Abnormal Epithelial Differentiation and Tear Film

Alteration in Pinguecula
Abnormal Diferensiasi Epitel dan Perubahan film Air Mata Pada Pinguecula
PURPOSE. To investigate the differentiation of conjunctival epithelium and tear film function in pingueculae.
METHODS. Twelve patients (12 eyes) who underwent simple excision for pingueculae were enrolled in the study.
Immunostaining for K1! K1"! K1#! $%&'(&! )ax*! )*+! Ki*,! and
K1* was performed on the pinguecula epithelium and normal conjunctival epithelium. -chirmer I test results and
tear film .rea/0up time (T12%T) were evaluated just .efore and 1 month after surgery.
RESUTS. (.normal epidermal differentiation of pinguecula tissue! as evidenced .y a decline in or a.sence of )ax*
expression! was accompanied .y a decline in or a.sence of K1" /eratin and $%&'(&! and an increase in K1# and
K1 /eratin. 1urthermore! the pinguecula epithelium was actively proliferating! as evidenced .y positive
expression of Ki*,! )*+! and K1* /eratin. The -chirmer I test results did not indicate any significant differences
.efore and after surgery. 3owever! the T12%T was significantly prolonged 1 month after surgery compared
with .efore surgery.
!O"!US#O"S. )inguecula is a condition of a.normal epithelial differentiation. It is characteri4ed .y s5uamous
proliferation and metaplasia! resulting in insta.ility of tear film with normal
.asic tear secretion. (Invest Ophthalmol Vis Sci. 2##"6'#7 2,1#82,1') 9:I71#.11*,;iovs.#<02"#'
TUJUAN . Untuk Selidiki diferensiasi epitel konungti!a dan fungsi film air mata pada
pingueculae .
MET"DE . Dua belas pasien # $% mata & 'ang menalani eksisi tunggal untuk pingueculae
Terdaftar dalam penelitian ini . #mmuno$taining untuk ($) * ($+ * ($, * MU-.A- * PA/0 * P01
* (i02 * dan ($0 dilakukan pada epitel pinguecula dan epitel normal konungti!a . 3asil tes
Schirmer 4 dan tear film break5 up time #T67UT& dilakukan pada saat sebelum operasi dan $ bulan
setelah operasi .
3AS48 . Diferensiasi epidermal 'ang abnormal dari aringan pinguecula* sebagaimana dibuktikan
oleh penurunan atau tidak adan'a ekspresi PA/0* bersamaan dengan penurunan atau tidak adan'a
($+ keratin dan MU-.A-* dan peningkatan ($, dan ($) keratin. Selain itu* epitel pinguecula
'ang aktif berkembang biak* sebagaimana dibuktikan oleh ekspresi positif (i02* P01* dan ($0
keratin. 3asil tes Schirmer 4 tidak memiliki perbedaan 'ang signifikan antara sebelum dan setelah
operasi. Namun* Secara signifikan T67UT memanang pada $ bulan setelah operasi dibandingkan
dengan sebelum operasi .
(ES4MPU8AN . Pingueculae adalah kondisi diferensiasi epitel abnormal. 3al ini ditandai oleh
proliferasi skuamosa dan metaplasia* mengakibatkan ketidakstabilan film air mata dengan sekresi
normal air mata.
)
inguecula is a common ocular surface disorder! with reported prevalence rates of 22.'= to "#=.
18+
)ingueculae
are .enign! yellowish! slightly raised! interpalpe.ral lipid0li/e deposits in the nasal and temporal lim.al
conjunctiva.
1
(lthough pinguecula is a relatively .enign condition! it can manifest as ocular irritation (e.g.! foreign0
.ody sensation! pain! and tearing)

and represents a significant eye health pro.lem in many communities.
(lthough the etiogenesis of pingueculae is still not fully understood! prolonged exposure to ultraviolet02 radiation
is thought to promote its development.
'8,
:ther factors (e.g.! p'+ mutation!
<
increased cholesterol meta.olism!
"
development of a.normal elastotic materials!
1#
age!
11!12
and inade5uate tear film sta.ility
1+
) may also play roles in
pinguecula formation. 3owever! to the .est of our /nowledge! there are no pu.lished studies comparing
proliferation and differentiation of pinguecula epithelium to those of normal conjunctival epithelium.
To .etter understand the alterations of the conjunctival epithelium in this condition! we compared pinguecula
samples to normal conjunctiva via immunostaining with monoclonal anti.odies to K1! K1"! K1#! $%&'(&!
)ax*! Ki*,! )*+! and K1* proteins! and examined the extent and distri.ution of their expressions. $oreover! we
evaluated the effect of pinguecula vis0a>0vis tear function! .ased on tear film .rea/0up time (T12%T) and the
results of pre0 and postoperative -chirmer I tests.
Pingueculae adalah gangguan permukaan bola mata umum* dengan tingkat pre!alensi 'ang
dilaporkan dari %%*.9 sampai +,9. Pingueculae bersifat inak* kekuningan* sedikit terangkat*
deposit interpalpebral seperti lipid di limbus konungti!a nasal dan temporal. Meskipun
pinguecula adalah bersifat inak* dapat men'ebabkan iritasi mata #misaln'a sensasi 5 benda asing*
n'eri* dan mata berair& dan menunukkan masalah kesehatan mata 'ang signifikan di ban'ak
komunitas.
Meskipun etiogenesis dari pingueculae masih belum sepenuhn'a dipahami* kontak 'ang terlalu
lama dengan radiasi ultra!iolet 5 7 diperkirakan meningkatkan perkembangann'a. 6aktor5faktor
lain #misaln'a* p.1 mutasi* perkembangan abnormal material ela$totic% usia* dan stabilitas film air
mata 'ang tidak memadai& mungkin uga memainkan peran dalam pembentukan pinguecula.
