Apoptosis of Ocular Surface Cells in Experimentally
`Induced Dry Eye
`
`Steven Yeh,1 Xiu Jun Song,1,2 William Farley,1 De-Quan Li,1 Michael E. Stern,3 and
`Stephen C. Pflugfelder1
`
`PURPOSE. To evaluate to effect of experimental dry eye on
`ocular surface apoptosis.
`METHODS. Aqueous tear production and clearance were inhib-
`ited by systemic administration of scopolamine and exposure
`to an air draft for 12 days in 4- to 6-week-old 129SvEv/CD-1
`mixed white mice. Eyes and ocular adnexa were excised,
`cryosectioned, and evaluated for apoptosis by terminal deoxy-
`nucleotidyl transferase-mediated dUTP-digoxigenin nick end
`labeling (TUNEL) assay,
`immunohistochemical assay for
`caspase-3 and poly(ADP-ribose) phosphate (PARP), and exam-
`ination of nuclear morphologic changes by Hoechst DNA nu-
`clear staining and transmission electron microscopy.
`RESULTS. The number of TUNEL-positive cells in the mice with
`induced dry eye was significantly increased compared with
`control mice in the following ocular regions: central corneal
`(P ⬍ 0.0014), peripheral corneal (P ⬍ 0.0001), bulbar con-
`junctival (P ⬍ 0.0021), and tarsal conjunctival (P ⬍ 0.0046)
`epithelia; tarsal conjunctival stroma (P ⬍ 0.0274); and lid margin
`(P ⬍ 0.0219, n ⫽ 4 in all cases). There were no significant
`differences observed between treated and control groups in
`the central corneal, peripheral corneal, or bulbar conjunctival
`stroma; meibomian glands; skin; retina-choroid; or episcleral
`regions. Immunohistochemistry for caspase-3 and poly(ADP-
`ribose) polymerase p85 fragment revealed increased immuno-
`reactivity in regions of increased TUNEL positivity, particularly
`in the corneal and conjunctival epithelial cells. Ultrastructural
`morphologic changes consistent with apoptosis were observed
`in the conjunctival epithelial cells.
`CONCLUSIONS. Experimentally induced dry eye in mice causes
`apoptosis of cells in ocular surface tissues including the central
`and peripheral corneal epithelium, bulbar and tarsal conjunc-
`tival epithelia, tarsal conjunctival stroma, and lid margin. Apo-
`ptosis may play a key role in the pathogenesis of keratocon-
`junctivitis sicca and may be a therapeutic target for this
`condition. (Invest Ophthalmol Vis Sci. 2003;44:124 –129) DOI:
`10.1167/iovs.02-0581
`
`From the 1Ocular Surface Center, Cullen Eye Institute, Baylor
`College of Medicine, Department of Ophthalmology, Houston, Texas;
`3Allergan, Inc., Irvine, California; and the 2Third Hospital of Hebei
`Medical University, Shijiazhuang, China.
`Supported in part by Allergan, Inc, an unrestricted grant from
`Research to Prevent Blindness, The Oshman Foundation, and The
`William Stamps Farish Fund.
`Submitted for publication June 13, 2002; accepted July 30, 2002.
`Disclosure: S. Yeh, None; X.J. Song, None; W. Farley, None;
`D.-Q. Li, None; M.E. Stern, Allergan, Inc. (E); S.C. Pflugfelder,
`Allergan, Inc. (C, F)
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be marked “advertise-
`ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`Corresponding author: Stephen C. Pflugfelder, Baylor College of
`Medicine, 6565 Fannin, NC-205 Ocular Surface Center, Houston, TX
`77030; stevenp@bcm.tmc.edu.
`
`124
`
`D ysfunction of the integrated ocular surface–lacrimal
`
`gland functional unit causes the ocular surface disease
`keratoconjunctivitis sicca (KCS).1 The ocular surface in this
`condition is poorly lubricated because of decreased produc-
`tion of mucins by the stratified epithelia and conjunctival
`goblet cells and has altered barrier function manifesting
`clinically as increased permeability to fluorescein dye.2 In-
`stability of the precorneal tear film and punctate epitheli-
`opathy cause blurred and fluctuating vision. The cause of
`these pathologic changes is currently unknown; however,
`there is mounting evidence that inflammation may play an
`important role.
