisms but notmammals showed many different functions In anhydro-biosis that is the capability of surviving prolonged periodsof dessication a better resistance to dryness conditionswas obtained by increasing the intracellular trehalose levelsin animal cells [15] in this way proteins and membraneswere protected from denaturation [20] A reduced deathby desiccation of human corneal epithelium in culture wasobtained with the pretreatment of the cells with a 100mMsolution of trehalose [16] Furthermore trehalosemight act asfree radicals scavenger reducing the oxidative damage of thecornea caused byUVB rays and suppressing the expression ofproinflammatory cytokines [21] Finally trehalose based eyedrops were found to be effective in the treatment of moderateto severe human dry eye [17] as it reduced the number ofdead cells on the ocular surface through the suppression ofapoptosis [18]
In the present study trehalose dosage was chosen on thebasis of previous experiences [16] which demonstrated theeffect of the exposure to trehalose for 151015840 on corneal epithelialcells maintained in cultureThis procedure of administrationwas adopted as it fitted appropriately the timing of thesurgical procedures
The positive action of trehalose on the corneal epitheliumcould be demonstrated by the reduction of the morphologicchanges in TTE and by the evaluation of the morphometricdata
As to the morphological aspects structural changes wereevident in TUE In fact a reduced number and length ofthe apical microfolds of the superficial cells a dark andvacuolated cytoplasm of the basal cells a lower number ofHd and a diffuse enlargement of the intercellular spaceswere observed On the contrary in TTE superficial cellsshowed well represented microfolds and basal cells hadclear cytoplasm with few vesicles and showed less evidentintercellular spaces
Basal cells with clear cytoplasm and euchromatic nucleusare considered the normal cells of the corneal epithelium[22] Noxae acting on the cornea were shown to transformthe structural and ultrastructural morphology of the basalcells which become darker with more condensed chromatinand cytoplasm andwider intercellular spacesThesemorpho-logical aspects have been described either in experimentalsuch as induced vitamin A deficiency [23] or in pathologicalconditions such as macular corneal dystrophy [24] andhereditary Thiel-Behnke corneal dystrophy [25] and wererelated to the concomitant deficiencies in Bowmanrsquos layer[24]
It is well known that following ethanol treatment theintracellular water is removed and replaced by alcohol itselfwith consequent changes in the tertiary structure of proteinsand cellular shrinkage [14] On the contrary when the cornealepithelium was pretreated with trehalose this molecule bysubstituting water molecules and forming hydrogen bonds
8 The Scientific World Journal
could stabilize the three-dimensional structure of the pro-teins [26]
The protective role of trehalose on the corneal epitheliumwas also supported by our morphometric results In factin both corneal epithelial groups the number of layers wassuperimposable and the epithelial thickness was within thenormal values [22] However in TTE the epithelium wasstatistically significantly thicker than TUE FurthermoreTTE showed statistically significantly higher values thanTUEfor basal cells area and cytoplasmatic density It was thuspossible to propose that the disaccharide could preserve thephysiological morphology of the epithelial cells in contrastwith the darker vacuolated appearance induced by theethanol alone
The morphometric analysis showed also that the numberof De between adjacent superficial cells was statisticallysignificantly higher in TTE In addition in both groupsDe of adjacent basal cells were statistically significantlyless numerous between dark cells when compared to thejunctions between clear cells or cleardark cells Even if nostatistical data are currently available as far as we know onDe distribution in the intact corneal epithelium these datacould indicate a better adhesion between cellular membranesin TTE and explain the morphological findings of widerintercellular spaces observed between dark cells
As to the Hd found in the inferior pole of the basalcells their mean number was calculated in 10 120583m of basalmembrane in order to obtain data from single basal cellsIn fact their ultrastructural aspect was uneven being char-acterized by the coexistence of clear and dark cells In thisway the analysis of a single cell could be performed and thevalues could be referred to as the specific morphological celltype Clear cells in the TTE showed normal values of Hdwhen compared to what reported in a previous work [27]whereas clear cells in TUE and dark cells in both groupsshowed statistically significantly lower values In clear cellsHd maintained their regular connection with the laminalucida of the basement membrane thus confirming that thecleavage plane of ethanol-induced corneal epithelial flaps waslocated between the lamina lucida and the lamina densa ofthe basement membrane [7] On the contrary in dark cellsmembrane-bound blebs of the basal pole and an extracellulargranular-filamentous material similar to that observed inpatients with recurrent corneal erosions [12] were presentthus indicating an irregular cleavage of these cells
5 Conclusions
The evidence reported in previous studies indicating thattrehalose is able to improve the corneal epithelial conditionsin course of diseases such as dry eye together with ourfindings shows that it has a direct therapeutic role on theepithelial cells therefore its use could be advantageous inthe treatment of patients with epithelial damage Howeverfurther studies about the clinical outcome are needed toconfirm the validity of its use
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
Theauthors are indebted toMr Sebastiano Brunetto from theDepartment of Biomedical Sciences and MorphofunctionalImaging for the technical assistance
References
[1] D T Azar R T Ang J-B Lee et al ldquoLaser subepithelialkeratomileusis electronmicroscopy and visual outcomes of flapphotorefractive keratectomyrdquo Current Opinion in Ophthalmol-ogy vol 12 no 4 pp 323ndash328 2001
[2] J B Lee J A Javier J H Chang C C Chen T Kato andD T Azar ldquoConfocal and electron microscopic studies oflaser subepithelial keratomileusis (LASEK) in the white leghornchick eyerdquo Archives of Ophthalmology vol 120 no 12 pp 1700ndash1706 2002
[3] S Y KimW J Sah YW Lim and TWHahn ldquoTwenty percentalcohol toxicity on rabbit corneal epithelial cells electronmicroscopic studyrdquo Cornea vol 21 no 4 pp 388ndash392 2002
[4] I K Song andCK Joo ldquoMorphological and functional changesin the rat cornea with an ethanol-mediated epithelial flaprdquoInvestigative Ophthalmology and Visual Science vol 45 no 2pp 423ndash428 2004
[5] C C Chen J H Chang J B Lee J Javier and D TAzar ldquoHuman corneal epithelial cell viability and morphologyafter dilute alcohol exposurerdquo Investigative Ophthalmology andVisual Science vol 43 no 8 pp 2593ndash2602 2002
[6] B Gabler CWinkler VonMohrenfels A K Dreiss J Marshalland C P Lohmann ldquoVitality of epithelial cells after alcoholexposure during laser-assisted subepithelial keratectomy flappreparationrdquo Journal of Cataract and Refractive Surgery vol 28no 10 pp 1841ndash1846 2002
[7] E M Espana M Grueterich A Mateo et al ldquoCleavage ofcorneal basement membrane components by ethanol exposurein laser-assisted subepithelial keratectomyrdquo Journal of Cataractand Refractive Surgery vol 29 no 6 pp 1192ndash1197 2003
[8] I G Pallikaris I I Naoumidi M I Kalyvianaki and V J Kat-sanevaki ldquoEpi-LASIK comparative histological evaluation ofmechanical and alcohol-assisted epithelial separationrdquo Journalof Cataract and Refractive Surgery vol 29 no 8 pp 1496ndash15012003
[9] A C Browning S Shah H S Dua S V Maharajan T Grayand M A Bragheeth ldquoAlcohol debridement of the cornealepithelium in PRK and LASEK an electron microscopic studyrdquoInvestigative Ophthalmology and Visual Science vol 44 no 2pp 510ndash513 2003
[10] J Y Oh J M Yu and J H Ko ldquoAnalysis of ethanol effectson corneal epitheliumrdquo Investigative Ophthalmology and VisualScience vol 54 no 6 pp 3852ndash3856 2013
[11] H S Dua R Lagnado D Raj et al ldquoAlcohol delamination ofthe corneal epithelium an alternative in the management ofrecurrent corneal erosionsrdquo Ophthalmology vol 113 no 3 pp404ndash411 2006
[12] R Mencucci I Paladini B Brahimi U Menchini H S Duaand P Romagnoli ldquoAlcohol delamination in the treatmentof recurrent corneal erosion an electron microscopic studyrdquo
The Scientific World Journal 9
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
Figure 1 (a b) Light micrographs of trypan blue dye test for epithelial cells vitality In both TUE (a) and TTE (b) positive dead cells (arrow)can be observed Scale bar 80 120583m (c d) Light micrographs of TUNEL reaction of corneal epithelium In both TUE (c) and TTE (d) nopositive cells can be observed Scale bar 30 120583m
Table 1 Morphometric parameters of the corneal epithelium in trehalose-untreated eyes (TUE) and trehalose-treated eyes (TTE)