7agaimanapun* untuk 'ang terbaik bagi pengetahuan kami* tidak ada penelitian 'ang
dipublikasikan membandingkan proliferasi dan diferensiasi epitel pinguecula terhadap epitel
konungti!a normal .
Untuk lebih memahami perubahan dari epitel konungti!a dalam kondisi ini* kami
membandingkan sampel pinguecula dan konungti!a 'ang normal melalui immuno$taining
dengan mono&lonal antibodi terhadap ($) * ($+ * ($, * MU-.A- * PA/0 * (i02 * P01 * dan
Protein ($0* dan Menelaah luas serta distribusi ekspresin'a. Selain itu* kami menge!aluasi efek
'i$ (a ('i$ fungsi air mata pada pingueculae* didasarkan pada tear film brea&( up time )TF*UT+
dan hasil dari pra5 dan pasca operasi ui Schirmer 4.
M
ATER#AS A"D
M
ETHODS
The following materials were used7 hydrogen peroxide! 9()I! 3oechst0 +++2 dye! acetone! Triton ?01##! .ovine serum
al.umin (2-()! and 1IT&0conjugated anti0mouse Ig@s (-igma0(ldrich! -t. Aouis! $:)6 mouse anti0cyto/eratin 1# (K1#)! 0K1"!
0)*+! and 0Ki*, anti.odies (9a/o 2iotechnology! @lostrup! 9enmar/)6 mouse anti0$%&'(& monoclonal anti.ody ((.cam
2iotechnology! &am.ridge! %K)6 mouse anti0K1! 0K1*! and 0)ax* monoclonal anti.odies (-anta &ru4 2iotechnology! -anta
&ru4! &()6 diamino.en4idine (9(26 9a/o &ytomation! &arpinteria! &()6 and the (2& /it for mouse (Bectastain Clite6 Bector
Aa.oratories! 2urlingame! &().
Patient$ and Specimen$
De studied pinguecula specimens from 12 patients who had had elective outpatient pinguecula surgery. &linical diagnosis of
pinguecula was .ased on previously reported criteria.
1
(ll investigations were conducted in accordance with the tenets of the
9eclaration of 3elsin/i and were approved .y the Cthics &ommittee of ?iamen Cye &enter. Informed consent was o.tained from
each patient .efore inclusion in
the study. (ll study patients were examined at the &ornea and :cular -urface &linic of ?iamen Cye &enter (?iamen! 1ujian!
&hina)! .etween 9ecem.er 2##, and 1e.ruary 2##<. (ll pingueculae were surgically removed .y one surgeon (3D). -u.jects
with previous ocular surgery! contact lens wear! pemphigoid! -joEgrenFs syndrome! or any other type of conjunctivitis or /eratitis
were excluded from the study. The patients were o.served for at least 1 month after surgery. Gormal conjunctiva o.tained from
human donors at ?iamen Cye &enter and Cye Institute served as the control.
E'aluation of Tear Film Function
(ll examinations were performed in the morning! and all measured varia.les
for each patient were evaluated during a single office visit in the
same dar/ened room 1 day .efore and 1 month after surgery. T12%T
measurements with fluorescein were ta/en and the -chirmer I test (without
topical anesthesia) was performed. T12%T was recorded as the average
of three successive measurements. The -chirmer I test result was
expressed as the wet length of the strip measured after ' minutes.
7A3AN DAN MET"DE
7ahan 'ang digunakan sebagai berikut : hidrogen peroksida* DAP4* pe;arna 3oechst5111)%*
aseton* Triton /5$,,* bo!ine serum albumin #7SA&* dan 64T-5terkonugasi anti5tikus 4g<
#Sigma5Aldrich* St. 8ouis* M" &= anti5c'tokeratin tikus $, # ($, &* 5($+* 5P01* dan 5(i02 antibodi
#Dako 7ioteknologi* <lostrup* Denmark&* tikus anti5MU-.A- antibodi monoklonal #Abcam
7ioteknologi* -ambridge* U(&* tikus anti5($)* 5($0* dan 5PA/0 antibodi monoklonal #Santa
-ru> 7iotechnolog'* Santa -ru>* -A&* diaminoben>idin #DA7* Dako -'tomation* -arpinteria*
-A& dan &it A*! untuk tikus #?ectastain Elite * ?ector 8aboratories* 7urlingame* -A&.
Pasien dan Spesimen
(ami mempeari spesimen pinguecula dari $% pasien operasi pinguecula ra;at alan 'ang telah
terpilih. Diagnosis klinis pinguecula didasarkan pada kriteria laporan sebelum'a. Semua
4n!estigasi dilakukan sesuai dengan prinsip5prinsip Deklarasi 3elsinki dan sudah disetuui oleh
(omite Etika /iamen E'e -enter. 4nformed consent dari setiap pasien diperoleh sebelum
dimasukkan ke dalam penelitian. Semua pasien penelitian diperiksa di klinik kornea dan
permukaan okuler Pusat mata /iamen #/iamen* 6uian* -hina&* antara Desember %,,2 dan
6ebruari %,,@. Semua pingueculae dioperasi oleh satu dokter bedah #3A&. Sub'ek dengan operasi
mata sebelumn'a* memakai lensa kontak* pemphigoid* Sogren sindrom* atau tiap enis
konungti!itis atau keratitis dikeluarkan dari penelitian. Para pasien diamati selama minimal $
bulan setelah operasi. (onungti!a 'ang normal diperoleh dari donor manusia di /iamen E'e
-enter dan E'e 4nstitute berperan sebagai kontrol .
E!aluasi 6ungsi dari 6ilm Air Mata
Semua pemeriksaan dilakukan di pagi hari* dan semua !ariabel pengukuran untuk setiap pasien
die!aluasi selama sekali kunungan di ruangan gelap 'ang sama $ hari sebelum dan $ bulan
setelah operasi . Pengukuran T67UT 'ang dilakukan dengan fluorescein dan tes Schirmer 4 #tanpa
anestesi topikal& sudah dilakukan. T67UT dicatat berdasrkan rata5rata tiga pengukuran berturut5
turut 'ang berhasil. 3asil ui Schirmer 4 din'atakan sebagai panang strip basah diukur setelah .
menit .