`Evidence for immune-based inflammation in dry eye in-
`cludes increased density of inflammatory cells and elevated
`inflammatory cytokines3–5 and proapoptotic fac-
`levels of
`tors6 –9 in the ocular surface and tear film. The relationship
`between ocular surface inflammation and apoptosis has also
`been the subject of much investigation. Expression of proapop-
`totic markers (Fas, Fas ligand, APO2.7, CD40, and CD40 ligand)
`by the conjunctival epithelium in KCS has been found to be
`significantly higher than in normal eyes and is positively cor-
`related with expression of HLA-DR class II antigen, an immune
`activation marker.6,7 After 6 months of therapy with cyclo-
`sporin A , the levels of cell membrane markers for apoptosis
`(i.e., Fas) and inflammation, such as HLA-DR, were significantly
`reduced.8
`In studies of chronic idiopathic canine KCS, increased ap-
`optosis was observed in epithelial cells and decreased apopto-
`sis in lymphocytes in the conjunctiva and lacrimal glands.9
`Immunohistochemistry studies demonstrated that p53, Fas,
`and Fas ligand were elevated in lacrimal acinar cells and con-
`junctival epithelial cells in dogs with dry eye.9 In contrast, low
`levels of expression of Bcl-2, an antiapoptosis marker, was
`observed in these tissues. After treatment with topical cyclo-
`sporin A, induction of apoptosis in lymphocytes and suppres-
`sion of apoptosis in conjunctival epithelial cells was observed
`in these animals.9 In lacrimal acinar cells, a decrease in p53 and
`an increase in Bcl-2 was also observed after treatment with
`cyclosporin A.9
`Although apoptosis has been demonstrated in chronic dry
`eye in humans and dogs, no studies have been undertaken to
`evaluate the occurrence and kinetics of apoptosis on the ocular
`surface after acute induction of dry eye. We investigated
`whether apoptosis develops on the ocular surface after exper-
`imental induction of dry eye in a mouse model of KCS, using
`three different techniques: the TUNEL assay, which detects
`DNA fragmentation; immunodetection of two cellular markers
`of apoptosis: activated caspase-3 (a downstream effector pro-
`tease) and cleaved poly(ADP-ribose) polymerase (PARP; a nu-
`clear DNA-binding protein); and examination of cell nuclei for
`morphologic changes characteristic of apoptosis by nuclear
`staining with a DNA-binding dye, and transmission electron
`microscopy.
`
`Investigative Ophthalmology & Visual Science, January 2003, Vol. 44, No. 1
`Copyright © Association for Research in Vision and Ophthalmology
`
`APOTEX 1035, pg. 1
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`IOVS, January 2003, Vol. 44, No. 1
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`Apoptosis in Dry Eye
`
`125
`
`TABLE 1. Quantitation of TUNEL-Positive Ocular Surface Cells
`
`Ocular Surface Tissue
`
`Dry Eye Mice (n ⴝ 4)
`
`Untreated Mice (n ⴝ 4)
`
`P
`
`Central corneal epithelium
`Central corneal stroma
`Peripheral corneal epithelium
`Peripheral corneal stroma
`Bulbar conjunctive epithelium
`Bulbar conjunctive stroma
`Tarsal conjunctive epithelium
`Tarsal conjunctive stroma
`Lid margin
`Meibomian glands
`Skin
`Retinal choroid
`Episclera
`
`32.5 ⫾ 9.7
`3.75 ⫾ 4.5
`28.0 ⫾ 6.2
`3.25 ⫾ 3.4
`31.3 ⫾ 11
`5.0 ⫾ 1.8
`55.8 ⫾ 25
`4.25 ⫾ 2.2
`27.5 ⫾ 15
`29.3 ⫾ 25
`24.8 ⫾ 22
`1.25 ⫾ 1.3
`0.50 ⫾ 1.0
`
`Values are mean ⫾ SD. Probabilities by t-test.
`* Indicates statistical significance (P ⬍ 0.05).