TTE 68 plusmn 07 577 plusmn 41lowast 271 plusmn 31lowast 244 plusmn 51 94 plusmn 16daggerdagger 63 plusmn 08 = 57 plusmn 1lowast 24 plusmn 21lowast 209 plusmn 24lowastDaggerlowast119875 lt 00001 versus TUEdagger119875 lt 00001 versus the other cell junctions within the TUE groupdaggerdagger119875 lt 00001 versus the other cell junctions in TTE group=119875 = 004 versus darkdark cell junctions in TTE groupDagger119875 lt 00001 versus clear cells of the same group
33 Morphometric Data The results of the morphometricanalysis within each group showed (Table 1) that for basalcells in TUE the number of De was 92 plusmn 11 betweenclearclear cells 57 plusmn 1 between cleardark cells and 42 plusmn1 between darkdark cells (clearclear versus cleardark119875 lt 00001 clearclear versus darkdark 119875 lt 00001and cleardark versus darkdark 119875 lt 00001) For Hda statistically significant difference was observed in theirnumber when clear and dark basal cells were compared(219 plusmn 29 and 181 plusmn 15 resp 119875 lt 00001) InTTE the number of De was 94 plusmn 16 between clearclearcells 63 plusmn 08 between cleardark cells and 57 plusmn 1between darkdark cells (clearclear versus cleardark 119875 lt00001 clearclear versus darkdark 119875 lt 00001 andcleardark versus darkdark 119875 = 004) For Hd a statis-tically significant difference was observed in their number
when clear and dark basal cells were compared (24 plusmn 21 and209 plusmn 24 resp 119875 lt 00001)
Comparing the two groups (Table 1) a statistically sig-nificant higher epithelial thickness in TTE was demonstrated(577 plusmn 41 120583m in TTE and 554 plusmn 35 120583m in TUE resp 119875 lt00001) As the number of De their number was statisticallysignificantly higher in TTE between superficial cells (271 plusmn31 in TTE and 201 plusmn 2 in TUE resp 119875 lt 00001) andbetween dark basal cells (57 plusmn 1 in TTE and 42 plusmn 1 in TUEresp 119875 lt 00001) As to the number of Hd present in theinferior pole of the basal cells it was statistically significantlyhigher in TTE in both the clear (24plusmn21 in TTE and 219plusmn29in TUE resp 119875 lt 00001) and the dark cells (209 plusmn 24 inTTE and 181 plusmn 15 in TUE resp 119875 lt 00001)
No statistically significant differences between bothgroups were found for the number of the cellular layers and
The Scientific World Journal 5
S
W
N
S
W
V
V
VN
B
B
lowast
lowastlowast
lowast
lowast
(a) (c)
(b) (d)
Figure 2 (a) Light micrograph from a semithin section of the corneal epithelium obtained with alcohol delamination in TUE Superficialcells show variable shape and optical density (arrow) basal cells have hyperchromic nuclei and large intercellular spaces (double arrow)Scale bar 25 120583m (b) TEM micrograph of the corneal epithelium obtained with alcohol delamination in TUE Superficial cells (S) show areduced number of apical microfolds (arrow) In the wing cells (W) intracellular vesicles and slightly dilated intercellular spaces (lowast) areevident Basal cells (B) are irregular in their shape and size and show large intercellular spaces (lowast) and a cytoplasm filled with vesicle (V)Many blebs (arrowhead) surrounded by granular material are evident Scale bar 5 120583m (c) Light micrograph from a semithin section of thecorneal epithelium in TTE Both superficial and wing cells show normal shape and well-evident intercellular borders (arrow) basal cells havepolygonal shape andmany small intracytoplasmatic vesicles (double arrow) Scale bar 25120583m (d) TEMmicrograph of the corneal epitheliumin TTE Superficial cells (S) show a normal flattened shape with well-evident apical microfolds (arrow)Wing cells (W) have uniform electrondensity and normal intercellular spaces (arrowhead) Basal cells (B) have variable electron density polygonal shape few cytoplasmatic vesicles(V) round nuclei (N) and small intercellular spaces (lowast) Scale bar 5 120583m
for the number of De between wing cells clearclear basalcells and cleardark basal cells
When the basal cells area was evaluated a statisticallysignificant difference was found between TUE and TTE(1994 plusmn 421 120583m2 and 2583 plusmn 515 120583m2 resp 119875 lt 00001)(Figure 4)
Considering the optical density of the cytoplasm of thebasal cells a statistically significant difference was foundbetween TUE and TTE (1255 plusmn 24OU and 781 plusmn 214 resp119875 lt 00001) (Figure 5)
4 Discussion
Diluted ethanol is currently used in patients undergoingLASEK to reduce the adhesion of the corneal epitheliumto Bowmanrsquos layer in order to expose the stroma for thesubsequent PRK treatment The theoretical advantage ofLASEK is derived from the repositioning of the epithelialflap over the laser-ablated corneal surface so facilitating thecorneal epithelial healing reducing pain and inflammationand decreasing the stromal haze [4]
6 The Scientific World Journal
N
NN
NN
t
n
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 3 (a) TEM micrograph of superficial cells from the corneal epithelium obtained with alcohol delamination on TUE The cells areflat and show short and irregular microfolds (arrow) and dilated intercellular spaces (lowast) Scale bar 5 120583m (b) TEM micrograph of wing cellsfrom the corneal epithelium obtained with alcohol delamination on TUE The nucleus (N) and the cytoplasm have normal appearanceintercellular spaces are moderately dilated (lowast) Scale bar 2 120583m (c) TEM micrograph of basal cells from the corneal epithelium obtainedwith alcohol delamination on TUEThe cells show elliptical nuclei (N) with evident nucleoli (n) dark and vacuolated cytoplasm and dilatedintercellular spaces (lowast) Scale bar 2 120583m (d) TEM micrograph of the inferior pole of a basal cell from the corneal epithelium obtained withalcohol delamination on TUE The cytoplasm is dense and few hemidesmosomes (arrows) are present Note the presence of cellular blebs(arrowhead) and of an irregular granular material Scale bar 05 120583m (e) TEM micrograph of superficial cells from the corneal epitheliumof TTE The cells are flat show regular microfolds (arrow) and are well-preserved intercellular borders (arrowhead) Scale bar 5120583m (f)TEMmicrograph of wing cells from the corneal epithelium of TTEMany tonofilaments (t) are evident around the apparently double nucleus(N1-N2) intercellular borders are normal with well-evident desmosomes (arrowhead) Scale bar 2 120583m (g) TEM micrograph of basal cells
from corneal epithelium in TTE Basal cells have round euchromatic nucleus (N) clear cytoplasm with only few vesicles small intercellularwidenings (lowast) and some desmosomes (arrow) Scale bar 2120583m (h) TEM micrograph of the inferior pole of a basal cell from the cornealepithelium in TTE The cytoplasm is clear and the hemidesmosomes (arrows) are numerous lowast = lamina lucida of the basement membraneScale bar 05 120583m
The Scientific World Journal 7
350
300
250
200
150
100
50
0 Trehalose-untreated eyes
Trehalose-treated eyes
Basa
l cel
ls ar
ea (e
xpre
ssed
in120583
m2)
1994 plusmn 421 2583 plusmn 515
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Figure 4Morphometric data of the basal cells area in TUE andTTEexpressed in 120583m2 lowast = 119875 lt 00001
255240225210195180165150135120105
907560453015
0 Trehalose-untreated eyes
Trehalose-treated eyes
781 plusmn 214 1255 plusmn 24
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Mea
n op
tical
den
sity
of b
asal
cells
cyto
plas
m(g
ray
scal
e OU
)
Figure 5 Morphometric data of the optical density of the basalcells cytoplasm in TUE and TTE expressed within the levels from0 (black) to 255 (white) optical units (OU) lowast = 119875 lt 00001
However adverse effects of alcohol on the corneal epithe-lial cells have been shown In particular the following changeswere described flattening of apical microvilli [3] and darkcytoplasm in the superficial cells [11] diffuse interruptions ofthe intercellular junctions with enlargement of the intercel-lular spaces [3 8 9] cellular edema [3] blebs of the cellularmembrane [5 8 9] autophagic vacuoles [5] damages of Hdandof the basementmembrane [4 5] and coexistence of clearand dark cytoplasm in the basal cells [5 8 11] Furthermorethe treatmentwith alcohol resulted in an increase of apoptoticbasal cells in both laboratory animals [2 4] and humans [5]
Therefore a treatment able to reduce the structural andultrastructural changes induced by the exposition to dilutedethanol characterized by the absence of side effects canbe considered useful in maintaining a healthier corneal
epithelium thus possibly improving its morphological andfunctional recovery after the surgical procedure
Trehalose a naturally occurring alpha-linked disaccha-ride formed by two molecules of glucose was consideredfor its well-known protective effects in eukaryotic cells [15]Trehalose synthesized by many living organisms but notmammals showed many different functions In anhydro-biosis that is the capability of surviving prolonged periodsof dessication a better resistance to dryness conditionswas obtained by increasing the intracellular trehalose levelsin animal cells [15] in this way proteins and membraneswere protected from denaturation [20] A reduced deathby desiccation of human corneal epithelium in culture wasobtained with the pretreatment of the cells with a 100mMsolution of trehalose [16] Furthermore trehalosemight act asfree radicals scavenger reducing the oxidative damage of thecornea caused byUVB rays and suppressing the expression ofproinflammatory cytokines [21] Finally trehalose based eyedrops were found to be effective in the treatment of moderateto severe human dry eye [17] as it reduced the number ofdead cells on the ocular surface through the suppression ofapoptosis [18]