#mmuno$taining
1ro4en sections of *0 m thic/ness were fixed in acetone at 2#H& for 1#
minutes. 1or immunofluorescence staining! fixed sections were incu.ated
in #.2= Triton ?01## for 1# minutes. (fter three rinses with )2- for '
minutes each and preincu.ation with 2= 2-( to .loc/ nonspecific staining!
the sections were incu.ated overnight at H& with various dilutions of
primary anti.odies7 $%&'(& and K1* (17'#)6 K1#! K1! and K1" (172##).
(fter three washes with )2- for 1' minutes each! sections were incu.ated
with an 1IT&0conjugated secondary anti.odyIra..it anti0mouse Ig@ (17
'#)Ifor 1 hour. (fter three additional '0minute washes in )2-! the
sections were o.served .y microscope (TC02###% Cclipse epifluorescence
microscope6 Gi/on! To/yo! Japan). 1or immunohistochemical staining!
fixed sections were placed in #.*= hydrogen peroxide for 1# minutes
to .loc/ endogenous peroxidase activity. Gonspecific staining was
.loc/ed .y 1= normal horse serum for +# minutes. -ections were then
incu.ated overnight at H& with various dilutions of primary anti.odies7
)*+ (17'#)! Ki*, (171##)! and )ax* (172##). (fter three 1'0minute washes
with )2-! sections were incu.ated with .iotinylated anti0mouse Ig@ (17
1##) for 1 hour and incu.ated with (2& reagent for +# minutes. The
reaction product was developed with 9(2 for 2 minutes. The sections
were photographed with a camera mounted on a light microscope (.oth
.y Gi/on).
#mage Anal,$i$
1or analysis of integrated optical density (I:9) expression of positive
immunostaining in the epithelial layer! images from immunostained
(K1! K1"! K1#! $%&'(&! )ax*! Ki*,! )*+! and K1* proteins) sections
were processed (Image )ro )lus ver. *.#6 $edia &y.ernetics! -ilver
-pring! $9). In .rief! correction of une5ual illumination (shading
correction) and the cali.ration of the measurement system were performed
with a reference slide after the images were recorded. 1or each
sample! different areas of + to 1# sections were scored. The images of
immunostained slides were converted to gray scale! and the I:9 was
measured as a linear com.ination .etween the average gray intensity
and the relative area occupied .y positive cells.
immunostaining
7agian beku dari ketebalan 05Bm ditetapkan dalam aseton pada suhu 5%,C- selama $, menit.
Untuk pe;arnaan immunofluore$cence* bagian tetap diinkubasi di ,*%9 Triton /5$,, selama $,
menit. Setelah tiga bilasan dengan P7S masing5masing selama . menit dan preinkubasi dengan
%9 7SA untuk memblokir pe;arnaan nonspesifik* diinkubasi semalam pada suhu )C- dengan
berbagai pengenceran antibodi primer : MU-.A- dan ($0 #,$:.,&= ($,* ($)* dan ($+ # $:%,, &.
Setelah tiga pencucian dengan P7S selama masing5masing $. menit* diinkubasi dengan 64T-5
terkonugasi antibodi sekunder 54g< kelinci anti5mouse #$ :.,&5 selama $ am. Setelah tambahan
tiga kali mencuci selama . menit di P7S* bagian diamati dengan mikroskop #TE5%,,,U Eclipse
epifluorescence mikroskop= Nikon* Tok'o* Jepang &. Untuk pe;arnaan imunohistokimia* bagian
'ang sudah tetap ditempatkan di ,*09 hidrogen peroksida selama $, menit untuk memblokir
akti!itas endogen peroksidase. Pe;arnaan nonspesifik telah diblokir oleh $9 serum kuda normal
selama 1, menit. 7agian kemudian diinkubasi semalam pada suhu )C- dengan pengenceran
berbagai antibodi primer : P01 #,$:.,&* (i02 #$:$,,&* dan PA/0 #$:%,,&. Setelah tiga kali mencuci
selama $. menit dengan P7S* bagian diinkubasi dengan terbiotinilasi 4g< anti5mouse #$:$,,&
selama $ am dan diinkubasi dengan A7- reagen selama 1, menit. Produk reaksi dikembangkan
dengan DA7 selama % menit. 7agian difoto dengan kamera 'ang dipasang pada mikroskop caha'a
#keduan'a oleh Nikon&.
Analisis gambar
Untuk analisis kepadatan optik terpadu #4"D& ekspresi positif immunostaining di lapisan epitel*
gambar dari bagian immuno$tained #($)* ($+* ($,* MU-.A-* PA/0* (i02* P01* dan ($0
protein& diproses #4mage Pro Plus !er. 0.,= Media -'bernetics* Sil!er Spring* MD&. Secara
singkat* koreksi pencaha'aan 'ang tidak merata #$hading &ore&$i& dan kalibrasi sistem
pengukuran dilakukan dengan slide referensi setelah gambar direkam. Untuk masing5masing
sampel* daerah 'ang berbeda dari 1 sampai $, bagian dicetak. <ambar5gambar dari slide
immuno$tained dikon!ersi ke skala abu5abu* dan 4"D telah diukur sebagai kombinasi linear
antara intensitas rata5rata abu5abu dan daerah relatif 'ang ditempati oleh sel positif .
Stati$tic$
-ummary data were reported as the mean -9. @roup mean data
were compared .y using the appropriate version of the t0test! where
P #.#' was considered statistically significant.
R
ESUTS
!linical E'aluation
)ingueculae from 12 eyes (four right! eight left) of 12 patients (2
men! 1# women6 average age! ,.2' ,.,' years6 range! +8'*)
were studied (Ta.le 1). )ingueculae were located nasally (n <) and temporally (n )! as shown in 1igure 1. Go
ocular surface
complication was o.served in any eye after surgery.
