A Mou Model for the Study of Recurrent Corneal Epithelial Erosions:␣91Integrin Implicated in Progression of the Dia
Sonali Pal-Ghosh,1Ahdeah Pajoohesh-Ganji,1,2Marcus Brown,1and Mary Ann Stepp1,3
P URPOSE.To describe an in vivo mou model for the study of recurrent corneal erosion syndrome(RCES)in mice and to characterize the changes in␣9integrin expression during wound healing.
M ETHODS.Corneal epithelial debridement wounds of two sizes (1.5and2.5mm)were made on the ocular surface of BALB/c mice and were evaluated at various times after wounding. Corneas were procesd either as whole mounts and stained with propidium iodide and an antibody against␣9integrin or for bromodeoxyuridine analys of cell proliferation.A pa-rate study involved analys of corneal wound healing over time in individual mice with large and small debridement wounds.Mice were anesthetized once per week and their corneas stained withfluorescein to asss the quality of the corneal epithelium.After6weeks,mice were killed and eyes procesd for study by immunofluorescence in either whole mounts or frozen ctions.
R ESULTS.Whole mount confocal microscopy showed open wounds on the ocular surface of mice at
1and2weeks after large wounds were created,but not after small wounds.In addition,␣9integrin was upregulated during healing,and changes were obrved in␣9integrin localization at the limbus with large wounds but not with small wounds.Although only 1of16corneas with small wounds had erosions at1and2 weeks,11of16with large wounds had erosions.However,by 6weeks,13of16eyes showed signs of erosion whether wounds were small or large.With large wounds,RCES corneas frequently showed numerous goblet cells adjacent to a limbus lacking␣9integrin.Corneas from mice with documented RCES showed both retention of␣9integrin and tenascin-C expres-sion at the anterior stromal–epithelial interface as well as impaired relocalization of␣31integrin to the bament mem-brane zone.
C ONCLUSIONS.The data show that spontaneous recurrent cor-neal erosions occur in a mou model after manual creation of a single wound by debridement.Differences between the heal-ing of small(1.5mm)and large(2.5mm)wounds were ob-rved.Large wounds often resulted in the prence of goblet cells on the central cornea and a loss of␣9integrin at the limbus.Small wounds never showed differences in the local-ization of␣9integrin at the limbus,and no goblet cells were obrved in the central cornea.More studies are needed to understand the caus of erosions in the mice.(Invest Ophthal-mol Vis Sci.2004;45:1775–1788)DOI:10.1167/iovs.03-1194 P atients with
recurrent corneal erosion syndrome(RCES) experience pain,discomfort,and a reduced quality of life.1,2The two major caus of RCES are trauma or various forms of epithelial bament membrane dystrophy,and the dia process can involve small erosions that heal within a few hours(microcytic)or large erosions that take days or weeks to heal(macrocytic).3,4Most erosions can be treated nonsurgically and generally heal well,but some persist and require intervention.Current surgical interventions include phototherapeutic keratectomy,5diamond burr superficial ker-atectomy,6and intrastromal puncture.7,8However,if the dis-ea is caud by a bament membrane dystrophy,the patient is likely to experience chronic episodes of painful erosions. Trauma to the ocular surface is the most frequent ophthalmic complaint in emergency rooms,and some studies have esti-mated erosions to occur after trauma as frequently as1in every 150cas.9The most common site for erosions is the inferior third of the cornea,and women are treated for RCES more often than men.3
The apparent cau of RCES is failure of the epithelial cells to regain tight adhesive contacts with the underlying corneal stroma after trauma.In epithelial bament membrane dia, it is the failure of the epithelial cells to maintain tight adhesion to a defective bament membrane.Cell adhesion molecules of the integrin family are in large part responsible for the adhe-sion of the corneal epitheliu
m to the stroma.Integrins function as␣heterodimers and are involved in mediating actin-bad cell adhesion and migration and intermediatefilament-bad cell–substrate adhesion through hemidesmosomes(HDs).HDs are the adhesion complexes that mediate tight adhesion of skin and corneal epithelium to their underlying bament mem-branes.10,11They are compod—from inside to outside the cell—of keratin intermediatefilaments,plectin,BP230,BP180,␣64integrin,laminin-5anchoringfilaments,and type VII collagen anchoringfibrils.12,13Plectin and BP230form the intracellular plaque of the HD and mediate interaction with keratins.BP180and␣64integrin are both integral membrane components of HDs,laminin-5is a specialized component of the bament membrane,and type VII collagenfibrils pene-trate deep into the corneal stroma to ensurefirm adhesion of the epithelium to the underlying connective tissue.In the cornea the optical clarity esntial to allow light to focus on the retina requires an optically smooth surface.This anatomic necessity results in an expod,sheer surface vulnerable to debridement by friction and rubbing.During migration after an injury,HDs disasmble,and epithelial sheet migration allows open wounds to be covered quickly and efficiently.11,14Once migration is complete,the HDs must reasmble to restore tight epithelial adhesion.