In the present study trehalose dosage was chosen on thebasis of previous experiences [16] which demonstrated theeffect of the exposure to trehalose for 151015840 on corneal epithelialcells maintained in cultureThis procedure of administrationwas adopted as it fitted appropriately the timing of thesurgical procedures
The positive action of trehalose on the corneal epitheliumcould be demonstrated by the reduction of the morphologicchanges in TTE and by the evaluation of the morphometricdata
As to the morphological aspects structural changes wereevident in TUE In fact a reduced number and length ofthe apical microfolds of the superficial cells a dark andvacuolated cytoplasm of the basal cells a lower number ofHd and a diffuse enlargement of the intercellular spaceswere observed On the contrary in TTE superficial cellsshowed well represented microfolds and basal cells hadclear cytoplasm with few vesicles and showed less evidentintercellular spaces
Basal cells with clear cytoplasm and euchromatic nucleusare considered the normal cells of the corneal epithelium[22] Noxae acting on the cornea were shown to transformthe structural and ultrastructural morphology of the basalcells which become darker with more condensed chromatinand cytoplasm andwider intercellular spacesThesemorpho-logical aspects have been described either in experimentalsuch as induced vitamin A deficiency [23] or in pathologicalconditions such as macular corneal dystrophy [24] andhereditary Thiel-Behnke corneal dystrophy [25] and wererelated to the concomitant deficiencies in Bowmanrsquos layer[24]
It is well known that following ethanol treatment theintracellular water is removed and replaced by alcohol itselfwith consequent changes in the tertiary structure of proteinsand cellular shrinkage [14] On the contrary when the cornealepithelium was pretreated with trehalose this molecule bysubstituting water molecules and forming hydrogen bonds
8 The Scientific World Journal
could stabilize the three-dimensional structure of the pro-teins [26]
The protective role of trehalose on the corneal epitheliumwas also supported by our morphometric results In factin both corneal epithelial groups the number of layers wassuperimposable and the epithelial thickness was within thenormal values [22] However in TTE the epithelium wasstatistically significantly thicker than TUE FurthermoreTTE showed statistically significantly higher values thanTUEfor basal cells area and cytoplasmatic density It was thuspossible to propose that the disaccharide could preserve thephysiological morphology of the epithelial cells in contrastwith the darker vacuolated appearance induced by theethanol alone
The morphometric analysis showed also that the numberof De between adjacent superficial cells was statisticallysignificantly higher in TTE In addition in both groupsDe of adjacent basal cells were statistically significantlyless numerous between dark cells when compared to thejunctions between clear cells or cleardark cells Even if nostatistical data are currently available as far as we know onDe distribution in the intact corneal epithelium these datacould indicate a better adhesion between cellular membranesin TTE and explain the morphological findings of widerintercellular spaces observed between dark cells
As to the Hd found in the inferior pole of the basalcells their mean number was calculated in 10 120583m of basalmembrane in order to obtain data from single basal cellsIn fact their ultrastructural aspect was uneven being char-acterized by the coexistence of clear and dark cells In thisway the analysis of a single cell could be performed and thevalues could be referred to as the specific morphological celltype Clear cells in the TTE showed normal values of Hdwhen compared to what reported in a previous work [27]whereas clear cells in TUE and dark cells in both groupsshowed statistically significantly lower values In clear cellsHd maintained their regular connection with the laminalucida of the basement membrane thus confirming that thecleavage plane of ethanol-induced corneal epithelial flaps waslocated between the lamina lucida and the lamina densa ofthe basement membrane [7] On the contrary in dark cellsmembrane-bound blebs of the basal pole and an extracellulargranular-filamentous material similar to that observed inpatients with recurrent corneal erosions [12] were presentthus indicating an irregular cleavage of these cells
5 Conclusions
The evidence reported in previous studies indicating thattrehalose is able to improve the corneal epithelial conditionsin course of diseases such as dry eye together with ourfindings shows that it has a direct therapeutic role on theepithelial cells therefore its use could be advantageous inthe treatment of patients with epithelial damage Howeverfurther studies about the clinical outcome are needed toconfirm the validity of its use
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
Theauthors are indebted toMr Sebastiano Brunetto from theDepartment of Biomedical Sciences and MorphofunctionalImaging for the technical assistance
References
[1] D T Azar R T Ang J-B Lee et al ldquoLaser subepithelialkeratomileusis electronmicroscopy and visual outcomes of flapphotorefractive keratectomyrdquo Current Opinion in Ophthalmol-ogy vol 12 no 4 pp 323ndash328 2001
[2] J B Lee J A Javier J H Chang C C Chen T Kato andD T Azar ldquoConfocal and electron microscopic studies oflaser subepithelial keratomileusis (LASEK) in the white leghornchick eyerdquo Archives of Ophthalmology vol 120 no 12 pp 1700ndash1706 2002
[3] S Y KimW J Sah YW Lim and TWHahn ldquoTwenty percentalcohol toxicity on rabbit corneal epithelial cells electronmicroscopic studyrdquo Cornea vol 21 no 4 pp 388ndash392 2002
[4] I K Song andCK Joo ldquoMorphological and functional changesin the rat cornea with an ethanol-mediated epithelial flaprdquoInvestigative Ophthalmology and Visual Science vol 45 no 2pp 423ndash428 2004
[5] C C Chen J H Chang J B Lee J Javier and D TAzar ldquoHuman corneal epithelial cell viability and morphologyafter dilute alcohol exposurerdquo Investigative Ophthalmology andVisual Science vol 43 no 8 pp 2593ndash2602 2002
[6] B Gabler CWinkler VonMohrenfels A K Dreiss J Marshalland C P Lohmann ldquoVitality of epithelial cells after alcoholexposure during laser-assisted subepithelial keratectomy flappreparationrdquo Journal of Cataract and Refractive Surgery vol 28no 10 pp 1841ndash1846 2002
[7] E M Espana M Grueterich A Mateo et al ldquoCleavage ofcorneal basement membrane components by ethanol exposurein laser-assisted subepithelial keratectomyrdquo Journal of Cataractand Refractive Surgery vol 29 no 6 pp 1192ndash1197 2003
[8] I G Pallikaris I I Naoumidi M I Kalyvianaki and V J Kat-sanevaki ldquoEpi-LASIK comparative histological evaluation ofmechanical and alcohol-assisted epithelial separationrdquo Journalof Cataract and Refractive Surgery vol 29 no 8 pp 1496ndash15012003
[9] A C Browning S Shah H S Dua S V Maharajan T Grayand M A Bragheeth ldquoAlcohol debridement of the cornealepithelium in PRK and LASEK an electron microscopic studyrdquoInvestigative Ophthalmology and Visual Science vol 44 no 2pp 510ndash513 2003
[10] J Y Oh J M Yu and J H Ko ldquoAnalysis of ethanol effectson corneal epitheliumrdquo Investigative Ophthalmology and VisualScience vol 54 no 6 pp 3852ndash3856 2013
[11] H S Dua R Lagnado D Raj et al ldquoAlcohol delamination ofthe corneal epithelium an alternative in the management ofrecurrent corneal erosionsrdquo Ophthalmology vol 113 no 3 pp404ndash411 2006
[12] R Mencucci I Paladini B Brahimi U Menchini H S Duaand P Romagnoli ldquoAlcohol delamination in the treatmentof recurrent corneal erosion an electron microscopic studyrdquo
The Scientific World Journal 9
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
Figure 2 (a) Light micrograph from a semithin section of the corneal epithelium obtained with alcohol delamination in TUE Superficialcells show variable shape and optical density (arrow) basal cells have hyperchromic nuclei and large intercellular spaces (double arrow)Scale bar 25 120583m (b) TEM micrograph of the corneal epithelium obtained with alcohol delamination in TUE Superficial cells (S) show areduced number of apical microfolds (arrow) In the wing cells (W) intracellular vesicles and slightly dilated intercellular spaces (lowast) areevident Basal cells (B) are irregular in their shape and size and show large intercellular spaces (lowast) and a cytoplasm filled with vesicle (V)Many blebs (arrowhead) surrounded by granular material are evident Scale bar 5 120583m (c) Light micrograph from a semithin section of thecorneal epithelium in TTE Both superficial and wing cells show normal shape and well-evident intercellular borders (arrow) basal cells havepolygonal shape andmany small intracytoplasmatic vesicles (double arrow) Scale bar 25120583m (d) TEMmicrograph of the corneal epitheliumin TTE Superficial cells (S) show a normal flattened shape with well-evident apical microfolds (arrow)Wing cells (W) have uniform electrondensity and normal intercellular spaces (arrowhead) Basal cells (B) have variable electron density polygonal shape few cytoplasmatic vesicles(V) round nuclei (N) and small intercellular spaces (lowast) Scale bar 5 120583m