Tear Film Function
Gone of the patients in this study had dry eye. The average
T12%T was '.+ +.# seconds. This figure increased significantly
to 12.* 1.< seconds .y one month after surgery (t
*.*<"! P #.#'! paired0sample t0test6 1ig. 2().
The -chirmer I test result! which is important for evaluating
the a5ueous phase of the tear film! was 12.< +. mm .efore
surgery and increased to 1.2 2. mm after surgery! a
nonsignificant difference (t #.""! P #.+*+! pairedsample
t0test6 1ig. 22).
statistika
Dingkasan data dilaporkan dengan mean E SD. <rup data mean dibandingkan menggunakan !ersi
t 5test* di mana P F ,*,. secara statistik dianggap signifikan.
3AS48
E!aluasi klinis
Pingueculae dari $% mata #empat mata kanan* delapan mata kiri& dari $% pasien #% pria* $, ;anita*
usia rata5rata* )2*%. E 2*2. tahun* kisaran* 1)5.0& telah ditelilti #Tabel $&. Pingueculae terletak
pada bagian nasal mata # n G @ & dan temporal mata # n G ) &* seperti 'ang ditunukkan pada
<ambar $ . Tidak ada ditemukan komplikasi pada permukaan mata pada tiap mata setelah operasi.
6ilm 6ungsi Air Mata
Tak satu pun dari pasien dalam penelitian ini memiliki mata kering. rata5rata nilai pengukuran
T67UT adalah .*1 E 1*, detik. Angka ini meningkat signifikan menadi $%*0 E $*@ detik pada satu
bulan setelah operasi #t G 50*0@+ * P F ,.,. * paired5sample t5test= <ambar %A&.
3asil ui Schirmer 4* 'ang penting untuk menge!aluasi fa$e encer film air mata* adalah $%*@ E 1*)
mm sebelum operasi dan $)*% E %*) mm setelah operasi* perbedaan 'ang tidak signifikan # t G
,*+)+* P G ,*101* paired($ample t 5test= <ambar %7&.
Epithelial Differentiation in Pinguecula
To investigate whether the conjunctival epithelial phenotype is
maintained in pingueculae! immunostaining was performed for
K1! K1" /eratin! and epidermis0specific K1# /eratin. K1
/eratin! a .asal epithelial cell mar/er that has .een reported to
.e confined to the .asal cell compartment in normal conjunctiva
1'!1*
and upregulated in pterygia!
1,!1<
was expressed in the
.asal layer of normal conjunctiva in our study (1ig. +(). 3owever!
K10positive cells dramatically increased in the supra.asal
and superficial layers of all pingueculae tissues (1ig. +2).
Cxpression of K1" /eratin! one of the major components of the
conjunctival epithelium and which is reported to .e uniformly
expressed!
1'!1*
was demonstrated in all normal conjunctival
epithelial cells (1ig. +&)! whereas K1" expression was dramatically
decreased in all pinguecula samples and was even negative
in some areas of full0thic/ness pinguecula epithelium (1ig.
+9). (s reported!
1'!1*
expression of K1# /eratin! an epidermal
/eratinocyte0specific intermediate filament! was negative in
normal conjunctiva (1ig. +C). -urprisingly! K1#0positive cells
emerged in the supra.asal to superficial cell layers of all
pinguecula tissues (1ig. +1).
To further investigate go.let cell differentiation in pinguecula
epithelium! we used immunofluorescence to examine
$%&'(&! the most a.undant mucin of the ocular surface.
1"821
Kesults showed that $%&'(& expression was prominent in
normal conjunctiva (1ig. +@)! .ut was significantly downregulated
in the epithelial cells of pinguecula (1ig. +3) and was
totally a.sent in five samples.
:ne distinguishing characteristic of the ocular surface epithelium!
as opposed to the epidermal epithelium! is expression
of )ax*! a transcription factor that plays a /ey role in eye
morphogenesis.
22!2+
To determine whether the aforementioned
a.normal epidermal differentiation was associated with
the loss of differentiation of ocular surface lineage in pinguecula!
we performed immunohistochemical staining for )ax*.
)ax*0positive nuclei were present throughout the full thic/ness
of the epithelium in normal conjunctiva (1igs. +I)! .ut was
decreased in all samples of pinguecula epithelium! regardless
of level. )ax* expression was lost in some .asal epithelial cells!
and was even negative in some full0thic/ness areas of epithelium
(1igs. +J). :ur previous study showed that a.normal
epithelial differentiation correlated highly with downregulation
of )ax* expression in severe ocular surface diseases.
2
Kesults of the present study indicate that )ax* downregulation
also correlates with a.normal epithelial differentiation in mild
ocular surface a.normalities such as pinguecula.
To 5uantify the expression levels of the a.ove0mentioned
cell differentiation mar/ers! the I:9 of positive immunostaining
in the epithelial layer was analy4ed. Kesults showed that
I:9s of K1# (+#+."# +2#.21)0 and K1 ('1*+.<"'
1*<,.+1<)0positive cells were higher in pinguecula than in
normal conjunctiva (P #.#'! 1ig. +K). 3owever! the I:9s of
K1" (2#<". 12!222.<') and $%&'(& ('1,., <'".*1) were significantly downregulated in pingueculae
compared
with normal conjunctiva (P #.#')! respectively (1ig. +K).
These results further confirm that a.normal epidermal differentiation
occurs in pinguecula.