`
`3.75 ⫾ 3.4
`1.0 ⫾ 0.82
`0.25 ⫾ 0.50
`0.25 ⫾ 0.50
`2.75 ⫾ 2.5
`2.25 ⫾ 1.7
`0.5 ⫾ 1.0
`0.75 ⫾ 0.96
`4.0 ⫾ 2.2
`0.75 ⫾ 1.5
`3.75 ⫾ 2.5
`0.5 ⫾ 1.0
`0.5 ⫾ 1.0
`
`⬍0.0014*
`⬍0.3893
`⬍0.0001*
`⬍0.1317
`⬍0.0021*
`⬍0.0701
`⬍0.0046*
`⬍0.0274*
`⬍0.029*
`⬍0.0615
`⬍0.1017
`⬍0.3867
`⬍1.000
`
`MATERIALS AND METHODS
`
`Animal Treatment and Tissue Collection
`All animal research protocols were approved by the Baylor College of
`Medicine Center for Comparative Medicine and conformed to the
`standards in the ARVO Statement for the Use of Animals in Ophthalmic
`and Vision Research.
`Aqueous tear production and clearance were inhibited by subcu-
`taneous injection of scopolamine (1 mg in 0.2 mL) three times daily in
`the flanks of 4- to 6-week-old 129SvEv/CD-1 white mice. Dry eye was
`induced in mice with a modification of a previously described tech-
`nique.10 Mice were exposed to a continuous air draft from a fan placed
`6 in. in front of the cage in an environmentally controlled room (50%
`humidity, 18°C) for 10 hours a day for 12 consecutive days. These
`treatments cause a statistically significant decrease in aqueous tear
`production and tear clearance that results in increased corneal epithe-
`lial permeability to fluorescein dye, conjunctival squamous metaplasia,
`and loss of goblet cells.10
`Mice were killed and their eyes and adnexa were excised, embed-
`ded in optimal cutting temperature (OCT) compound (VWR, Swannee,
`GA), flash frozen in liquid nitrogen, and sectioned with a cryostat (HM
`500; Micron, Waldorf, Germany) into 5-␮m sagittal slices that were
`placed on microscope slides (Superfrost/Plus; Fisher, Houston, TX).
`These were stored at ⫺80°C until they were used in the studies of
`apoptosis.
`
`Apoptosis Evaluation
`A combination of immunohistochemical and morphologic studies
`were used to study apoptosis in our mouse model. Specifically, assays
`for DNA fragmentation (TUNEL), caspase 3 activation, and PARP cleav-
`age were performed for immunohistochemical characterization of ap-
`optosis; transmission electron microscopy and Hoechst dye nuclear
`staining were used for morphologic characterization of nuclear
`changes observed in apoptosis.
`The terminal deoxynucleotidyl transferase-mediated dUTP-nick end
`labeling (TUNEL) assay, which detects 3⬘ hydroxyl ends in fragmented
`DNA as an early event in the apoptotic cascade,11 was performed with
`a kit (ApopTag; Intergen Co., Purchase, NY), using a modification of
`the manufacturer’s protocol as previously described.11,12 Cryosections
`of whole mouse eyes were fixed in 1% paraformaldehyde, and cell
`membranes were permeabilized with 2:1 ethanol:acetic acid solution.
`The samples were incubated with TdT enzyme and 11-digoxigenin
`dUTP at 37°C for 90 minutes. After quenching the reaction, samples
`were blocked with blocking solution and incubated with anti-digoxi-
`genin FITC-conjugated antibody for 60 minutes at room temperature.
`One cryosection was incubated with 1 ␮g/mL DNase I in TdT buffer
`
`for 30 minutes at room temperature before incubation with TdT
`enzyme and 11-digoxigenin dUTP as a positive reaction control.
`After completion of the initial TUNEL procedure, the cryosections
`were evaluated for expression of activated caspase-3. After three
`washes in phosphate-buffered saline (PBS, pH 7.2), tissue samples
`were incubated with 5 ␮g/mL polyclonal rabbit anti-active caspase-3
`primary antibody (PharMingen, San Diego, CA) or PBS as a primary
`antibody negative control at 4°C overnight. Samples were then
`blocked with 10% goat serum for 30 minutes at room temperature and
`incubated with goat anti-rabbit conjugated antibody (Alexa Fluor 594;
`Molecular Probes, Eugene, OR) for 45 minutes at room temperature,
`followed by three washes in PBS. Nuclei were then counterstained
`using 0.5 ␮g/mL Hoechst 33342 dye (Sigma, St. Louis, MO) in approx-
`imately 30 ␮L mounting gel (Gel Mount; Fisher) and a 22 ⫻ 50-mm
`coverslip (Fisher) then applied.