for the number of De between wing cells clearclear basalcells and cleardark basal cells
When the basal cells area was evaluated a statisticallysignificant difference was found between TUE and TTE(1994 plusmn 421 120583m2 and 2583 plusmn 515 120583m2 resp 119875 lt 00001)(Figure 4)
Considering the optical density of the cytoplasm of thebasal cells a statistically significant difference was foundbetween TUE and TTE (1255 plusmn 24OU and 781 plusmn 214 resp119875 lt 00001) (Figure 5)
4 Discussion
Diluted ethanol is currently used in patients undergoingLASEK to reduce the adhesion of the corneal epitheliumto Bowmanrsquos layer in order to expose the stroma for thesubsequent PRK treatment The theoretical advantage ofLASEK is derived from the repositioning of the epithelialflap over the laser-ablated corneal surface so facilitating thecorneal epithelial healing reducing pain and inflammationand decreasing the stromal haze [4]
6 The Scientific World Journal
N
NN
NN
t
n
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 3 (a) TEM micrograph of superficial cells from the corneal epithelium obtained with alcohol delamination on TUE The cells areflat and show short and irregular microfolds (arrow) and dilated intercellular spaces (lowast) Scale bar 5 120583m (b) TEM micrograph of wing cellsfrom the corneal epithelium obtained with alcohol delamination on TUE The nucleus (N) and the cytoplasm have normal appearanceintercellular spaces are moderately dilated (lowast) Scale bar 2 120583m (c) TEM micrograph of basal cells from the corneal epithelium obtainedwith alcohol delamination on TUEThe cells show elliptical nuclei (N) with evident nucleoli (n) dark and vacuolated cytoplasm and dilatedintercellular spaces (lowast) Scale bar 2 120583m (d) TEM micrograph of the inferior pole of a basal cell from the corneal epithelium obtained withalcohol delamination on TUE The cytoplasm is dense and few hemidesmosomes (arrows) are present Note the presence of cellular blebs(arrowhead) and of an irregular granular material Scale bar 05 120583m (e) TEM micrograph of superficial cells from the corneal epitheliumof TTE The cells are flat show regular microfolds (arrow) and are well-preserved intercellular borders (arrowhead) Scale bar 5120583m (f)TEMmicrograph of wing cells from the corneal epithelium of TTEMany tonofilaments (t) are evident around the apparently double nucleus(N1-N2) intercellular borders are normal with well-evident desmosomes (arrowhead) Scale bar 2 120583m (g) TEM micrograph of basal cells
from corneal epithelium in TTE Basal cells have round euchromatic nucleus (N) clear cytoplasm with only few vesicles small intercellularwidenings (lowast) and some desmosomes (arrow) Scale bar 2120583m (h) TEM micrograph of the inferior pole of a basal cell from the cornealepithelium in TTE The cytoplasm is clear and the hemidesmosomes (arrows) are numerous lowast = lamina lucida of the basement membraneScale bar 05 120583m
The Scientific World Journal 7
350
300
250
200
150
100
50
0 Trehalose-untreated eyes
Trehalose-treated eyes
Basa
l cel
ls ar
ea (e
xpre
ssed
in120583
m2)
1994 plusmn 421 2583 plusmn 515
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Figure 4Morphometric data of the basal cells area in TUE andTTEexpressed in 120583m2 lowast = 119875 lt 00001
255240225210195180165150135120105
907560453015
0 Trehalose-untreated eyes
Trehalose-treated eyes
781 plusmn 214 1255 plusmn 24
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Mea
n op
tical
den
sity
of b
asal
cells
cyto
plas
m(g
ray
scal
e OU
)
Figure 5 Morphometric data of the optical density of the basalcells cytoplasm in TUE and TTE expressed within the levels from0 (black) to 255 (white) optical units (OU) lowast = 119875 lt 00001
However adverse effects of alcohol on the corneal epithe-lial cells have been shown In particular the following changeswere described flattening of apical microvilli [3] and darkcytoplasm in the superficial cells [11] diffuse interruptions ofthe intercellular junctions with enlargement of the intercel-lular spaces [3 8 9] cellular edema [3] blebs of the cellularmembrane [5 8 9] autophagic vacuoles [5] damages of Hdandof the basementmembrane [4 5] and coexistence of clearand dark cytoplasm in the basal cells [5 8 11] Furthermorethe treatmentwith alcohol resulted in an increase of apoptoticbasal cells in both laboratory animals [2 4] and humans [5]
Therefore a treatment able to reduce the structural andultrastructural changes induced by the exposition to dilutedethanol characterized by the absence of side effects canbe considered useful in maintaining a healthier corneal
epithelium thus possibly improving its morphological andfunctional recovery after the surgical procedure
Trehalose a naturally occurring alpha-linked disaccha-ride formed by two molecules of glucose was consideredfor its well-known protective effects in eukaryotic cells [15]Trehalose synthesized by many living organisms but notmammals showed many different functions In anhydro-biosis that is the capability of surviving prolonged periodsof dessication a better resistance to dryness conditionswas obtained by increasing the intracellular trehalose levelsin animal cells [15] in this way proteins and membraneswere protected from denaturation [20] A reduced deathby desiccation of human corneal epithelium in culture wasobtained with the pretreatment of the cells with a 100mMsolution of trehalose [16] Furthermore trehalosemight act asfree radicals scavenger reducing the oxidative damage of thecornea caused byUVB rays and suppressing the expression ofproinflammatory cytokines [21] Finally trehalose based eyedrops were found to be effective in the treatment of moderateto severe human dry eye [17] as it reduced the number ofdead cells on the ocular surface through the suppression ofapoptosis [18]
In the present study trehalose dosage was chosen on thebasis of previous experiences [16] which demonstrated theeffect of the exposure to trehalose for 151015840 on corneal epithelialcells maintained in cultureThis procedure of administrationwas adopted as it fitted appropriately the timing of thesurgical procedures
The positive action of trehalose on the corneal epitheliumcould be demonstrated by the reduction of the morphologicchanges in TTE and by the evaluation of the morphometricdata
As to the morphological aspects structural changes wereevident in TUE In fact a reduced number and length ofthe apical microfolds of the superficial cells a dark andvacuolated cytoplasm of the basal cells a lower number ofHd and a diffuse enlargement of the intercellular spaceswere observed On the contrary in TTE superficial cellsshowed well represented microfolds and basal cells hadclear cytoplasm with few vesicles and showed less evidentintercellular spaces
Basal cells with clear cytoplasm and euchromatic nucleusare considered the normal cells of the corneal epithelium[22] Noxae acting on the cornea were shown to transformthe structural and ultrastructural morphology of the basalcells which become darker with more condensed chromatinand cytoplasm andwider intercellular spacesThesemorpho-logical aspects have been described either in experimentalsuch as induced vitamin A deficiency [23] or in pathologicalconditions such as macular corneal dystrophy [24] andhereditary Thiel-Behnke corneal dystrophy [25] and wererelated to the concomitant deficiencies in Bowmanrsquos layer[24]
It is well known that following ethanol treatment theintracellular water is removed and replaced by alcohol itselfwith consequent changes in the tertiary structure of proteinsand cellular shrinkage [14] On the contrary when the cornealepithelium was pretreated with trehalose this molecule bysubstituting water molecules and forming hydrogen bonds
8 The Scientific World Journal
could stabilize the three-dimensional structure of the pro-teins [26]
The protective role of trehalose on the corneal epitheliumwas also supported by our morphometric results In factin both corneal epithelial groups the number of layers wassuperimposable and the epithelial thickness was within thenormal values [22] However in TTE the epithelium wasstatistically significantly thicker than TUE FurthermoreTTE showed statistically significantly higher values thanTUEfor basal cells area and cytoplasmatic density It was thuspossible to propose that the disaccharide could preserve thephysiological morphology of the epithelial cells in contrastwith the darker vacuolated appearance induced by theethanol alone