Diferensiasi Epitel Pada Pinguecula
Untuk Selidiki Apakah fenotipe epitel konungti!a dipertahankan dalam pingueculae*
immunostaining dilakukan untuk ($) * ($+ keratin* dan epidermis 5 spesifik keratin ($,. ($)
keratin* penanda sel epitel basal 'ang telah dilaporkan terbatas pada kompartemen sel basal di
normal conuncti!a dan mengalami penimgkatan komponen sel dalam pter'gia* telah ditemukan
dalam lapisan basal konungti!a normal dalam penelitian kami #<ambar 1A &. Namun* sel ($)
positif ditemukan meningkat pada suprabasal dan lapisan permukaan semua aringan pingueculae
#<ambar 17&. Ekspresi ($+ keratin* salah satu komponen utama dari epitel konungti!a dan
dilaporkan terekspresi secara seragam* ditunukkan pada semua epitel konungti!a normal
#<ambar 1-&* sedangkan ekspresi ($+ mengalami penurunan dramatis pada semua sampel
pinguecula dan bahkan negatif di beberapa daerah full5thickness epitel pinguecula #<ambar 1D&.
Dilaporkan* ekspresi ($, keratin* sebuah keratinosit epidermal spesifik dengan intermediate
filamen* negatif dalam konungti!a normal #<ambar 1E&. Anehn'a* sel ($,5positif muncul dalam
lapisan suprabasal sel sampai lapisan permukaan sel pada semua aringan pinguecula #<ambar
16&.
Untuk men'elidiki diferensiasi sel goblet di epitel pinguecula epitel* kami menggunakan
immunofluore$cence untuk memeriksa MU-.A-* musin 'ang paling berlimpah pada permukaan
bola mata. 3asil Menunukkan bah;a ekspresi MU-.A- menonol di konungti!a normal
#<ambar 1<&* tetapi secara signifikan mengalami penurunan komponen sel dalam sel epitel
pinguecula #<ambar 13& dan benar5benar absen dalam lima sampel.
Salah satu ciri 'ang membedakan dari epitel permukaan bola mata* dibandigkan dengan epitel
epidermis* adalah ekspresi dari PA/0* faktor transkripsi memainkan peran kunci dalam
morphogenesis mata. Untuk menentukan apakah diferensiasi epidermal abnormal terkait dengan
hilangn'a diferensiasi permukaan bola mata pada pinguecula* kami melakukan pe;arnaan
imunohistokimia untuk PA/0. 4nti PA/05positif ditemukan pada epitel dengan ketebalan penuh
dalam konungti!a normal #<ambar 14&* tapi ditemukan penurunan dalam semua sampel epitel
pinguecula* apapun tingkatn'a. Ekspresi PA/0 hilang dalam beberapa sel epitel basal* dan bahkan
negatif di beberapa daerah epitel dengan ketebalan penuh #<ambar 1J&. Penelitian kami
sebelumn'a menunukkan diferensiasi epitel abnormal sangat berkorelasi dengan berkurangn'a
komponen sel PA/0 pada pen'akit permukaan bola mata 'ang parah. 3asil dari penelitian ini
mengindikasikan do;nregulasi PA/0 uga berkorelasi dengan diferensiasi epitel abnormal pada
kelainan permukaan mata ringan sebagai pinguecula .
Untuk mengukur tingkat ekspresi dari penanda diferensiasi sel 'ang telah disebutkan di atas* 4"D
dari immunostaining positif pada lapisan epitel dianalisis. 3asiln'a menunukkan bah;a 4"D dari
($, # 1),1.+, E 1%,).%$& dan ($) #.$01.@+. E $0@2.1$@& 5 sel positif di pinguecula lebih tinggi
dari pada konungti!a normal #P F ,.,. <ambar 1(&. Namun* 4"D dari ($+ #%,@+.) E $%*%%%.@.&
dan MU-.A- #.$2*)2 E @.+.0$& secara signifikan mengalami do;nregulasi pada pingueculae
dibandingkan konungti!a normal dengan #P F ,*,.&* berturut5turut #<ambar 1(&. 3asil ini lebih
lanut mengkonfirmasi diferensiasi epidermal 'ang abnormal teradi pada pinguecula .
F#-URE .. (.normal epithelial differentiation in pinguecula. K1 /eratin was confined to the .asal cell compartment in normal
conjunctiva (A). K10positive cells were present in almost all epithelial cells in pinguecula (*). K1" /eratin was homogeneously
distri.uted throughout all layers of normal conjunctiva (!). K1" expression declined or .ecame negative in pinguecula (D). K1#
/eratin expression was totally negative in normal conjunctiva (E)! .ut was positive in superficial cell layers in the epithelium of
pinguecula (F). $%&'(& was present in normal conjunctiva (-) .ut not in pinguecula (H). )ax* was expressed in almost all
epithelial cells in normal conjunctiva (#)! whereas )ax* nuclear staining was mar/edly attenuated and even a.sent in .asal
epithelial cells of the pinguecula (/). I:9 results (0) showed significant differences of K1#! K1! K1"! $%&'(&! and )ax*
expression .etween pinguecula and normal conjunctiva (LP < #.#'6 LLP < #.#1). -cale .ars7 (A8H) 1## Bm6 (#! /) 1## Bm.
Epithelial Proliferation in Pinguecula
To further characteri4e the proliferation status of pinguecula epithelial
cells! we performed immunostaining for Ki*,! p*+! and
K1* /eratin. (ll negative control samples without primary anti.ody
showed no staining (data not shown). Guclear Ki*, staining!
indicative of cell proliferation! was detected in the .asal and
supra.asal layers of normal conjunctival epithelium (1ig. () and
pinguecula (1ig. 2). Cxpression of Ki*, was significantly increased
in pinguecula samples (,*,.2 '.") compared with
normal tissue (1*1.+2 1+1.2<)! as shown .y the I:9s (1ig. @).
)ositive nuclear staining of p*+! a mar/er originally thought to .e
expressed in .asal epithelial progenitor cells of all stratified epithelia!
including normal conjunctiva (1ig. &)!