Efforts to determine the molecular origin of and to develop more effective treatments for RCES have been slowed by the
From the Departments of1Anatomy and Cell Biology and2Biolog-
ical Sciences,The George Washington University,Washington,DC;
and the3Department of Ophthalmology,The George Washington
University Medical Center,Washington,DC.
Supported by the National Eye Institute/National Institutes of
Health Grant EY08512–14(MAS).
Submitted for publication October31,2003;revid January21,
2004;accepted February4,2004.
Disclosure:S.Pal-Ghosh,None;A.Pajoohesh-Ganji,None;M.
网络营销的定义Brown,None;M.A.Stepp,None
The publication costs of this article were defrayed in part by page
charge payment.This article must therefore be marked“adverti-
ment”in accordance with18U.S.C.§1734solely to indicate this fact.
Corresponding author:Mary Ann Stepp,2300I Street NW,Wash-
ington DC,20037;mastepp@gwu.edu.
Investigative Ophthalmology&Visual Science,June2004,Vol.45,No.6
Copyright©Association for Rearch in Vision and Ophthalmology1775
lack of rodent animal models.Studies of spontaneous erosions have typically ud various purebred dogs known to be sus-ceptible to RCES.15,16Other studies have looked at chronic corneal erosion in animals by repetitive mechanical debride-ment of the cornea.17,18For a number of years,we have studied the role of cell adhesion molecules in regulating reepi-thelialization after mechanical debridement of rat and mou corneas,11,19–22using a model in which a circular area of epithelial cells was removed with a dulled scalpel.The sizes of the wounds varied,depending on the study;either a1.5-or a 2.5-mm diameter region of corneal epithelial cells was rou-tinely removed from the central corneas of8-week-old mice. The epithelial bament membrane was left intact at the time
of injury and was not denatured by chemical or thermal means after wounding.