The morphometric analysis showed also that the numberof De between adjacent superficial cells was statisticallysignificantly higher in TTE In addition in both groupsDe of adjacent basal cells were statistically significantlyless numerous between dark cells when compared to thejunctions between clear cells or cleardark cells Even if nostatistical data are currently available as far as we know onDe distribution in the intact corneal epithelium these datacould indicate a better adhesion between cellular membranesin TTE and explain the morphological findings of widerintercellular spaces observed between dark cells
As to the Hd found in the inferior pole of the basalcells their mean number was calculated in 10 120583m of basalmembrane in order to obtain data from single basal cellsIn fact their ultrastructural aspect was uneven being char-acterized by the coexistence of clear and dark cells In thisway the analysis of a single cell could be performed and thevalues could be referred to as the specific morphological celltype Clear cells in the TTE showed normal values of Hdwhen compared to what reported in a previous work [27]whereas clear cells in TUE and dark cells in both groupsshowed statistically significantly lower values In clear cellsHd maintained their regular connection with the laminalucida of the basement membrane thus confirming that thecleavage plane of ethanol-induced corneal epithelial flaps waslocated between the lamina lucida and the lamina densa ofthe basement membrane [7] On the contrary in dark cellsmembrane-bound blebs of the basal pole and an extracellulargranular-filamentous material similar to that observed inpatients with recurrent corneal erosions [12] were presentthus indicating an irregular cleavage of these cells
5 Conclusions
The evidence reported in previous studies indicating thattrehalose is able to improve the corneal epithelial conditionsin course of diseases such as dry eye together with ourfindings shows that it has a direct therapeutic role on theepithelial cells therefore its use could be advantageous inthe treatment of patients with epithelial damage Howeverfurther studies about the clinical outcome are needed toconfirm the validity of its use
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
Theauthors are indebted toMr Sebastiano Brunetto from theDepartment of Biomedical Sciences and MorphofunctionalImaging for the technical assistance
References
[1] D T Azar R T Ang J-B Lee et al ldquoLaser subepithelialkeratomileusis electronmicroscopy and visual outcomes of flapphotorefractive keratectomyrdquo Current Opinion in Ophthalmol-ogy vol 12 no 4 pp 323ndash328 2001
[2] J B Lee J A Javier J H Chang C C Chen T Kato andD T Azar ldquoConfocal and electron microscopic studies oflaser subepithelial keratomileusis (LASEK) in the white leghornchick eyerdquo Archives of Ophthalmology vol 120 no 12 pp 1700ndash1706 2002
[3] S Y KimW J Sah YW Lim and TWHahn ldquoTwenty percentalcohol toxicity on rabbit corneal epithelial cells electronmicroscopic studyrdquo Cornea vol 21 no 4 pp 388ndash392 2002
[4] I K Song andCK Joo ldquoMorphological and functional changesin the rat cornea with an ethanol-mediated epithelial flaprdquoInvestigative Ophthalmology and Visual Science vol 45 no 2pp 423ndash428 2004
[5] C C Chen J H Chang J B Lee J Javier and D TAzar ldquoHuman corneal epithelial cell viability and morphologyafter dilute alcohol exposurerdquo Investigative Ophthalmology andVisual Science vol 43 no 8 pp 2593ndash2602 2002
[6] B Gabler CWinkler VonMohrenfels A K Dreiss J Marshalland C P Lohmann ldquoVitality of epithelial cells after alcoholexposure during laser-assisted subepithelial keratectomy flappreparationrdquo Journal of Cataract and Refractive Surgery vol 28no 10 pp 1841ndash1846 2002
[7] E M Espana M Grueterich A Mateo et al ldquoCleavage ofcorneal basement membrane components by ethanol exposurein laser-assisted subepithelial keratectomyrdquo Journal of Cataractand Refractive Surgery vol 29 no 6 pp 1192ndash1197 2003
[8] I G Pallikaris I I Naoumidi M I Kalyvianaki and V J Kat-sanevaki ldquoEpi-LASIK comparative histological evaluation ofmechanical and alcohol-assisted epithelial separationrdquo Journalof Cataract and Refractive Surgery vol 29 no 8 pp 1496ndash15012003
[9] A C Browning S Shah H S Dua S V Maharajan T Grayand M A Bragheeth ldquoAlcohol debridement of the cornealepithelium in PRK and LASEK an electron microscopic studyrdquoInvestigative Ophthalmology and Visual Science vol 44 no 2pp 510ndash513 2003
[10] J Y Oh J M Yu and J H Ko ldquoAnalysis of ethanol effectson corneal epitheliumrdquo Investigative Ophthalmology and VisualScience vol 54 no 6 pp 3852ndash3856 2013
[11] H S Dua R Lagnado D Raj et al ldquoAlcohol delamination ofthe corneal epithelium an alternative in the management ofrecurrent corneal erosionsrdquo Ophthalmology vol 113 no 3 pp404ndash411 2006
[12] R Mencucci I Paladini B Brahimi U Menchini H S Duaand P Romagnoli ldquoAlcohol delamination in the treatmentof recurrent corneal erosion an electron microscopic studyrdquo
The Scientific World Journal 9
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
Figure 3 (a) TEM micrograph of superficial cells from the corneal epithelium obtained with alcohol delamination on TUE The cells areflat and show short and irregular microfolds (arrow) and dilated intercellular spaces (lowast) Scale bar 5 120583m (b) TEM micrograph of wing cellsfrom the corneal epithelium obtained with alcohol delamination on TUE The nucleus (N) and the cytoplasm have normal appearanceintercellular spaces are moderately dilated (lowast) Scale bar 2 120583m (c) TEM micrograph of basal cells from the corneal epithelium obtainedwith alcohol delamination on TUEThe cells show elliptical nuclei (N) with evident nucleoli (n) dark and vacuolated cytoplasm and dilatedintercellular spaces (lowast) Scale bar 2 120583m (d) TEM micrograph of the inferior pole of a basal cell from the corneal epithelium obtained withalcohol delamination on TUE The cytoplasm is dense and few hemidesmosomes (arrows) are present Note the presence of cellular blebs(arrowhead) and of an irregular granular material Scale bar 05 120583m (e) TEM micrograph of superficial cells from the corneal epitheliumof TTE The cells are flat show regular microfolds (arrow) and are well-preserved intercellular borders (arrowhead) Scale bar 5120583m (f)TEMmicrograph of wing cells from the corneal epithelium of TTEMany tonofilaments (t) are evident around the apparently double nucleus(N1-N2) intercellular borders are normal with well-evident desmosomes (arrowhead) Scale bar 2 120583m (g) TEM micrograph of basal cells
from corneal epithelium in TTE Basal cells have round euchromatic nucleus (N) clear cytoplasm with only few vesicles small intercellularwidenings (lowast) and some desmosomes (arrow) Scale bar 2120583m (h) TEM micrograph of the inferior pole of a basal cell from the cornealepithelium in TTE The cytoplasm is clear and the hemidesmosomes (arrows) are numerous lowast = lamina lucida of the basement membraneScale bar 05 120583m
The Scientific World Journal 7
350
300
250
200
150
100
50
0 Trehalose-untreated eyes
Trehalose-treated eyes
Basa
l cel
ls ar
ea (e
xpre
ssed
in120583
m2)
1994 plusmn 421 2583 plusmn 515
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Figure 4Morphometric data of the basal cells area in TUE andTTEexpressed in 120583m2 lowast = 119875 lt 00001
255240225210195180165150135120105
907560453015
0 Trehalose-untreated eyes
Trehalose-treated eyes
781 plusmn 214 1255 plusmn 24
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Mea
n op
tical
den
sity
of b
asal
cells
cyto
plas
m(g
ray
scal
e OU
)
Figure 5 Morphometric data of the optical density of the basalcells cytoplasm in TUE and TTE expressed within the levels from0 (black) to 255 (white) optical units (OU) lowast = 119875 lt 00001
However adverse effects of alcohol on the corneal epithe-lial cells have been shown In particular the following changeswere described flattening of apical microvilli [3] and darkcytoplasm in the superficial cells [11] diffuse interruptions ofthe intercellular junctions with enlargement of the intercel-lular spaces [3 8 9] cellular edema [3] blebs of the cellularmembrane [5 8 9] autophagic vacuoles [5] damages of Hdandof the basementmembrane [4 5] and coexistence of clearand dark cytoplasm in the basal cells [5 8 11] Furthermorethe treatmentwith alcohol resulted in an increase of apoptoticbasal cells in both laboratory animals [2 4] and humans [5]
Therefore a treatment able to reduce the structural andultrastructural changes induced by the exposition to dilutedethanol characterized by the absence of side effects canbe considered useful in maintaining a healthier corneal
epithelium thus possibly improving its morphological andfunctional recovery after the surgical procedure
Trehalose a naturally occurring alpha-linked disaccha-ride formed by two molecules of glucose was consideredfor its well-known protective effects in eukaryotic cells [15]Trehalose synthesized by many living organisms but notmammals showed many different functions In anhydro-biosis that is the capability of surviving prolonged periodsof dessication a better resistance to dryness conditionswas obtained by increasing the intracellular trehalose levelsin animal cells [15] in this way proteins and membraneswere protected from denaturation [20] A reduced deathby desiccation of human corneal epithelium in culture wasobtained with the pretreatment of the cells with a 100mMsolution of trehalose [16] Furthermore trehalosemight act asfree radicals scavenger reducing the oxidative damage of thecornea caused byUVB rays and suppressing the expression ofproinflammatory cytokines [21] Finally trehalose based eyedrops were found to be effective in the treatment of moderateto severe human dry eye [17] as it reduced the number ofdead cells on the ocular surface through the suppression ofapoptosis [18]