2'82,
.ut now recogni4ed
as a mar/er of cells with proliferation potential! was
found in almost all .asal and some supra.asal epithelial cells in
pinguecula (1ig. 9). 3owever! there was no significant difference
(t .++6 P #.22,) in the I:9 of p*+0positive cells
.etween pinguecula (2#'+.* 1#1.,") and normal conjunctiva
(1**1.*1##.,"). K1* /eratin is indicative of an alternative
pathway of /eratinocyte proliferation and is also /nown for the
mar/ed enhancement of its expression in stratified s5uamous
epithelium showing hyperproliferation or a.normal differentiation.
2<
K1* /eratin was negative in normal conjunctiva (1ig. C)!
.ut was expressed in supra.asal and superficial epithelial cells in
pinguecula (1ig. 1). &ollectively! these results indicate that a.normal
epithelial differentiation is indeed associated with hyperproliferation
in pinguecula.
Proliferasi Epitel Pada Pinguecula
Untuk mencirikan lebih lanut status proliferasi sel epitel pada pinguecula* dilakukan
immunostaining untuk (i02* p01* dan ($0 keratin. Semua sampel kontrol 'ang negatif tanpa
antibodi primer menunukkan tidak ada pe;arnaan #data tidak ditampilkan&. Pe;arnaan inti (i02*
menunukkan proliferasi sel* terdeteksi di lapisan basal dan suprabasal epitel normal konungti!a
#<ambar )A& dan pinguecula #<ambar )7& . Ekspresi (i02 'ang meningkat secara signifikan
dalam sampel pinguecula #202.%) E ).).+& Dibandingkan dengan aringan normal # $0$.1% E
$1$*%@ &* seperti 'ang ditunukkan oleh 4"D #<ambar )< &. Pe;arnaan inti positif dari p01*
sebagai penanda a;al 'ang diekspresikan sel progenitor epitel basal dari semua epitel berlapis*
termasuk konungti!a normal #<ambar )-&* tapi sekarang diakui sebagai penanda sel dengan
proliferasi potensial* ditemukan di hampir semua sel epitel basal dan beberapa di suprabasal pada
pinguecula #<ambar )D&. Namun* tidak ada perbedaan 'ang signifikan #t G 5).)11= P G ,.%%2&
pada 4"D sel p015positif antara pinguecula #%,.1.)0 E $,$).2+& dan konungti!a 'ang normal
# $00$.0$ E ),,.2+&. ($0 keratin merupakan indikasi alur alternatif proliferasi keratinosit dan
uga dikenal sebagai penanda ika teradi peningkatan ekspresi dalam epitel skuamosa berlapis
menunukkan h'perproliferation atau diferensiasi abnormal. ($0 keratin negatif pada konungti!a
normal #<ambar )E&* tapi ditemukan dalam epitel sel suprabasal dan epitel sel permukaan di
pinguecula #<ambar )6&. Secara kolektif* hasil ini menunukkan diferensiasi abnormal epitel
memang terkait dengan h'perproliferation di pinguecula .
D
#S!USS#O"
The results of this study show! for the first time! that pinguecula
is a condition of a.normal differentiation characteri4ed .y
s5uamous metaplasia with proliferation.
-5uamous metaplasia has .een defined as the pathologic
transition of a non/eratini4ed stratified secretory epithelium to a /eratini4ed nonsecretory epithelium.
2"!+#
It is the
hallmar/
of a variety of severe ocular surface disorders manifesting
dry eye caused .y the lac/ of lacrimal gland secretion such
as -joEgren syndrome! -tevens0Johnson syndrome! mucous
mem.rane pemphigoid! chemical;thermal .urns! and vitamin
( deficiency.
2"!+1
&han et al.
+2
used impression cytology to
evaluate ocular surface a.normalities in four eyes with
pinguecula and pterygium and found that the surface cells in
pinguecula exhi.ited s5uamous metaplasia. In the present
study! we thoroughly analy4ed epithelial differentiation of
pinguecula .y means of immunostaining! which permitted
visuali4ation of full0thic/ness changes rather than only superficial
cell profiles from impression cytology. De confirmed that
s5uamous metaplasia! as evidenced .y expression of K1#! was
accompanied .y loss of $%&'(& and K1" expression in all
pinguecula samples (1igs. +&83). :ur recent study descri.ed
s5uamous metaplasia induced .y air exposure of human lim.al
explant cultures.
++
The pathogenesis of s5uamous metaplasia
in pinguecula may .e also interpreted .y using this model.
1irst! pinguecula is a manifestation if lift0up from normal con0
junctiva that mimic airlift culture. -econd! T12%T is shorter in
pinguecula! which can cause more exposure time of the a.normal
tissue than normal ocular surface. In the urothelial
model of s5uamous metaplasia! Aiang et al.
+
proposed five
models to explain the etiogenesis of s5uamous metaplasia7
transdifferentiation at the terminal differentiated cell level! deand
redifferentiation! pluripotency or plasticity of stem cells!
selected expansion of different stem cells! and expansion and
replacement .y a different lineage. In our study! we performed
immunostaining of )ax* to determine the differentiation lineage
of epithelial cells in pinguecula tissue. )ostnatal )ax*
expression is restricted to corneal! conjunctival! lens! and iris
epithelia and amacrine cells of the retina.
+'!+*
:ur previous
study clearly showed that )ax* helps maintain the normal
postnatal corneal epithelial phenotype. 9ownregulation of
)ax* is associated with a.normal epidermal differentiation in
severe ocular surface diseases.
2
In the present study! we also
found downregulation of )ax* in conjunctival epithelium of
pinguecula. $oreover! )ax* expression was attenuated and
even a.sent in .asal epithelial cells! in which conjunctival
progenitor cells were present! suggesting that a.normal differentiation
in pingueculae may occur in some progenitor cells.
:ther than )ax*! which is related to in vivo phenotype maintenance
of ocular surface epithelial cells! our previous study
showed that p+< signaling pathway is involved in a.normal
differentiation of lim.al epithelial cells in ex vivo explant
culture++. 1urther investigation is warranted to determine
whether p+< signaling pathway is also related to the a.normal
differentiation in pinguecula epithelium.