Among the proteins who localizations change after wound-ing is␣9integrin.22In the normal,unwounded adult mou limbus␣9integrin localizes to the apical aspect of the basal cells where it is found both in the cytoplasm and in the cell membrane.23Studies of the developing mou eye have shown that this limbal basal cell localization is a relatively late event in the development of the ocular surface.When evaluated at1 week after birth,mice have a uniform distribution of␣9inte-grin over the entire ocular surface.By2weeks,this distribu-tion pattern changes dramatically,and the central corneal ep-ithelial cells express less␣9integrin and the limbal basal cells more.The adult pattern is established by between6and8 weeks.23
Becau the focus of our past work has been reepithelial-ization,our efforts have focud primarily on the study of healing over short time spans—generally,from hours to v-eral days.However,to provide strain-specific mou control corneas for studies of knockout mice,we began to asss corneas at time points from1to12weeks after wounding. During the cour of tho studies,we obrved that,after a single,initial manual debridement wound to an8-week-old BALB/c mou cornea,corneal epithelial erosions occurred spontaneously,at times ranging from1to8weeks after wounding.The results that follow—from studies of wild-type healthy BALB/c mice—are an extension
of initial obrvations after studies of genetically altered mice.24
In the current studies,we characterize,for thefirst time,an in vivo mou model of erosions that spontaneously developed in respon to a single,initial corneal debridement wound.We show that the RCES epithelium was characterized by both a higher cell proliferation rate than that of unwounded corneal epithelium and by altered localization of␣9integrin.Goblet cells were obrved on the corneal surface with large wounds but not with small wounds.We then assd the healing history of the individual eyes over time(1–6weeks after wounding),usingfluorescein to demonstrate that we were describing RCES rather than chronic open wounds.We were thus able to obrve recurrent erosions in the BALB/c mou after creating both small and large wounds.With the healing history documented for individual eyes,we demonstrated that after the large wounds were made,most RCES eyes had less␣9 integrin at the limbus and numerous goblet cells prent over the corneal surface.Finally,we show that,in RCES eyes,␣31 integrin localization was disrupted,but␣64was localized primarily at the basal aspect of the basal cells.The data will improve our understanding of RCES in both animals and hu-mans.Furthermore,having an experimental model to test hy-pothes about caus and possible treatments for RCES should,in time,reduce the pain and improve the quality of life of people with this condition.M ETHODS
Materials and Chemicals
Most of the chemicals ud in the studies were obtained from
Sigma-Aldrich(St.Louis,MO),including EGTA,bovine rum albumin (BSA),sodium dodecyl sulfate(SDS),and hor rum.Ketamine was purchad from Fort Dodge Veterinary Supplies(Fort Dodge,IA), xylazine from Vedco(St.Joph,MO),and sodium pentobarbital from Veterinary Laboratories,Inc.(Lenexa,KS).Optimal cutting tempera-ture(OCT)compound(Tissue Tek II)was obtained from Laboratory Tek(Napierville,IL),and proparacaine eye drops and erythromycin ophthalmic ointment were obtained from Bausch&Lomb(Tampa,FL). We purchad coverslips and paraformaldehyde from Fisher Scientific (Somerville,NJ),slides from Shandon(Pittsburgh,PA),and the pap pen and mounting medium(Fluormount)from Electron Microscopy Sci-ences(Washington,PA).Cell proliferation was studied using a5-bro-mo-2Ј-deoxy-uridine(BrdU)labeling and detection kit(catalog no.1 296736;Roche Diagnostics,Indianapolis,IN).For detecting mou primary antibodies on mou tissue,another kit(MOM;FMK-2201; Vector Laboratories,Burlingame,CA)was ud.For primary antibod-ies,we ud a rat anti-mou antibody against tenascin-C(Mtn-12, T-3413;Sigma-Aldrich),a rat anti-mou4monoclonal antibody(346-11A,catalog no.09491D;BD-Pharmingen,San Diego,CA),and a monoclonal anti-mou antibody agai
nst mucin5AC Ab-1(45M1,cat-alog no.MS-145-P1;NeoMarkers,Fremont,CA).For␣3and␣9,we ud polyclonal antibodies we had previously characterized.25Pro-pidium iodide(PI;P-1304),Alexa Fluor488anti-rabbit(A-11,006), anti-mou(A-11,001),and Alexa Fluor594anti-rat(A-11,007)cond-ary antibodies were obtained from Molecular Probes(Eugene,OR). Manual Corneal Debridement