`Apoptotic cells in different regions of the ocular surface were
`assessed by epifluorescence microscope (Eclipse E400; Nikon, Garden
`City, NY) . Photographs at 400⫻ magnification were taken of repre-
`sentative areas of the cornea, bulbar conjunctiva, and tarsal conjunc-
`tiva. All TUNEL-positive cells were counted in the conjunctiva in
`100-␮m length ⫻ 100-␮m width areas in the sagittal sections and in the
`cornea in 100-␮m length ⫻ 20-␮m width areas in the sagittal sections.
`A Student’s t-test was used to analyze statistical significance of the data
`collected.
`Immunodetection of the PARP p85 fragment was performed by
`immunofluorescent staining of cryosections that were fixed in 100%
`methanol at 4°C for 10 minutes. After three washes in PBS, samples
`were blocked with 10% goat serum for 30 minutes at room tempera-
`ture and incubated for 2 hours at room temperature with a 1:50
`dilution of rabbit anti-mouse PARP p85 fragment antibody (Promega,
`Madison, WI) or PBS, as a primary antibody negative control. After
`three washes in PBS, samples were incubated with goat anti-rabbit
`conjugated antibody (Alexa Fluor 488; Molecular Probes) for 45 min-
`utes at room temperature, followed by three washes in PBS and nuclei
`counterstaining with 0.5 ␮g/mL Hoechst 33342 dye (Sigma).
`For transmission electron microscopy, the cornea and conjunctiva
`were excised from treated and control mice. The tissues were diced
`carefully into 1-mm cubes, to avoid crushing, and fixed in 3% glutar-
`aldehyde buffered to pH 7.2 with 0.01 M piperazine-N,N⬘-bis(2-ethane
`sulfonic acid [PIPES]) for 1 hour. They were rinsed in buffer and
`postfixed in PIPES-buffered osmium tetroxide (pH 7.2) for 1 hour at
`room temperature, rinsed in several changes of distilled water, and
`dehydrated through a graded series of ethanol. The dehydrated tissues
`were incubated in two 45-minute changes of propylene oxide followed
`by a 1:1 mixture of propylene oxide and Spurr resin for 1.5 hours. The
`tissue pieces were then incubated in pure resin for 1.5 hours, after
`
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`

`126 Yeh et al.
`
`IOVS, January 2003, Vol. 44, No. 1
`
`FIGURE 1. TUNEL-caspase-3 dual-la-
`bel assay. In the conjunctiva (A) or
`cornea (B). (A) Conjunctiva. Top left:
`TUNEL-positive cells in bulbar con-
`junctival epithelium in treated mouse.
`Top center: Hoechst 33342 nuclear
`stain in treated mouse. Top right: ac-
`tive caspase-3 staining in treated
`mouse. Bottom left: TUNEL staining
`of bulbar and tarsal conjunctiva in
`untreated control mouse. Bottom
`center: Hoechst 33342 nuclear stain-
`ing of bulbar and tarsal conjunctiva
`in untreated control mouse. Bottom
`right: active caspase-3 staining of bul-
`bar and tarsal conjunctiva in un-
`treated control mouse. (B) Central
`cornea. Top left: TUNEL-positive cells
`in central corneal epithelium in
`treated mouse. Top center: Hoechst
`33342 nuclear stain in treated mouse.
`Top right: active caspase-3 staining
`of corneal epithelium in treated
`mouse. Bottom left: TUNEL-positive
`cells in superficial corneal epithe-
`lium of untreated control mouse.
`Bottom center: Hoechst 33342 nuclear
`stain in untreated control mouse.
`Bottom right: active caspase-3 stain-
`ing in untreated control mouse.