Traditionally! pingueculae have .een considered to .e degenerative
lesions! descri.ed histologically as having elastotic
degeneration of its collagen.
+,
3owever! pingueculae also exhi.it
features associated with p'+ mutation and increased cholesterol
meta.olism! in agreement with the hypothesis that a
pinguecula comprises a potentially proliferative tissue.
"!+<
In
the present study! expressions of Ki*, and K1* were significantly
greater in pingueculae than in normal conjunctiva. This
o.servation also supports the notion that there is hyperproliferation
in pinguecula epithelium. -ince overexpression of p*+
is associated with the development of conjunctival neoplasms!
including conjunctival s5uamous cell carcinoma
+"
and
pterygium!
#
we performed )*+ immunostaining to investigate
the proliferative capacity of pinguecula. (lthough the
difference was not statistically significant! there were more
p*+0positive nuclei in pinguecula than in normal conjunctiva!
further indicating that pingueculae exhi.it characteristics
of s5uamous proliferative diseases.
( previous study has demonstrated shortened T12%T .ut
normal tear secretion in pingueculae.
1+
In our study! the
-chirmer I test result was in the normal range in 1# patients!
whereas T12%T was shorter than normal! consistent with the
prior study.
1+
$oreover! T12%T was significantly prolonged
after a single excision! further indicating that pingueculae are
associated with a significant pertur.ation of tear film sta.ility.
There are several interpretations of the shortened T12%T in
pinguecula7 a.normal eyelid .lin/ing! presence of an irregular
formation! and mucus deficiency. (nother explanation may .e
the presence of s5uamous metaplasia in the surface epithelium
of pinguecula! which could compromise the sta.ility of tear
film. (lthough s5uamous metaplasia is common in ocular surface
epithelium of dry eye!
2"!+1
further study is needed to
reveal whether there is a causative relation .etween s5uamous
metaplasia and tear function in pingueculae.
In summary! our study confirmed the deleterious effect of
pingueculae on tear film sta.ility and ocular surface health
status! as evidenced .y decreased mucosal defense capa.ility
and damage to the ocular surface epitheliaIthat is! s5uamous
metaplasia! resulting in tear film insta.ility. ( correlation may
exist .etween s5uamous metaplasia and ocular tear dysfunction
in the pathogenesis of pingueculae. 1urthermore! it strategies
of restoring )ax* expression! as well as use of a.normal
differentiation inhi.itors and artificial tears! could .e .eneficial
in the prevention and treatment of pingueculae.
PEM7A3ASAN
3asil penelitian ini menunukkan* untuk pertama kalin'a* pinguecula adalah kondisi abnormal
diferensiasi ditandai dengan metaplasia skuamosa dan proliferasi.
Metaplasia skuamosa telah didefinisikan sebagai transisi patologis dari epitel sekretori berlapis
nonkeratin menadi epitel keratin non sekretori. 4ni adalah ciri dari berbagai gangguan permukaan
bola mata 'ang parah mengakibatkan mata kering disebabkan oleh kurangn'a sekresi kelenar air
mata seperti pada sindrom Sogren* sindrom Ste!ens 5 Johnson* mukus membran pemfigoid* luka
bakar karena kimiaHtermal * dan defisiensi !itamin A. -han et al. menggunakan pengaruh sitologi
untuk menge!aluasi kelainan permukaan bola mata pada empat mata dengan pinguecula dan
pter'gium dan menemukan sel5sel permukaan di pinguecula memperlihatkan metaplasia
skuamosa. Pada penelitian saat ini* dilakukan analisa diferensiasis epitel pinguekula secara
men'eluruh dengan cara immunostaining* 'ang mem!isualisasikan perubahan pada ketebalan
penuh tidak han'a dari profil sel permukaan menggunakan kesan sitologi . (ami mengkonfirmasi
adan'a metaplasia skuamosa* sebagaimana dibuktikan oleh ekspresi ($,* disertai dengan
hilangn'a ekspresi ($+ dan MU-.A- ekspresi dalam semua sampel pinguecula #<ambar 1-5 3&.
Penelitian sebelumn'a menelaskan metaplasia skuamosa disebabkan oleh paparan udara e&$plan
&ultur limbal manu$ia. Patogenesis metaplasia skuamosa pada pinguecula uga dapat
diinterpretasikan dengan menggunakan model ini. Pertama* pinguecula adalah sebuah manifestasi
ika diangkat dari normal conuncti!a meniru &ultur airlift. (edua* T67UT memendek pada
pinguecula* dapat men'ebabkan ;aktu pemaparan lebih lama pada aringan abnormal
dibandingkan permukaan mata normal. Metaplasia skuamosa pada model urothelial* 8iang et
al.1) mengemukakan lima contoh untuk menelaskan etiogenesis metaplasia skuamosa :
tran$diferen$ia$i pada diferen$ia$i terminal ting&at $el% dediferen$ia$i dan rediferen$ia$i%
pluripoten dan pla$ti$ita$ $el $tem% e&$pan$i ,ang dipilih dari $el($el indu& ,ang berbeda%
$erta perlua$an dan penggantian oleh gari$ &eturunan ,ang berbeda. Dalam penelitian kami*
kami melakukan immunostaining dari PaI0 untuk menentukan diferensiasi lineage sel epitel pada
aringan pinguecula. Ekspresi PaI0 postnatal terbatas untuk kornea* konungti!a* lensa* dan epitel
iris dan sel5sel amacrine retina. Penelitian kami sebelumn'a menunukkan PaI0 membantu
menaga fenotip epitel kornea postnatal. Do;nregulasi PaI0 berhubungan diferensiasi epidermis
abnormal pada pen'akit permukaan bola mata 'ang parah. Pada penelitian saat ini* do;nregulasi
PaI0 uga ditemukan pada epitel konungti!a pinguecula. Selain itu* ekspresi PaI0 melemah
bahkan menghilang pada epitel sel basal* dimana terdapat sel progenitor konungti!a*
menunukkan bah;a abnormal diferensiasi pada pinguecula bisa teradi di beberapa sel progenitor.