All experiments described in this article were conducted in voluntary compliance with the ARVO Statement for the U of Animals in Oph-thalmic and Vision Rearch,and all procedures were approved by the George Washington University Animal Care and U Committee.Eight to10-week-old male and female BALB/c mice(22to25g)were anesthetized with250L of a1:10dilution of a1:1mixture of ketamine(100mg/mL;Aveco Co.,Inc.,Fort Dodge,IA)and xylazine (20mg/mL;Miles Inc.,Shawnee Mission,KS).Once the animals were anesthetized,a topical anesthetic(proparacaine ophthalmic solution) was applied to their ocular surfaces until the blink nsation was lost, and their corneas were scraped with a dull scalpel to remove the corneal epithelium.For small wounds,a1.5-mm central corneal area was demarcated with a trephine and removed manually by gentle scraping with a dulled blade.For large wounds,a2.5-mm area of epithelial tissue was removed,with care taken to avoid limbal blood vesls.After they were wounded,all eyes were treated with erythro-mycin ophthalmic ointment to minimize inflammation and to keep the ocular surface moist while the mice w
ere under anesthesia.Corneas with large wounds were allowed to heal in vivo for1,1.5,2,and3 days,and1,2,and4weeks and tho with small wounds for6,12,18, 24,48,and72hours,and1and2weeks.At least three eyes were assd for each time point for the data prented in Figures1to5. After mice were killed by lethal injection using sodium pentobarbital, eyes were enucleated and either frozen in OCT compound for immu-nofluorescence(IF)orfixed as whole mounts.
Whole mounts
承诺书有法律效力吗At the indicated time points,mice were killed and eyes were removed andfixed for2hours in a4:1dilution of prechilled100%methanol and dimethyl sulfoxide atϪ20°C and then stored in100%methanol.All eyes were transferred into phosphate-buffered saline(PBS)in a graded methanol ries(70%,50%,and30%methanol and PBS,30minutes each).PBS(10ϫ)was made as follows:14.4g Na
2
HPO
4
长白山旅游攻略, 2.4g
KH
2作文心愿
HPO
4
,2g KCl,80g NaCl,in a total volume of1liter of water(pH 7.4).All incubations were performed with gentle shaking and at room temperature,unless otherwi specified.After the eyes were washed
1776Pal-Ghosh et al.IOVS,June2004,Vol.45,No.6
in PBS twice,for30minutes each,they were incubated with blocking buffer for2hours.Blocking buffer was made as follows:to100mL1ϫPBS,1g of BSA was added,the mixture was stirred for30minutes,1 mL of hor rum was added,and the mixture was stirred for an additional minute.The tissues were then incubated overnight with primary antibody,at4°C.The next day,the tissues were washedfive times with PBS and0.02%Tween20(PBST)for1hour each,blocked for2hours,and then incubated with
中元节八大禁忌goat anti-rabbit Alexa488c-ondary antibody overnight at4°C.The next day,the eyes were washed three times with PBST for1hour each,followed by nuclear staining with PI for5minutes,and three washes with double-distilled water (Millipore,Bedford,MA)for5minutes each.For the goblet cell stain-ing,the blocking buffer from the antibody detection kit(MOM;Vector Laboratories)was ud.Then,under a discting microscope(Model SZ40;Olympus,Lake Success,Melville,NY),the retina,lens,and iris were discarded,and four incisions were made in each cornea.To achieve the bestflattening,the corneas were placed epithelial-side-up on a blackfilter(25mm,0.45m,HABG02500;Millipore),mounting medium was then added,and they were coverslipped.The images were then captured with a confocal microscope.
Immunofluorescence Microscopy on
Tissue Sections
For ctions,adult BALB/c mice were killed with a lethal injection,and eyes were frozen in OCT compound.Eight-micrometer cryostat c-tions were cut from the eyes and baked overnight at37°C.The ctions were then rehydrated in PBS for15minutes,blocked with blocking buffer,and incubated with primary antibodies(␣3,␣9,and4integrin and tenascin-C)for1hour in a moist chamber
at room temperature. After they were washed with PBS,the ctions were treated with blocking buffer for15minutes and were incubated with the appropri-ate Alexa condary antibodies for45minutes in a moist chamber. They were then washed with blocking buffer for15minutes,covered in mounting medium,and coverslipped.The images were then cap-tured with a confocal microscope.
