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Fluorescein staining and physiological state of corneal epithelial cells
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Fluorescein staining and physiological state of corneal epithelial cells Kalika L. Bandamwar a,b,∗ , Eric B. Papas a,b,c , Qian Garrett a,b a
Brien Holden Vision Institute, Sydney, Australia School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia c Vision Cooperative Research Centre, Sydney, Australia b
a r t i c l e
i n f o
Article history: Received 7 April 2013 Received in revised form 1 September 2013 Accepted 20 November 2013 Keywords: Fluorescein Micropunctate staining Corneal epithelium Apoptosis Live-dead cells
a b s t r a c t Purpose: To evaluate the physiological status of corneal epithelial cells exhibiting fluorescein staining. Methods: Fluorescein staining properties of corneal epithelial cells under normal and stressed conditions were studied using cell-culture (human corneal limbal epithelial cells – HCLE) and organ-culture (rabbit) models. Stress stimuli comprised exposure to hypotonicity, hypertonicity, preservatives, scratch, and alkaline wounding. In addition to fluorescein, cells were stained with Hoechst-33342 (HO), Propidiumiodide (PI), and Annexin-V (AN-V) to identify live, dead and apoptotic cells. Clinical-slit-lamp and fluorescence confocal-microscopic (FCM) observations were performed. FCM images were quantified for fluorescence intensity using Image-J software. Results: Healthy HCLE cells uniformly took up fluorescein to a moderate degree with a mean grey value of 62 ± 24 (mean ± SD) on a scale of 0–256 (no unit). Fluorescence levels similar to those observed prior to stress were associated with healthy cells. Apoptotic cells showed the highest fluorescence (138 ± 38). Dead cells showed minimal fluorescence (23 ± 7) that was similar to the background (20 ± 11, p > 0.05). Observations in whole rabbit eyes were in general agreement with these cell culture findings. Conclusions: The clinical observation of corneal staining with fluorescein suggests the presence of epithelial cells that are undergoing apoptosis but does not indicate dead cells. Under in vitro or ex vivo conditions, healthy cells took up fluorescein at levels that were lower than those of apoptotic cells and thus, are not likely to be perceived as exhibiting staining during clinical observation. Sodium fluorescein may be considered as a probe for apoptotic epithelial cells. © 2013 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.
1. Introduction The use of dyes to aid in evaluation of the ocular surface is common clinical practice. Several examples have been used over the last 130 years [1], including rose bengal [2], lissamine green [3] and most commonly, sodium fluorescein, which is also known as “fluorescein” [4]. The application of fluorescein during ocular examination was first reported by Pfluger [1] for staining epithelial abrasions on rabbit corneas. Since then, its use has been widely reported in the ophthalmic literature and it is now an important part of ocular surface assessment. Despite its well accepted clinical utility, the exact interpretation of corneal staining (we use the term “staining” to refer to superficial micropunctate fluorescein staining unless stated otherwise) is not
∗ Corresponding author at: Brien Holden Vision Institute Research Centre, Level 4, B V Raju Bhavan, Banjara Hills, Road No. 2, Hyderabad 500034, India. Tel.: +91 814 250 32 11. E-mail addresses: [email protected], [email protected] (K.L. Bandamwar).