In the present study trehalose dosage was chosen on thebasis of previous experiences [16] which demonstrated theeffect of the exposure to trehalose for 151015840 on corneal epithelialcells maintained in cultureThis procedure of administrationwas adopted as it fitted appropriately the timing of thesurgical procedures
The positive action of trehalose on the corneal epitheliumcould be demonstrated by the reduction of the morphologicchanges in TTE and by the evaluation of the morphometricdata
As to the morphological aspects structural changes wereevident in TUE In fact a reduced number and length ofthe apical microfolds of the superficial cells a dark andvacuolated cytoplasm of the basal cells a lower number ofHd and a diffuse enlargement of the intercellular spaceswere observed On the contrary in TTE superficial cellsshowed well represented microfolds and basal cells hadclear cytoplasm with few vesicles and showed less evidentintercellular spaces
Basal cells with clear cytoplasm and euchromatic nucleusare considered the normal cells of the corneal epithelium[22] Noxae acting on the cornea were shown to transformthe structural and ultrastructural morphology of the basalcells which become darker with more condensed chromatinand cytoplasm andwider intercellular spacesThesemorpho-logical aspects have been described either in experimentalsuch as induced vitamin A deficiency [23] or in pathologicalconditions such as macular corneal dystrophy [24] andhereditary Thiel-Behnke corneal dystrophy [25] and wererelated to the concomitant deficiencies in Bowmanrsquos layer[24]
It is well known that following ethanol treatment theintracellular water is removed and replaced by alcohol itselfwith consequent changes in the tertiary structure of proteinsand cellular shrinkage [14] On the contrary when the cornealepithelium was pretreated with trehalose this molecule bysubstituting water molecules and forming hydrogen bonds
8 The Scientific World Journal
could stabilize the three-dimensional structure of the pro-teins [26]
The protective role of trehalose on the corneal epitheliumwas also supported by our morphometric results In factin both corneal epithelial groups the number of layers wassuperimposable and the epithelial thickness was within thenormal values [22] However in TTE the epithelium wasstatistically significantly thicker than TUE FurthermoreTTE showed statistically significantly higher values thanTUEfor basal cells area and cytoplasmatic density It was thuspossible to propose that the disaccharide could preserve thephysiological morphology of the epithelial cells in contrastwith the darker vacuolated appearance induced by theethanol alone
The morphometric analysis showed also that the numberof De between adjacent superficial cells was statisticallysignificantly higher in TTE In addition in both groupsDe of adjacent basal cells were statistically significantlyless numerous between dark cells when compared to thejunctions between clear cells or cleardark cells Even if nostatistical data are currently available as far as we know onDe distribution in the intact corneal epithelium these datacould indicate a better adhesion between cellular membranesin TTE and explain the morphological findings of widerintercellular spaces observed between dark cells
As to the Hd found in the inferior pole of the basalcells their mean number was calculated in 10 120583m of basalmembrane in order to obtain data from single basal cellsIn fact their ultrastructural aspect was uneven being char-acterized by the coexistence of clear and dark cells In thisway the analysis of a single cell could be performed and thevalues could be referred to as the specific morphological celltype Clear cells in the TTE showed normal values of Hdwhen compared to what reported in a previous work [27]whereas clear cells in TUE and dark cells in both groupsshowed statistically significantly lower values In clear cellsHd maintained their regular connection with the laminalucida of the basement membrane thus confirming that thecleavage plane of ethanol-induced corneal epithelial flaps waslocated between the lamina lucida and the lamina densa ofthe basement membrane [7] On the contrary in dark cellsmembrane-bound blebs of the basal pole and an extracellulargranular-filamentous material similar to that observed inpatients with recurrent corneal erosions [12] were presentthus indicating an irregular cleavage of these cells
5 Conclusions
The evidence reported in previous studies indicating thattrehalose is able to improve the corneal epithelial conditionsin course of diseases such as dry eye together with ourfindings shows that it has a direct therapeutic role on theepithelial cells therefore its use could be advantageous inthe treatment of patients with epithelial damage Howeverfurther studies about the clinical outcome are needed toconfirm the validity of its use
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
Theauthors are indebted toMr Sebastiano Brunetto from theDepartment of Biomedical Sciences and MorphofunctionalImaging for the technical assistance
References
[1] D T Azar R T Ang J-B Lee et al ldquoLaser subepithelialkeratomileusis electronmicroscopy and visual outcomes of flapphotorefractive keratectomyrdquo Current Opinion in Ophthalmol-ogy vol 12 no 4 pp 323ndash328 2001
[2] J B Lee J A Javier J H Chang C C Chen T Kato andD T Azar ldquoConfocal and electron microscopic studies oflaser subepithelial keratomileusis (LASEK) in the white leghornchick eyerdquo Archives of Ophthalmology vol 120 no 12 pp 1700ndash1706 2002
[3] S Y KimW J Sah YW Lim and TWHahn ldquoTwenty percentalcohol toxicity on rabbit corneal epithelial cells electronmicroscopic studyrdquo Cornea vol 21 no 4 pp 388ndash392 2002
[4] I K Song andCK Joo ldquoMorphological and functional changesin the rat cornea with an ethanol-mediated epithelial flaprdquoInvestigative Ophthalmology and Visual Science vol 45 no 2pp 423ndash428 2004
[5] C C Chen J H Chang J B Lee J Javier and D TAzar ldquoHuman corneal epithelial cell viability and morphologyafter dilute alcohol exposurerdquo Investigative Ophthalmology andVisual Science vol 43 no 8 pp 2593ndash2602 2002
[6] B Gabler CWinkler VonMohrenfels A K Dreiss J Marshalland C P Lohmann ldquoVitality of epithelial cells after alcoholexposure during laser-assisted subepithelial keratectomy flappreparationrdquo Journal of Cataract and Refractive Surgery vol 28no 10 pp 1841ndash1846 2002
[7] E M Espana M Grueterich A Mateo et al ldquoCleavage ofcorneal basement membrane components by ethanol exposurein laser-assisted subepithelial keratectomyrdquo Journal of Cataractand Refractive Surgery vol 29 no 6 pp 1192ndash1197 2003
[8] I G Pallikaris I I Naoumidi M I Kalyvianaki and V J Kat-sanevaki ldquoEpi-LASIK comparative histological evaluation ofmechanical and alcohol-assisted epithelial separationrdquo Journalof Cataract and Refractive Surgery vol 29 no 8 pp 1496ndash15012003
[9] A C Browning S Shah H S Dua S V Maharajan T Grayand M A Bragheeth ldquoAlcohol debridement of the cornealepithelium in PRK and LASEK an electron microscopic studyrdquoInvestigative Ophthalmology and Visual Science vol 44 no 2pp 510ndash513 2003
[10] J Y Oh J M Yu and J H Ko ldquoAnalysis of ethanol effectson corneal epitheliumrdquo Investigative Ophthalmology and VisualScience vol 54 no 6 pp 3852ndash3856 2013
[11] H S Dua R Lagnado D Raj et al ldquoAlcohol delamination ofthe corneal epithelium an alternative in the management ofrecurrent corneal erosionsrdquo Ophthalmology vol 113 no 3 pp404ndash411 2006
[12] R Mencucci I Paladini B Brahimi U Menchini H S Duaand P Romagnoli ldquoAlcohol delamination in the treatmentof recurrent corneal erosion an electron microscopic studyrdquo
The Scientific World Journal 9
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
Figure 4Morphometric data of the basal cells area in TUE andTTEexpressed in 120583m2 lowast = 119875 lt 00001
255240225210195180165150135120105
907560453015
0 Trehalose-untreated eyes
Trehalose-treated eyes
781 plusmn 214 1255 plusmn 24
lowast
lowastP lt 00001 versus trehalose-untreated eyes
Mea
n op
tical
den
sity
of b
asal
cells
cyto
plas
m(g
ray
scal
e OU
)
Figure 5 Morphometric data of the optical density of the basalcells cytoplasm in TUE and TTE expressed within the levels from0 (black) to 255 (white) optical units (OU) lowast = 119875 lt 00001
However adverse effects of alcohol on the corneal epithe-lial cells have been shown In particular the following changeswere described flattening of apical microvilli [3] and darkcytoplasm in the superficial cells [11] diffuse interruptions ofthe intercellular junctions with enlargement of the intercel-lular spaces [3 8 9] cellular edema [3] blebs of the cellularmembrane [5 8 9] autophagic vacuoles [5] damages of Hdandof the basementmembrane [4 5] and coexistence of clearand dark cytoplasm in the basal cells [5 8 11] Furthermorethe treatmentwith alcohol resulted in an increase of apoptoticbasal cells in both laboratory animals [2 4] and humans [5]