BrdU Cell Proliferation Analys
Cell proliferation was assd using the BrdU labeling and detection kit,as recommended by the manufacturer.All incubations were at room temperature unless specified.We assd time points at1.5and 3days,and1,2,and3weeks for large wounds,and4and8weeks for small wounds.Corneas were wounded as described earlier.Control (unwounded)eyes were obtained from mice that had not been sub-jected to wounding of either eye.At the given time points,the mice were killed and their eyes enucleated and placed immediately into a BrdU-containing labeling solution with complete minimum esntial medium(MEM).22After30minutes in the labeling solution,the eyes were washed three times in MEM without BrdU and allowed to incu-bate for an additional15minutes in MEM.Whole eyes were embedded in OCT,and8-m ctions cut and baked overnight at37°C.Next, ctions were rehydrated in PBS andfixed in ice-cold70%methanol in 50mM glycine buffer(pH2)for20minutes atϪ20°C.Afterfixation, ctions were incubated in PBS twice,for2minutes each,then with 2.5%trypsin
in0.1%CaCl
2
in PBS,for3minutes.They were then briefly washed in PBS and transferred to a solution containing4M HCl, for3minutes.Sections were washed twice in PBS,for5minutes each, and then transferred to blocking buffer.Slides were procesd for IF as recommended by the manufacturer(Roche Diagnostics).At least three corneas and two slides per time point were ud—each slide contain-ing four to six corneal cross ctions.The number of labeled cells in at least three different ctions from each slide were counted and the data expresd as the number of cells labeled per unit length of basal cell basal membrane,as measured with image-analysis software(Im-agePro Plus ver.4.1software;Media Cybernetics,Silver Spring,MD). The data were then analyzed for significance on computer with the unpaired t-test(Instat1.1for Macintosh;GraphPad Software,San Di-ego,CA).Recurrent Erosion Study
To determine whether erosions were chronic or recurrent,we as-sd healing over time in individual animals.We wounded eyes bilaterally,with the small wound in the left eye and the large wound in the right.The purpo of wounding both eyes in the same animal was to correlate the qualit
y of healing of contralateral eyes.This experi-mental design was critical,becau it allowed us to determine whether individual animals were poor healers or whether one size of wound healed more poorly than the other.After initial wounding,the mice were allowed to heal for1,2,4,and6weeks.At each time point,the mice were placed under general anesthesia and their eyes stained with fluorescein and viewed using a discting microscope modified with blue-light illumination.Wounds were scored as open or clod bad onfluorescein staining.Care was taken to make sure that the eyes remained moist after asssment and,before anesthesia wore off;each eye was treated with a single application of erythromycin ophthalmic ointment.At the last time point(6weeks)wounds were assd (usingfluorescein)to be open or clod.Mice were killed by lethal injection,their eyes restained with a vital dye,and photographs taken using the discting microscope,to asss scaring and vascularization. After enucleation,eyes were carefully labeled and either procesd for whole mounts or frozen for tissue ctioning.
Confocal Microscopy
Confocal microscopy was performed at the Center for Microscopy and Image Analysis(CMIA)at the George Washington University Medical Center.A confocal lar scanning microscope(model MRC1024; Bio-Rad,Hercules,CA)equipped with a krypton-argon lar and an inverted microscope(m
odel IX-70;Olympus)was ud to image the localization of Alexa488(488-nm lar line excitation;522/35emission filter)and PI(568nm excitation;605/32emissionfilter).Optical ctions(zϭ0.5m)of confocal epifluorescence images were ac-quired quentially at1-m intervals with a20ϫobjective lens(NAϭ0.7)and/or a10ϫobjective lens(NAϭ0.3)with image acquisition software(LarSharp ver.3.2;Bio-Rad).Typically,six to eight optical ctions,each taken a1-m intervals,were merged and viewed en face.Image management software(Photoshop ver.7.0;Adobe Sys-tems,Mountain View,CA;with Bio-Rad plugins)was ud both to convert images from pic(Bio-Rad)into tifffiles and to create montages, and the images were prented.