well understood and has been the topic of continuing debate [5–9]. Early reports proposed that staining was the result of fluorescein accumulation in voids on the ocular surface or intercellular spaces [10] while others [2,11] suggested that normal desquamation of the corneal epithelium might create cavities on the ocular surface in which fluorescein could accumulate to cause a punctuate appearance. Recently however, these “pooling” based theories have been challenged by the observation that repeated saline rinsing does not substantially alter the appearance of corneas exhibiting superficial micro-punctate staining [12]. It is also unlikely that this specific clinical picture could be produced by the rapid stromal diffusion of fluorescein through disturbed cell–cell junctions [2], as has also been suggested. The hypothesis that epithelial cells might actually take up fluorescein was confirmed by Wilson et al. [13] who proposed that fluorescein uptake was probably secondary to cell damage, as had earlier been suggested by Tabery [14–16]. However, correlation of fluorescein staining with cell damage was not demonstrated. More recently, it has been suggested that certain types of staining, in particular those associated with contact lens multipurpose solution (MPS) (i.e. SICS – solution induced corneal staining), is not indicative of ocular surface damage but rather due to an interaction
1367-0484/$ – see front matter © 2013 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.clae.2013.11.003
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between fluorescein molecules, certain components of the lens care solution and the ocular surface [17]. This suggestion proposes that the appearance of clinically visible staining under these circumstances is an artefact produced by the interaction of fluorescein with certain compounds in the solution, specifically ophthalmic preservative molecules. These complexes then adhere to the ocular surface giving the appearance of punctuate staining. Thus, evaluation of the physiological state of cultured corneal epithelial cells or ex vivo corneal cells induced by various stress stimuli (mechanical or chemical) and confirmed using apoptotic or necrotic specific stains would help to indicate the association between the observed clinical appearance and the underlying status of the cells. Based on these observations, it is evident that there is a range of potential mechanisms by which fluorescein could interact with the ocular surface. Which of these theories accounts for the observed clinical appearance might vary depending on the particular circumstances. A corneal abrasion caused by mechanical injury might fluoresce in the presence of fluorescein due to a different mechanism than, for example, that associated with the use of a multipurpose disinfecting solution [12]. Understanding which of these responses describes the response of epithelial cells in any situation is important to correctly interpret the clinical picture, identify potential etiological factors, and determine appropriate remedial actions [7,18]. The purpose of the present study was to investigate the association between fluorescein staining and the physiological state of the cells. We used in vitro cell cultures and ex vivo organ culture models to study how various stress stimuli affect the response of corneal epithelial cells to fluorescein. By using both in vitro cell culture and ex vivo organ culture models, together with a series of well characterized alternative stains for detecting cellular apoptosis and necrosis, we aimed to establish how physiological alteration of epithelial cells affects the associated fluorescein staining characteristics. 2. Materials and methods 2.1. Reagents Sodium Fluorescein, Propidium iodide (PI) and Hoechst-33342 (HO) were obtained from Sigma–Aldrich, MO, USA. AnnexinV-Cy3 was purchased from Biovision (Mountain View, CA, USA). Eagle’s minimum essential medium (EMEM), foetal bovine serum (FBS), Keratinocyte serum free medium (K-SFM), antibiotics (Penicillin–Streptomycin) and rat tendon fibre (RTF) were obtained from Invitrogen-GIBCO, Carlsbad, CA, USA. Epithelial growth factor (EGF), trypsin–EDTA and bovine pituitary extract (BPE) were obtained from Invitrogen, Mount Waverley, VIC, Australia. Sodium chloride, calcium chloride and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were also obtained from Sigma. Polyhexamethylene biguanide (PHMB) (Cosmocil-CQ® ) was obtained from Snow Drift, South Tucson, AZ. Organ culture medium was prepared by adding 10% FBS (w/v), 1% RTF (v/v) and 0.1% antibiotic (v/v) in EMEM. Binding buffer was prepared in Milli-Q water and contained 140 mmol/l NaCl, 10 mmol/l HEPES and 2.5 mmol/l CaCl2 . 2.2. Cell culture Immortalized human corneal limbal epithelial (HCLE) cells were cultured using previously described methods [19–21]. Briefly, HCLE cells were maintained at 2 × 104 cells/cm2 in a K-SFM supplemented with 25 g/ml of BPE, 0.2 ng/ml of EGF and 0.4 mM CaCl2 and grown at 37 ◦ C in a 5% carbon dioxide humidified atmosphere. Once 80% confluence was reached, the cells were detached using