Therefore a treatment able to reduce the structural andultrastructural changes induced by the exposition to dilutedethanol characterized by the absence of side effects canbe considered useful in maintaining a healthier corneal
epithelium thus possibly improving its morphological andfunctional recovery after the surgical procedure
Trehalose a naturally occurring alpha-linked disaccha-ride formed by two molecules of glucose was consideredfor its well-known protective effects in eukaryotic cells [15]Trehalose synthesized by many living organisms but notmammals showed many different functions In anhydro-biosis that is the capability of surviving prolonged periodsof dessication a better resistance to dryness conditionswas obtained by increasing the intracellular trehalose levelsin animal cells [15] in this way proteins and membraneswere protected from denaturation [20] A reduced deathby desiccation of human corneal epithelium in culture wasobtained with the pretreatment of the cells with a 100mMsolution of trehalose [16] Furthermore trehalosemight act asfree radicals scavenger reducing the oxidative damage of thecornea caused byUVB rays and suppressing the expression ofproinflammatory cytokines [21] Finally trehalose based eyedrops were found to be effective in the treatment of moderateto severe human dry eye [17] as it reduced the number ofdead cells on the ocular surface through the suppression ofapoptosis [18]
In the present study trehalose dosage was chosen on thebasis of previous experiences [16] which demonstrated theeffect of the exposure to trehalose for 151015840 on corneal epithelialcells maintained in cultureThis procedure of administrationwas adopted as it fitted appropriately the timing of thesurgical procedures
The positive action of trehalose on the corneal epitheliumcould be demonstrated by the reduction of the morphologicchanges in TTE and by the evaluation of the morphometricdata
As to the morphological aspects structural changes wereevident in TUE In fact a reduced number and length ofthe apical microfolds of the superficial cells a dark andvacuolated cytoplasm of the basal cells a lower number ofHd and a diffuse enlargement of the intercellular spaceswere observed On the contrary in TTE superficial cellsshowed well represented microfolds and basal cells hadclear cytoplasm with few vesicles and showed less evidentintercellular spaces
Basal cells with clear cytoplasm and euchromatic nucleusare considered the normal cells of the corneal epithelium[22] Noxae acting on the cornea were shown to transformthe structural and ultrastructural morphology of the basalcells which become darker with more condensed chromatinand cytoplasm andwider intercellular spacesThesemorpho-logical aspects have been described either in experimentalsuch as induced vitamin A deficiency [23] or in pathologicalconditions such as macular corneal dystrophy [24] andhereditary Thiel-Behnke corneal dystrophy [25] and wererelated to the concomitant deficiencies in Bowmanrsquos layer[24]
It is well known that following ethanol treatment theintracellular water is removed and replaced by alcohol itselfwith consequent changes in the tertiary structure of proteinsand cellular shrinkage [14] On the contrary when the cornealepithelium was pretreated with trehalose this molecule bysubstituting water molecules and forming hydrogen bonds
8 The Scientific World Journal
could stabilize the three-dimensional structure of the pro-teins [26]
The protective role of trehalose on the corneal epitheliumwas also supported by our morphometric results In factin both corneal epithelial groups the number of layers wassuperimposable and the epithelial thickness was within thenormal values [22] However in TTE the epithelium wasstatistically significantly thicker than TUE FurthermoreTTE showed statistically significantly higher values thanTUEfor basal cells area and cytoplasmatic density It was thuspossible to propose that the disaccharide could preserve thephysiological morphology of the epithelial cells in contrastwith the darker vacuolated appearance induced by theethanol alone
The morphometric analysis showed also that the numberof De between adjacent superficial cells was statisticallysignificantly higher in TTE In addition in both groupsDe of adjacent basal cells were statistically significantlyless numerous between dark cells when compared to thejunctions between clear cells or cleardark cells Even if nostatistical data are currently available as far as we know onDe distribution in the intact corneal epithelium these datacould indicate a better adhesion between cellular membranesin TTE and explain the morphological findings of widerintercellular spaces observed between dark cells
As to the Hd found in the inferior pole of the basalcells their mean number was calculated in 10 120583m of basalmembrane in order to obtain data from single basal cellsIn fact their ultrastructural aspect was uneven being char-acterized by the coexistence of clear and dark cells In thisway the analysis of a single cell could be performed and thevalues could be referred to as the specific morphological celltype Clear cells in the TTE showed normal values of Hdwhen compared to what reported in a previous work [27]whereas clear cells in TUE and dark cells in both groupsshowed statistically significantly lower values In clear cellsHd maintained their regular connection with the laminalucida of the basement membrane thus confirming that thecleavage plane of ethanol-induced corneal epithelial flaps waslocated between the lamina lucida and the lamina densa ofthe basement membrane [7] On the contrary in dark cellsmembrane-bound blebs of the basal pole and an extracellulargranular-filamentous material similar to that observed inpatients with recurrent corneal erosions [12] were presentthus indicating an irregular cleavage of these cells
5 Conclusions
The evidence reported in previous studies indicating thattrehalose is able to improve the corneal epithelial conditionsin course of diseases such as dry eye together with ourfindings shows that it has a direct therapeutic role on theepithelial cells therefore its use could be advantageous inthe treatment of patients with epithelial damage Howeverfurther studies about the clinical outcome are needed toconfirm the validity of its use
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
Theauthors are indebted toMr Sebastiano Brunetto from theDepartment of Biomedical Sciences and MorphofunctionalImaging for the technical assistance
References
[1] D T Azar R T Ang J-B Lee et al ldquoLaser subepithelialkeratomileusis electronmicroscopy and visual outcomes of flapphotorefractive keratectomyrdquo Current Opinion in Ophthalmol-ogy vol 12 no 4 pp 323ndash328 2001
[2] J B Lee J A Javier J H Chang C C Chen T Kato andD T Azar ldquoConfocal and electron microscopic studies oflaser subepithelial keratomileusis (LASEK) in the white leghornchick eyerdquo Archives of Ophthalmology vol 120 no 12 pp 1700ndash1706 2002
[3] S Y KimW J Sah YW Lim and TWHahn ldquoTwenty percentalcohol toxicity on rabbit corneal epithelial cells electronmicroscopic studyrdquo Cornea vol 21 no 4 pp 388ndash392 2002
[4] I K Song andCK Joo ldquoMorphological and functional changesin the rat cornea with an ethanol-mediated epithelial flaprdquoInvestigative Ophthalmology and Visual Science vol 45 no 2pp 423ndash428 2004
[5] C C Chen J H Chang J B Lee J Javier and D TAzar ldquoHuman corneal epithelial cell viability and morphologyafter dilute alcohol exposurerdquo Investigative Ophthalmology andVisual Science vol 43 no 8 pp 2593ndash2602 2002
[6] B Gabler CWinkler VonMohrenfels A K Dreiss J Marshalland C P Lohmann ldquoVitality of epithelial cells after alcoholexposure during laser-assisted subepithelial keratectomy flappreparationrdquo Journal of Cataract and Refractive Surgery vol 28no 10 pp 1841ndash1846 2002
[7] E M Espana M Grueterich A Mateo et al ldquoCleavage ofcorneal basement membrane components by ethanol exposurein laser-assisted subepithelial keratectomyrdquo Journal of Cataractand Refractive Surgery vol 29 no 6 pp 1192ndash1197 2003
[8] I G Pallikaris I I Naoumidi M I Kalyvianaki and V J Kat-sanevaki ldquoEpi-LASIK comparative histological evaluation ofmechanical and alcohol-assisted epithelial separationrdquo Journalof Cataract and Refractive Surgery vol 29 no 8 pp 1496ndash15012003
[9] A C Browning S Shah H S Dua S V Maharajan T Grayand M A Bragheeth ldquoAlcohol debridement of the cornealepithelium in PRK and LASEK an electron microscopic studyrdquoInvestigative Ophthalmology and Visual Science vol 44 no 2pp 510ndash513 2003
[10] J Y Oh J M Yu and J H Ko ldquoAnalysis of ethanol effectson corneal epitheliumrdquo Investigative Ophthalmology and VisualScience vol 54 no 6 pp 3852ndash3856 2013
[11] H S Dua R Lagnado D Raj et al ldquoAlcohol delamination ofthe corneal epithelium an alternative in the management ofrecurrent corneal erosionsrdquo Ophthalmology vol 113 no 3 pp404ndash411 2006