R ESULTS
Evidence of Erosion after Manual Debridement at 1Week after Induction of Large Wounds
Becau the corneal surface in the adult mou is approxi-mately3mm in diameter,creating the large wound involved removal of most of the corneal epithelium.Despite this,initial reepithelialization of the wounds was complete between2 and3days(Fig.1).Although we have shown that␣9integrin expression is incread in cross ctions taken from open wounds2days after large wounds,22this time point occurred late in the reepithelialization process.Most of the corneal epithelium had been for
ced to disasmble its HDs to migrate, and the entire epithelial sheet was looly adherent.We have found that it is technically difficult to cut unfixed frozen c-tions at time points earlier than2days after large wounds are made22;therefore,we did not know how soon after wounding the increa in␣9integrin occurred or how it wasfirst mani-fested.It could have incread gradually over the entire corneal surface—first at the limbal region and later toward the leading edge—or it could have incread just at the leading edge.
To study the spatial and temporal differences in␣9integrin localization after imposing large wounds,we ud whole mounts of corneal tissues for immunofluorescence.In whole mounts of unwounded control cornea,␣9integrin was ob-
IOVS,June2004,Vol.45,No.6Altered Integrin Localization in Recurrent Erosions1777
rved as a patchy pattern among a subpopulation of cells in the limbal region and was abnt from the cells at the central cornea (Fig.2A).By sacri ficing mice immediately after large wounds were made,we con firmed that the wounds left the ␣9integrin –positive limbal basal cells intact at the limbus (Fig.2B).One day after imposition of large wounds,␣9integrin was upregulated in the limbal region,as well as in cells behind the leading edge (Fig.3A).Upregulation of ␣9integrin at the limbus at
1day was not uniform around the circumference of the cornea,and some regions of the limbus were more positive than others.The gment of the limbus shown in Figure 3A shows an intermediate level of ␣9integrin staining.At sites where there were more ␣9-positive cells in the limbus,there were fewer cells expressing ␣9integrin toward the leading edge.Two days after wounding (Fig.3B),more ␣9integrin was prent toward the leading edge but less localized in the limbus.The results indicate that,as a function of time after wounding,␣9integrin expression incread toward the lead-ing edge and decread at the limbal region (Fig.3B).All three wounds assd at 1and 2days were open (Fig.3,asterisks).By 3days,all large wounds assayed were clod,accompanied by decread ␣9integrin expression over the entire corneal surface (Fig.3D).In the limbal region,variability was en in the number of ␣9integrin –positive cells.Some regions had none (Fig.3D),whereas others had many ␣9integrin –positive cells (Fig.3C),similar to that obrved in unwounded corneas.The localization of ␣9integrin was expected to return to the pattern obrved in the unwounded control cornea after 3days.However,that was not the ca.At 1week after making the large wounds,we found that open wounds had
reappeared
春节作文英语
F IGURE 1.The initial reepithelializa-tion of large wounds was complete 3days after wounding.The number of eyes indicated were assd for the timing of wound closure after the eyes were stained (obtained after sac-ri fice)with a vital dye at 1,1.5,2,and 3days after wounding.Eyes were photographed under a discting mi-
croscope.