`
`which they were transferred to fresh resin in block molds and allowed
`to cure at 60°C overnight. Sections cut 1 ␮m thick from the hardened
`blocks were mounted on glass slides, stained with an alcoholic solution
`of toluidine blue and basic fuchsin and examined by light microscope.
`Areas of interest were trimmed, and 60-nm sections were cut and
`mounted on copper grids (300 mesh). The grids were stained with
`uranyl acetate and lead citrate and photographed with an electron
`microscope (model 100C Temscan; JEOL, Peabody, MA; and 4489 film;
`Eastman Kodak, Rochester, NY).
`
`RESULTS
`
`As previously reported, mice treated with systemic scopol-
`amine injections and subjected to an air draft and desiccating
`environment showed a significant decrease in aqueous tear
`production and clearance by day 4 that was sustained through-
`out the treatment period.10 Dryness was accompanied by de-
`velopment of KCS, with loss of conjunctival goblet cells and
`increased corneal epithelial permeability to carboxyfluores-
`cein.10 Apoptosis in the cornea and conjunctiva was assessed
`in mice treated in this fashion for 12 days and in untreated
`control animals.
`
`FIGURE 2. TUNEL assay positive control sections. Top left: Hoechst
`33342 nuclear stain of lid margin (L), tarsal conjunctiva (T), bulbar
`conjunctiva (B), and cornea (K). Top right: TUNEL-positive cells in lid
`margin,
`tarsal and bulbar conjunctiva, and cornea. Bottom left:
`Hoechst 33342 nuclear stain of retina and choroid. Bottom right:
`TUNEL-positive cells of retina and choroid.
`
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`IOVS, January 2003, Vol. 44, No. 1
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`Apoptosis in Dry Eye
`
`127
`
`tral and peripheral corneal and bulbar and tarsal conjunctival
`epithelia; tarsal conjunctival stroma; and lid margin (Table 1,
`Fig. 1). There were no significant differences between dry eye
`and control groups in the central corneal, peripheral corneal,
`or bulbar conjunctival stroma; meibomian glands; skin; retina-
`choroid; or episcleral regions (Table 1).
`Positive control experiments revealed TUNEL-positive cells
`throughout all regions of the eye including the cornea, bulbar
`and tarsal conjunctiva, retina, and choroid (Fig. 2).
`Immunofluorescent staining for active caspase 3 revealed
`increased immunoreactivity in regions of TUNEL positivity.
`
`FIGURE 3. PARP staining of ocular surface tissues in the tarsal con-
`junctiva (A) and cornea (B). (A) Tarsal conjunctiva. Top left: numerous
`PARP-positive cells were detected in the superficial tarsal conjunctival
`epithelium of treated mouse. Inset: higher magnification of boxed area
`(left) highlights PARP-positive cells in conjunctival epithelium. Top
`right: Hoechst 33342 nuclear counterstain of tarsal conjunctival re-
`gion. Bottom left: representative tarsal conjunctival section from un-
`treated control mouse. Inset: higher magnification of boxed area (left)
`highlights absence of PARP-positive cells in conjunctiva of untreated
`control mouse Bottom right: Hoechst 33342 nuclear counterstain of
`tarsal conjunctiva in untreated control mouse. (B) Cornea. Top left:
`PARP-positive cells in superficial corneal epithelium of treated mouse.
`Inset: higher magnification of boxed area (left) highlights PARP-posi-
`tive cells within the corneal epithelium. Top right: Hoechst 33342
`nuclear counterstain of treated mouse cornea. Bottom left: PARP stain-
`ing in cornea of untreated control mouse. Inset: higher magnification
`of boxed area (left) highlights absence of PARP-positive cells in cornea
`of untreated control mouse. Bottom right: Hoechst 33342 nuclear
`counterstain in cornea of untreated control mouse.