[12] R Mencucci I Paladini B Brahimi U Menchini H S Duaand P Romagnoli ldquoAlcohol delamination in the treatmentof recurrent corneal erosion an electron microscopic studyrdquo
The Scientific World Journal 9
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
could stabilize the three-dimensional structure of the pro-teins [26]
The protective role of trehalose on the corneal epitheliumwas also supported by our morphometric results In factin both corneal epithelial groups the number of layers wassuperimposable and the epithelial thickness was within thenormal values [22] However in TTE the epithelium wasstatistically significantly thicker than TUE FurthermoreTTE showed statistically significantly higher values thanTUEfor basal cells area and cytoplasmatic density It was thuspossible to propose that the disaccharide could preserve thephysiological morphology of the epithelial cells in contrastwith the darker vacuolated appearance induced by theethanol alone
The morphometric analysis showed also that the numberof De between adjacent superficial cells was statisticallysignificantly higher in TTE In addition in both groupsDe of adjacent basal cells were statistically significantlyless numerous between dark cells when compared to thejunctions between clear cells or cleardark cells Even if nostatistical data are currently available as far as we know onDe distribution in the intact corneal epithelium these datacould indicate a better adhesion between cellular membranesin TTE and explain the morphological findings of widerintercellular spaces observed between dark cells
As to the Hd found in the inferior pole of the basalcells their mean number was calculated in 10 120583m of basalmembrane in order to obtain data from single basal cellsIn fact their ultrastructural aspect was uneven being char-acterized by the coexistence of clear and dark cells In thisway the analysis of a single cell could be performed and thevalues could be referred to as the specific morphological celltype Clear cells in the TTE showed normal values of Hdwhen compared to what reported in a previous work [27]whereas clear cells in TUE and dark cells in both groupsshowed statistically significantly lower values In clear cellsHd maintained their regular connection with the laminalucida of the basement membrane thus confirming that thecleavage plane of ethanol-induced corneal epithelial flaps waslocated between the lamina lucida and the lamina densa ofthe basement membrane [7] On the contrary in dark cellsmembrane-bound blebs of the basal pole and an extracellulargranular-filamentous material similar to that observed inpatients with recurrent corneal erosions [12] were presentthus indicating an irregular cleavage of these cells
5 Conclusions
The evidence reported in previous studies indicating thattrehalose is able to improve the corneal epithelial conditionsin course of diseases such as dry eye together with ourfindings shows that it has a direct therapeutic role on theepithelial cells therefore its use could be advantageous inthe treatment of patients with epithelial damage Howeverfurther studies about the clinical outcome are needed toconfirm the validity of its use
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
Theauthors are indebted toMr Sebastiano Brunetto from theDepartment of Biomedical Sciences and MorphofunctionalImaging for the technical assistance
References
[1] D T Azar R T Ang J-B Lee et al ldquoLaser subepithelialkeratomileusis electronmicroscopy and visual outcomes of flapphotorefractive keratectomyrdquo Current Opinion in Ophthalmol-ogy vol 12 no 4 pp 323ndash328 2001
[2] J B Lee J A Javier J H Chang C C Chen T Kato andD T Azar ldquoConfocal and electron microscopic studies oflaser subepithelial keratomileusis (LASEK) in the white leghornchick eyerdquo Archives of Ophthalmology vol 120 no 12 pp 1700ndash1706 2002
[3] S Y KimW J Sah YW Lim and TWHahn ldquoTwenty percentalcohol toxicity on rabbit corneal epithelial cells electronmicroscopic studyrdquo Cornea vol 21 no 4 pp 388ndash392 2002
[4] I K Song andCK Joo ldquoMorphological and functional changesin the rat cornea with an ethanol-mediated epithelial flaprdquoInvestigative Ophthalmology and Visual Science vol 45 no 2pp 423ndash428 2004
[5] C C Chen J H Chang J B Lee J Javier and D TAzar ldquoHuman corneal epithelial cell viability and morphologyafter dilute alcohol exposurerdquo Investigative Ophthalmology andVisual Science vol 43 no 8 pp 2593ndash2602 2002
[6] B Gabler CWinkler VonMohrenfels A K Dreiss J Marshalland C P Lohmann ldquoVitality of epithelial cells after alcoholexposure during laser-assisted subepithelial keratectomy flappreparationrdquo Journal of Cataract and Refractive Surgery vol 28no 10 pp 1841ndash1846 2002
[7] E M Espana M Grueterich A Mateo et al ldquoCleavage ofcorneal basement membrane components by ethanol exposurein laser-assisted subepithelial keratectomyrdquo Journal of Cataractand Refractive Surgery vol 29 no 6 pp 1192ndash1197 2003
[8] I G Pallikaris I I Naoumidi M I Kalyvianaki and V J Kat-sanevaki ldquoEpi-LASIK comparative histological evaluation ofmechanical and alcohol-assisted epithelial separationrdquo Journalof Cataract and Refractive Surgery vol 29 no 8 pp 1496ndash15012003
[9] A C Browning S Shah H S Dua S V Maharajan T Grayand M A Bragheeth ldquoAlcohol debridement of the cornealepithelium in PRK and LASEK an electron microscopic studyrdquoInvestigative Ophthalmology and Visual Science vol 44 no 2pp 510ndash513 2003
[10] J Y Oh J M Yu and J H Ko ldquoAnalysis of ethanol effectson corneal epitheliumrdquo Investigative Ophthalmology and VisualScience vol 54 no 6 pp 3852ndash3856 2013
[11] H S Dua R Lagnado D Raj et al ldquoAlcohol delamination ofthe corneal epithelium an alternative in the management ofrecurrent corneal erosionsrdquo Ophthalmology vol 113 no 3 pp404ndash411 2006
[12] R Mencucci I Paladini B Brahimi U Menchini H S Duaand P Romagnoli ldquoAlcohol delamination in the treatmentof recurrent corneal erosion an electron microscopic studyrdquo
The Scientific World Journal 9
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
British Journal of Ophthalmology vol 94 no 7 pp 933ndash9392010
[13] I Eltoum J Fredenburgh R B Myers and W E GrizzleldquoIntroduction to the theory and practice of fixation of tissuesrdquoJournal of Histotechnology vol 24 no 3 pp 173ndash190 2001
[14] P K Gupta and ZW BalochCytohistology Essentials and BasicConcepts Cambridge University Press New York NY USA2011
[15] J Luyckx and C Baudouin ldquoTrehalose an intriguing disaccha-ride with potential for medical application in ophthalmologyrdquoClinical Ophthalmology vol 5 no 1 pp 577ndash581 2011
[16] T Matsuo ldquoTrehalose protects corneal epithelial cells fromdeath by dryingrdquo British Journal of Ophthalmology vol 85 no5 pp 610ndash612 2001
[17] T Matsuo Y Tsuchida and N Morimoto ldquoTrehalose eye dropsin the treatment of dry eye syndromerdquoOphthalmology vol 109no 11 pp 2024ndash2029 2002
[18] W Chen X Zhang M Liu et al ldquoTrehalose protects againstocular surface disorders in experimental murine dry eyethrough suppression of apoptosisrdquo Experimental Eye Researchvol 89 no 3 pp 311ndash318 2009
[19] M A Ekici O Uysal H I Cikriklar et al ldquoEffect of etanerceptand lithium chloride on preventing secondary tissue damagein rats with experimental diffuse severe brain injuryrdquo EuropeanReview For Medical and Pharmacological Sciences vol 18 no 1pp 10ndash27 2014
[20] N Guo I Puhlev D R Brown J Mansbridge and F LevineldquoTrehalose expression confers desiccation tolerance on humancellsrdquo Nature Biotechnology vol 18 no 2 pp 168ndash171 2000
[21] J Cejkova T Ardan C Cejka and J Luyckx ldquoFavorableeffects of trehalose on the development of UVB-mediatedantioxidantpro-oxidant imbalance in the corneal epitheliumproinflammatory cytokine andmatrixmetalloproteinase induc-tion and heat shock protein 70 expressionrdquo Graefes Archive forClinical and Experimental Ophthalmology vol 249 no 8 pp1185ndash1194 2011
[22] M J Hogan J A Alvarado and J E Weddel Histology of theHuman Eye Saunders Philadelphia Pa USA 1971
[23] N B K Shams L A Hanninen H V Chaves et al ldquoEffect ofvitamin A deficiency on the adhesion of rat corneal epitheliumand the basement membrane complexrdquo Investigative Ophthal-mology and Visual Science vol 34 no 9 pp 2646ndash2654 1993
[24] A Micali A Pisani D Puzzolo et al ldquoMacular corneal dystro-phy in vivo confocal and structural datardquo Ophthalmology vol121 no 6 pp 1164ndash1173 2014
[25] H C Gear K Ramaesh and F Roberts ldquoRecurrence of Thiel-Behnke corneal dystrophy an electron microscopic studyrdquoBritish Journal of Ophthalmology vol 89 no 4 p 518 2005
[26] N K Jain and I Roy ldquoEffect of trehalose on protein structurerdquoProtein Science vol 18 no 1 pp 24ndash36 2009
[27] M D Resch U Schlotzer-Schrehardt C Hofmann-RummeltF E Kruse and B Seitz ldquoAlterations of epithelial adhesionmolecules and basement membrane components in latticecorneal dystrophy (LCD)rdquo Graefes Archive for Clinical andExperimental Ophthalmology vol 247 no 8 pp 1081ndash10882009
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