F IGURE 2.␣9Integrin localizes to the limbus in the unwounded mou cornea and large wounds do not re-move the ␣9integrin –positive cells at the limbus.Whole mounts of un-wounded corneas were flattened as indicated in the diagram (A );the site of the limbus is indicated by the in-ner circle .Tissues were stained to reveal the distribution of ␣9integrin (green )within the epithelial cells of the ocular surface,which was also stained with the nuclear marker PI (red ).Note that the central cornea was negative for ␣9integrin,whereas the limbal region showed numerous cells with localization of ␣9integrin.A cornea wounded using the technique for large wounds and killed immediately after wounding (0hour)was also stained for ␣9integrin and PI (B ).Note that the wound did not remove any of the ␣9integrin –positive cells at the limbus.Images are oriented (L,limbus;C,cornea)as indicated by the arrow on the left .Bar,50m.
1778Pal-Ghosh et al.IOVS,June 2004,Vol.45,No.6
on the ocular surface,accompanied by incread expression of ␣9integrin in the peripheral cornea and toward the leading edge (Fig.4A –C).We hypothesized that by 1week after wounding,small patches of epithelial cells had eroded,and the cells at the periphery of the new wound site were force
d once more to migrate to cover the newly expod wound.All three large wounds studied showed signs of erosion at 1week.By 2weeks after wounding,we also obrved ␣9integrin on cells near the center of the cornea and around the corneal periphery (Figs.4D –F).While the specimen in Figure 4C showed an erosion at 1week,the one in Figure 4F did not show an eroded area but rather a depression on the corneal surface,which was covered with ␣9integrin-negative epithelial cells.In addition to obrving the erosions,we saw fewer cells staining posi-tively for ␣9integrin in the limbus (compare Figs.4A and 4D with Fig.3and with the unwounded limbus in Fig.2).
When we evaluated corneas 4weeks after making the large wounds,all corneas assd had intact clod epithelia (Figs.
5A,5B);however,the corneal epithelium did not appear to be normal.Although ␣9integrin was no longer obrved in the central cornea,the quality of the healed epithelium varied within and among eyes and the localization of ␣9integrin at the limbus was reduced or abnt.The images shown in Fig-ures 5A and 5B are from different regions of the same eye.In Figure 5A,the number of ␣9integrin –positive cells in the limbal region appeared to be almost back to normal levels (compare Fig.5A to the limbal region shown in Fig.2),and no ␣9-positive cells were en in the central cornea.However,in a different region of the same eye (Fig.5B),no ␣9integrin –positive cells were en at the limbal regio
n,and a diffu low level of staining for ␣9integrin was en among clusters of epithelial cells in the central cornea.In addition,numerous gobletlike cells (some indicated by white arrows)were
en.
F IGURE 4.A condary wave of cell migration was obrved in most of the corneas evaluated 1and 2weeks after creation of large wounds.The diagram between (A )and (D )indicates the orientation of each image prented relative to the entire cornea,with L,P,and C indi-cating limbus,peripheral cornea,and central cornea,respectively.Whole mount images from the limbus to the central cornea are shown from typical corneas taken 1(A –C )and 2(D –F )weeks after wounding and stained to reveal the distribution of ␣9integrin (green )within epithelial cells,which have been counterstained with the nuclear marker PI (red ).(B ,C ,E ,F ,ints )Higher magni fications of the regions indicated by the asterisks .(B )The cut made to flatten the cornea.Note that ␣9integrin was reduced at the limbal regions in (A )and (D )but was abundant on corneal epithelial cells 1and 2weeks after wounding.(C )Shows an erosion.(F )Shows a central depression covered by epithelial cells not expressing ␣9integrin.␣9integrin –positive cells were prent around the depression.␣9integrin was most abundant 50to 100m away from the edge of the migrating epithelial sheet at 1week.Bars:(B –F )100m;(A ,D ,ints )50优秀人才入境计划
m.