trypsin–EDTA and transferred to grow on glass cover slips, maintained in the same culture medium. Cells were then incubated overnight to allow attachment. 2.3. Organ culture Freshly excised eyes (n = 20) of New Zealand white rabbits were obtained within 1 h post-mortem. These rabbits were obtained from other research that did not require ocular tissue. All the procedures were conducted in accordance with ARVO statement for the use of animals in ophthalmic and vision research. Only clear corneas with no visible oedema or pre-existing staining were used. Two surgical needles were passed through the lateral or posterior conjunctiva of the eye globe to provide support and to aid handling. During imaging, these needles were temporarily inserted into a Styrofoam block, creating a stable fixture for the globes which could then be attached to the bio-microscope chin rest during imaging. In this way direct contact with the corneal surface was avoided. 2.4. Stress stimuli Near confluent monolayers of HCLE cells and freshly excised eyes were rinsed once with the respective culture media and then exposed to a single stress stimulus. Stimuli were chosen based on conditions which could reasonably be expected to mimic clinically observable corneal insults in a real life situation. Chosen stimuli included exposure to hypotonicity, hypertonicity, ophthalmic preservative, and scratch or alkaline wounding. All experiments were replicated at least three times. The time course for development of corneal staining arising from moderate hyperosmolar environment in dry eye or exposure to preservative in solution induced corneal staining (SICS) [22] ranged from minutes to hours. However, in ex vivo animal eyes it is important to reproduce a similar environment more rapidly, to avoid post-mortem changes. Hence stimulus strengths were increased above those typical of human, in vivo situations such as hyperosmolarity in dry eyes and preservative exposure in SICS. Milli-Q® water was introduced to create a hypotonic environment [23]. Likewise, hyper-osmolar solution (5% w/v NaCl in culture medium, >2000 mOsm) was used to produce hypertonicity [24]. A solution of 0.001% PHMB (w/v in phosphate buffered saline, PBS) (concentration ∼10 times higher than used in ophthalmic preparations) represented a commonly used ophthalmic preservative [25]. For all the above stimuli, cells were incubated with 1 ml of the stimulus solution for a period of 1 min PBS was used as a control. For rabbit corneas, 1 ml of test solution was administered, dropwise over period of 1 min. Confluent monolayer cultures were wounded by scratching with a blunt ended 200 l pipette tip, producing a wound of approximately 1 mm diameter [26]. Alkaline wounds were induced by placing a 2.6 mm circular disc of filter paper that had been freshly soaked for 20 s in 2 l of 0.2 N NaOH, on the centre of the cell sheet for 20 s [20,21]. Following exposure to the various stress stimuli, cultured cells and eye balls were rinsed with culture media prior to staining. As the depth of the injury could not be accurately limited to the superficial cornea epithelium, scratch and alkaline wounds were not performed in ex vivo rabbit eyes. 2.5. In vitro confocal microscopic evaluation of cultured cells under normal and stressed conditions Fluorescein staining of the cells in response to various stress stimuli and its association with cell physiological health status were evaluated using a confocal microscope (FluoView FV1000 Confocal
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Table 1 Criteria to distinguish live, apoptotic and dead cells based on staining with Propidium iodide (PI), Hoechst 33342 (HO) and Annexin-V-Cy3 (AN-V-Cy3) dyes. Dye
Fluorescence
Specific location
Normal live
Early apoptotic
Late apoptotic or early necrotic dead cells
PI HO AN-V-Cy3
Red Blue Red
Nucleus Nucleus Cell membrane
−ve +ve −ve
−ve +ve +ve
+ve +ve +ve
Microscope, Olympus, Japan) in conjunction with the use of specific dyes, Hoechst-3342 (HO), Propidium iodide (PI), and Annexin-V (AN-V) for live, dead or apoptotic cell identification. HO is a bright blue nucleic acid dye which fluoresces in contact with nucleic acid. PI is also a nucleic acid dye and fluoresces more brightly when it comes in contact with nucleic acids after the cell membrane it is permeabilized. This property of PI is used to identify permeable cells such as late apoptotic to late necrotic i.e. dead cells. Annexin-V (AN-V) is a probe to identify apoptotic cell membrane changes. It has calcium dependent specific affinity towards phosphotidylserine (PS), a cell membrane protein that flips from inside to outside of the membrane of cells which are undergoing early to late apoptosis [27–29]. Hence, Annexin-V stains early apoptotic to late stage apoptotic cells. It should be noted that AN-V is commercially available with various conjugated fluorophores, such as AN-V-FITC (bright green), AN-V-Cy5 (week blue-violet florescence) and AN-V-Cy3 (red), in the present study, we used the AN-V conjugated with Cy3 (red) in order to distinguish from the fluorescein staining (green). Although PI and AN-V-Cy3 both display red fluorescence, they have distinct locations on cells and hence were considered suitable for simultaneous use. The criteria for defining normal (live), apoptotic, and necrotic dead cells in the present study are described in Table 1. Optimal conditions for fluorescein staining of the cells were determined after a series of experiments were performed using varying concentrations of the fluorescein dye (in the range of 0.001–1.0%) and incubation times with cells (from 15 s to 10 min), as well as the number of rinses (effect of rinsing up to 6 times was tested). Table 2 summarize the resulting optimal microscopic settings which were then