`
`TUNEL and Caspase-3 Immunostaining
`Normal, untreated control mice exhibited minimal apoptosis
`assessed by TUNEL staining in the cornea and conjunctiva
`(Table 1, Fig. 1). The number of TUNEL-positive cells was
`significantly increased in mice with induced dry eye compared
`with control mice in the following ocular surface tissues: cen-
`
`FIGURE 4. Hoechst 33342 nuclear stain of conjunctiva (A) and cornea
`(B). (A) Tarsal conjunctiva. Top left: the superficial tarsal conjunctival
`epithelium in the treated mouse showed dysmorphic architecture and
`small, fragmented appearance of nuclei. Bottom left: higher magnifi-
`cation of boxed area (top left) shows multiple fragmented cells in tarsal
`conjunctival epithelium of treated mouse (arrows). Top right: tarsal
`conjunctival architecture of untreated control mouse. Bottom right:
`higher magnification of boxed area (top right) shows no nuclear or cell
`fragments in the untreated mouse. (B) Cornea. Top left: the corneal
`epithelium in the treated mouse appeared thinned and atrophic with
`dysmorphic architecture. Bottom left: higher magnification of boxed
`area (top left) shows clumped chromatin in multiple basal epithelial
`cells (arrows). This finding was also seen to some degree in the
`superficial corneal epithelium. Top right: thicker, regular architecture
`in cornea of untreated control mouse. Bottom right: higher magnifi-
`cation of boxed area (top right) shows peripheral clumping of nuclear
`material, which was not seen to the same degree in the untreated
`control cornea as in the treated cornea.
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`128 Yeh et al.
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`IOVS, January 2003, Vol. 44, No. 1
`
`FIGURE 5. Transmission electron mi-
`croscopy (TEM) of conjunctival epi-
`thelium of treated mouse. (A) Lower
`magnification TEM of bulbar con-
`junctiva epithelium. Multiple epithe-
`lial cells (arrows) exhibited chroma-
`tin condensation and peripheral
`aggregation of compacted chroma-
`tin. (B) Higher magnification TEM of
`bulbar conjunctiva epithelial cell
`showing chromatin condensation and
`peripheral aggregation of nuclear ma-
`terial, suggesting apoptotic cell death.
`
`Specifically, active caspase 3 activity was localized to the cen-
`tral corneal epithelium, peripheral corneal epithelium, super-
`ficial conjunctival epithelium, and lid margins of the mouse
`with induced dry eye. This staining was not observed in the
`negative control for which secondary antibody alone was used
`(data not shown).
`
`Anti-PARP p85 Fragment Immunostaining
`
`PARP p85 fragment staining revealed positive cells in the cen-
`tral and peripheral corneal and the bulbar and tarsal conjunc-
`tival epithelia and stroma in the treated mice. The punctate and
`diffuse pattern of PARP staining was similar to that observed in
`apoptotic liver epithelial cells.13 The cells showing the stron-
`gest staining for PARP in the tarsal conjunctival and central
`corneal epithelia of treated mice (Fig. 3). In contrast, only
`nonspecific staining of the conjunctival epithelial basement
`membrane and weak diffuse cytoplasmic staining was noted in
`untreated control mice (Fig. 3). No staining was observed in
`the secondary antibody alone control (data not shown).
`
`Hoechst 33342 Nuclear Staining
`Nuclear staining of the cornea and conjunctiva with Hoechst
`33342 revealed dysmorphic nuclear architecture and frag-
`mented nuclear material in the treated mice compared with
`control animals (Fig. 4).
`
`Transmission Electron Microscopy
`Ultrastructural nuclear changes consistent with apoptosis were
`observed in the cornea and conjunctiva of treated mice. Nu-
`clear chromatin condensation and peripheral migration of
`chromatin, as shown in a representative section in Figure 5,
`were noted in many conjunctival epithelial cells. These nuclear
`changes were rarely observed in the conjunctival stromal cells.
`Fewer apoptotic cells were noted in the corneal epithelium
`than in the conjunctiva. Although some nuclear condensation
`and peripheral margination of chromatin were observed in
`occasional corneal epithelial cells, nuclear fragmentation to the
`extent of that in the conjunctival epithelium was not observed.