(emudian PaI0* 'ang berhubungan dengan fenotip in !i!o menaga sel epitel permukaan okuler*
penelitian sebelumn'a menunukkan alur sin'al p1@ terlibat dalam diferensiasi abnormal dari sel
epitel limbal dalam e1 'i'o e1plant culture. Penelitian lebih lanut diperlukan untuk
menentukan apakah alur sin'al p1@ berhubungan dengan diferensiasi abnormal dalam epitel
pinguecula.
Secara tradisional* pingueculae telah dianggap sebagai degeneratif lesi* secara histologi
digambarkan memiliki degenerasi elastotic collagen. Namun* pingueculae uga menunukkan fitur
Terkait dengan mutasi p.1 dan peningkatan metabolisme kolesterol* sesuai dengan hipotesis
bah;a pinguecula berpotensi mengalami proliferatif aringan. Dalam penelitian ini* ekspresi (i02
dan ($0 lebih signifikan pada pingueculae dibanndingkan pada konungti!a normal. Pengamatan
ini uga mendukung gagasan bah;a ada h'perproliferasi di epitel pinguecula. Seak o!erekspresi
p01 terkait dengan perkembangan neoplasma konungti!a* termasuk karsinoma sel skuamous
konungti!a dan pterigium* dilakukan immunostaining P01 untuk selidiki kapasitas proliferasi
pinguecula. Meskipun perbedaan itu tidak signifikan secara statistik* ada lebih ban'ak inti p015
positif dalam pinguecula daripada di konungti!a normal* selanutn'a mengindikasikan bah;a
pinguecula menunukkan karakteristik pen'akit proliferasi skuamous.
Sebuah penelitian sebelumn'a telah menunukkan pemendekan T67UT tetapi teradi sekresi air
mata 'ang normal pada pingueculae. Dalam penelitian kami* 3asil tes Schirmer 4 berada di
kisaran normal pada $, pasien* Sedangkan T67UT lebih pendek dari biasan'a* konsisten dengan
penelitian sebelumn'a. Selain itu * T67UT secara signifikan memanang setelah eksisi tunggal*
lebih lanut dindikasikan bah;a pingueculae terkait dengan gangguan 'ang signifikan pada
stabilitas film air mata. Ada beberapa interpretasi dari T67UT 'ang memendek pada pinguecula :
kelopak mata 'ang abnormal berkedip* &ehadiran forma$i ,ang tida& teratur* dan kekurangan
mukus. Penelasan lain mungkin karena metaplasia skuamosa pada epitel permukaan dari
pinguecula* 'ang bisa membaha'akan stabilitas film air mata. Meskipun metaplasia skuamosa
umum teradi di epitel permukaan okuler pada mata kering* studi lebih lanut diperlukan untuk
mengungkapkan apakah ada hubungan pen'ebab antara skuamosa metaplasia dan fungsi air mata
di pingueculae.
Singkatn'a* penelitian kami menegaskan efek merusak dari pingueculae terhadapa stabilitas film
air mata dan status kesehatan permukaan okuler* sebagaimana dibuktikan oleh penurunan
kemampuan pertahanan mukosa dan kerusakan pada epitel permukaan mata okuler 5'aitu*
skuamosa metaplasia* sehingga teradi ketidakstabilan film air mata. Sebuah korelasi mungkin ada
antara metaplasia skuamosa dan disfungsi air mata okular dalam patogenesis pingueculae. Selain
itu* strategi memulihkan ekspresi PA/0* serta penggunaan inhibitor diferensiasi abnormal dan air
mata buatan* dapat bermanfaat dalam pencegahan dan pengobatan pingueculae .
Acknowledgments
The authors than/ Mueping Nhou and Jianxing $a for critical
review of the project proposal and Nhaosheng Aiu and -u4hen
?ie for generous support in the clinical study.
Ucapan Terima (asih
Para penulis mengucapkan terima kasih Jueping JianIing Khou dan Ma untuk kritis
penelaahan terhadap usulan pro'ek dan Khaosheng 8iu dan Su>hen
/ie atas dukungan dalam studi klinis.
6rom the 4magePro ;ebsite:
L4ntegrated "ptical Densit' #4"D&: Deports the a!erage intensit'Hdensit' of each obect. This
!alue ;ill be eIpressed in terms of the current intensit'Hdensit' mode and calibration.L
The best eIplanation 4 ha!e found: 4"D G area I a!erage densit'
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specificit' of antibodiesN to their antigenN to target fluorescentN d'esN to specific biomoleculeN
targets ;ithin a cell* and therefore allo;s !isualisation of the distribution of the target molecule
through the sample. 4mmunofluorescence is a ;idel' used eIample of immunostainingN and is a
specific eIample of immunohistochemistr'N that makes use of fluorophores to !isualise the
location of the antibodies.O$P
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such as DNA or protein* in response to an eIternal !ariable. An increase of a cellular component is
called upregulation.
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such as a hormoneN or neurotransmitterN* ;hich reduces the cellQs sensiti!it' to the molecule. This
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;hen Ienobiotic molecules such as dioIinN are administered #resulting in greater degradation of
these molecules&.
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method to detect a specific proteinN in a sample. The term immunostaining ;as originall' used to
refer to the immunohistochemical stainingN of tissue sections* as first described b' Albert -oonsN
in $+)$.O$P No; ho;e!er* immunostaining encompasses a broad range of techniMues used in
histolog'N* cell biolog'N* and molecular biolog'N that utilise antibod'5based staining methods.
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fluorescent methods using en>'mesN such as peroIidaseN #see immunoperoIidase stainingN& and
alkaline phosphataseN are no; used. These en>'mes are capable of catal'sing reactions that gi!e a
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