F IGURE 3.Changes in ␣9integrin localization during reepithelializa-tion after large wounding.The diagram above each image indicates the orientation of each montage below relative to the entire cornea;the innermost irregular circle in the schema above (A )and (B )indicates the remaining open wound.The montages in (A ),(B ),and (D )show typical regions of whole mount preparations of corneas obtained 1,2,and 3days after wounding,respectively.(C )The limbal region only,of a cornea 3days after wounding,to highlight variability in staining (compare C with the limbal region in D ).Images are oriented as indicated by the arrows ,with the central cornea toward the bottom and the limbus toward the top .(A ,B ,✽)Leading edge of migration.Note that the initial reepithelialization was complete by 3days.Bars,50m.
IOVS,June 2004,Vol.45,No.6Altered Integrin Localization in Recurrent Erosions 1779
To con firm that the gobletlike cells were in fact goblet cells,we stained similar corneas 4weeks after large wounds with anti-mucin antibody MUC5AC (Fig.5C).We routinely obrved more goblet cells on the corneal surface in regions without ␣9integrin in the limbus.
Evidence of Erosion after Manual Debridement in Small Wounds
Becau,as shown in Figure 1,initial reepithelialization was complete at 3days,we were surprid to e open wounds after 3days.It is possible that the results,suggesting recur-rent epithelial erosions,could be a side effect of removing more than 50%of the corneal epithelial cells during the initial wounding process.To determine whether similar erosions could be obrved after small wounds were made,involving removal of less than 40%of the corneal epithelium,we as-sd corneas for ␣9integrin localization and PI staining,using whole mounts at 6,12,18,24,48,and 72hours and at 1and 2weeks after the wounds were made (Fig.6).No fewer than three eyes were assd at each time point studied,and the data prented re flect typical results.There was no change in the number or the intensity of staining of ␣9integrin –positive cells in the limbal region,at any time point studied (data not shown).From 6to 12hours after wounding (Figs.6A,
床头6B),there was no expression of ␣9integrin at or near the leading edge;between 12and 24hours,integrin localization became abundant in and around the margins of the wound as it clod (Figs.6B –D).At 48hours after wounding,␣9integrin localization disappeared from the central cornea (Fig.6E);however,at 72hours (Fig.6F)and at 1week (Fig.6G),some ␣9integrin –positive cells were prent on the central cornea.The 2-week sample showed low but diffu staining for ␣9integrin on the corneal surface,but no signs of erosions or depressions (Fig.6H).The data suggest that erosio
ns of the corneal surface can occur even after small wounding,but appear to be less frequent initially.The small depression en in Figure 6F at 72hours was similar to the larger depression en in Figure 4F,2weeks after large wounding.The local-ized depression sites indicate thinning areas of the corneal stroma.
Incread Corneal Epithelial Cell Proliferation Obrved Several Weeks after Creation of Large and Small Wounds
If the corneal erosions occurred after debridement wounds initially clod,we hypothesized that the rate of corneal epi-thelial cell proliferation with both large and small
wounds
F IGURE 5.At 4weeks,wounds were clod but variability was obvious in the quality of the epithelial surface.The schema between (A )and (B )indicates the orientation of each image prented relative to the entire cornea (L,limbus;C,cornea).The two montages shown are whole mounts from different quadrants of the same eye taken 4weeks after a large wound was created.Data indicate that,although some limbal regions of the cornea reverted back to the ␣9integrin expression pro file obrved before wounding (A ),other regions (B )lacked ␣9integrin in the limbus and showed evidence of gobletlike cells all the way to the center of the cornea —some of which are indicated by arrows .When prent in the cornea,␣9integrin appeared diffu and cytoplasmic rather than membranous.Similar variability was obrved in all three corneas stained at 4weeks.To con firm that the cells on the central cornea,which appeared to be goblet cells in (B ),were goblet cells,corneas were stained with an antibody against the mucin MUC5AC (C ).Data indicate numerous goblet cells on the ocular surface 4weeks after wounding.(C ,✽)A goblet cell cluster shown at higher magni fication in the int .Images are oriented as indicated by the arrows on the left .Bars,100m.
1780Pal-Ghosh et al.IOVS,June 2004,Vol.45,No.6