`
`DISCUSSION
`
`Although apoptosis of ocular surface cells has been detected in
`chronic dry eye in dogs and humans, our experiments indicate
`that apoptosis is an acutely inducible event that accompanies
`the development of KCS in our experimental mouse model of
`dry eye. Apoptosis on the ocular surface was demonstrated by
`several markers and by characteristic nuclear morphologic
`changes. The TUNEL assay revealed DNA fragmentation sug-
`gestive of apoptosis in multiple ocular surface tissues, includ-
`ing the central and peripheral corneal epithelium, bulbar and
`tarsal conjunctival epithelia, tarsal conjunctival stroma, and lid
`margin in mice with induced dry eye. Immunohistochemistry
`for caspase 3 and PARP and ultrastructural studies using trans-
`mission electron microscopy suggest that the most marked
`apoptosis occurred in the superficial bulbar conjunctival epi-
`thelium. Although TUNEL staining was observed in the periph-
`eral and central corneal epithelium, less ultrastructural evi-
`dence of apoptosis was observed at these sites than in the
`conjunctival epithelial tissues. It is possible that the TUNEL
`assay may detect cells entering the apoptotic pathway before
`dissolving into irreversible nuclear fragmentation or it may
`detect other nonspecific DNA changes induced by ocular sur-
`face dryness.
`Apoptosis occurs through two pathways, an extrinsic path-
`way involving the interaction of death ligands (e.g., TNF-␣, Fas
`ligand) with their respective cell surface receptors and an
`intrinsic pathway that is initiated by insults that damage the
`DNA, such as ultraviolet light and chemotherapeutic agents.
`Both pathways eventually result in mitochondrial damage with
`release of cytochrome c and downstream activation of caspases,
`such as caspase 3. Activation of other downstream caspases
`results in cleavage of cellular proteins, such as PARP, cytoker-
`atin 18, and other caspases, that lead to the morphologic and
`biochemical changes of apoptosis (Fig. 6).14,15
`Poly(ADP-ribose) polymerase (PARP) is a nuclear DNA-bind-
`ing protein that functions in DNA base excision repair.16 PARP
`cleavage results in a decreased enzymatic repair function and
`contributes to the progression of apoptosis, although PARP
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`Apoptosis in Dry Eye
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`
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`
`FIGURE 6. Mechanisms and markers of apoptosis. Summary of current
`knowledge of the mediators of apoptosis.14,15
`
`cleavage is not absolutely necessary for apoptosis to proceed.17
`Caspase 3, a downstream effector caspase, is responsible for
`cleavage of several critical nuclear targets in the apoptotic
`cascade. These include the inhibitor of caspase-activated de-
`oxynuclease (ICAD), which results in nuclear fragmentation,
`and PARP, which results in a defective DNA repair function.18
`Detection of the activated caspase 3 and PARP p85 fragment in
`the bulbar and tarsal conjunctival epithelium and in the central
`and peripheral corneal epithelium of the treated mice in our
`study suggests that these apoptotic mediators may be involved
`in the pathogenesis of dry eye.
`Although the precise pathway by which apoptosis occurs in
`dry eye has not yet been elucidated, we believe that this
`phenomenon may play a crucial role in its pathogenesis and in
`the clinical manifestations of KCS. The proinflammatory ocular
`surface milieu that develops in dry eye could certainly activate
`extrinsic pathways of apoptosis. For example, increased ex-
`pression of the proapoptotic cytokines, TNF-␣ and IL-1, have
`been detected in the conjunctival epithelium and tear fluid of
`patients with KCS.19,20
`Whether the occurrence of apoptosis is a physiologic re-
`sponse to an ocular surface insult or a critical element in the
`pathogenesis of the ocular surface disease in dry eye may be
`delineated by further natural history studies correlating apo-
`ptosis with clinical progression of dry eye. However, the death
`of cells responsible for ocular surface health and protection,
`such as the conjunctival goblet cells, could contribute to pro-
`gression of KCS. Furthermore, the punctate epithelial erosions
`that are observed in the cornea and conjunctiva of patients
`with KCS could be attributed to sloughing of apoptotic epithe-
`lial cells.
`Our studies suggest that apoptosis may play a key role in the
`pathogenesis of KCS. Characterization of apoptotic pathways
`may provide therapeutic targets for the treatment of KCS. As
`previously reported, one of the therapeutic mechanisms of
`cyclosporin A in dry eye could be inhibition of mitochondrial
`pathways of apoptosis.9 Cyclosporin A has been reported to
`prevent the mitochondrial permeability transition that pro-
`ceeds apoptotic cell death.21,22
`
`APOTEX 1035, pg. 6
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