used throughout the experiments for acquiring images. (1) Concentration of fluorescein: 1% (w/v in culture medium). (2) Incubation time: 1 min. (3) Number of rinses: 3 (with 2 ml volume of culture medium). Following the treatment with or without stress, both stressed and control (unstressed/health) HCLE cells were stained with 1 ml fluorescein (1% w/v in culture medium) for 1 min followed by 3 rinses with culture medium. Cells were then stained with a mixture of PI, HO and AN-V-Cy3 (as per manufacturer’s recommendation). Briefly, the cells were incubated with PI (0.02 ng/ml w/v in culture medium, Sigma–Aldrich, MO, USA), HO (0.01 ng/ml w/v in culture medium, Sigma–Aldrich, MO, USA) and AN-V-Cy3 (20 l/ml v/v in culture medium) Biovision (Mountain View, CA) for 5 min at room temperature followed by one rinse with culture medium prior to imaging. To evaluate the effect of mixture of other dyes (PI, HO, AN-V) on fluorescein staining of the cells, the stressed cells were divided in two groups. One group was stained with fluorescein only and the other was stained with a mixture of fluorescein, PI, HO and AN-V. Quantification of low, moderate and hyper-fluoresce intensity: The fluorescence intensity of cells stained with fluorescein was obtained by extracting the green component (8 bits) from digitally acquired RGB, then using Image-J software to determine the relevant grey scale intensity values. All images were obtained at
200× magnification using constant viewing conditions. For each test condition and control, 3 separate samples were prepared and multiple images were obtained at various points across the cell field (10–15 images/sample). This was repeated on three separate days for repeatability. Of these, at least 10 pictures for each test and control condition were randomly selected. Using the HO/AN-V-Cy3/PI staining characteristics (Table 1) as a guide for cell physiological status, 10 cells for each of the dead, healthy and apoptotic states respectively were identified from each image and grey scale fluorescein intensity values measured. Although auto-fluorescence of HCLE cells was observed at high laser intensities, when the optimized confocal settings were employed, auto-fluorescent intensity did not differ appreciably from the background level of the culture surface that was devoid of cells. Hence, cover slips with no cells were measured on each day to establish the background fluorescence intensity. To eliminate the possibility of bias introduced as a consequence of the manual selection of cells for this analysis, an alternative means of establishing fluorescence characteristics was also conducted. All cells in the field of view of five randomly selected images from each test and control stimulus were analyzed for their grey scale fluorescein intensity values followed by classification of each cell as either live, apoptotic or dead based on their response to HO, AN-V and PI. Three distinct levels of fluorescence intensities were observed. Grey scale values in the range 0–40 were classified as low, those between 41 and 100 as moderate and 101 and 256 as high. In vitro light microscopic evaluation of the effect of stress stimuli on morphology of cultured cells and clinical slit-lamp microscopic evaluation of fluorescein staining of cultured corneal epithelial cells under healthy and stressed conditions: The stressed HCLE cells induced by various stimuli as described previously were further evaluated under light microscope for cell morphology. The fluorescein-staining of these HCLE cells was further assessed using a clinical slit-lamp bio-microscope (Telaval-31, Carl ZEISS, Germany) equipped with cobalt blue light, Kodak Wratten-12 filter and a camera attachment (Canon Power Shot A620). Images were obtained using slit-lamp standard settings as described elsewhere [22]. 2.6. Evaluation of fluorescein staining of the ex vivo corneas under stress Several images were captured from various focal planes to determine intensities of live, dead and apoptotic cells. No statistical analysis was performed for the ex vivo observations which were conducted primarily to provide visual confirmation of the in vitro findings. Prior to the in vitro confocal microscopic evaluations, the fluorescein stained corneas were evaluated and imaged using a clinical slit-lamp bio-microscope. Soon after slit-lamp bio-microscopic observation of fluorescein staining, corneas were incubated with 1 ml of culture medium containing 0.1 ng/ml of HO, 0.2 ng/ml of PI, and 10 l of AN-V (Annexin-V) reagent and incubated for 5 min in the dark. Corneas were then evaluated under the in vitro laboratory fluorescent confocal microscope (FluoView FV1000, OLYMPUS, Japan) at various magnifications.
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Table 2 Filter setting for visualization of PI, HO, AN-V, and fluorescein staining of HCLE cells. Dye
Excitation filter (nm)
Dichrome mirror (nm)
Barrier filter (nm)
Gain
Off set
HV
Laser power
PI HO AN-V-Cy3 Fluorescein
520–550 330–385 520–550 440–490
565 400 565 505
565–675 425–473 565–675 510–550
1 1 1 1
12 12 12 10
450 350 450 350
18 12 18 10
Fluorescent intensity values were compared between groups using analysis of variance. A p-value of less than 0.05 was considered to be statistically significant. 3. Results 3.1. Fluorescein staining of healthy HCLE cells Fluorescein staining of the healthy cells was imaged over 1 m depth intervals across a single cell using the confocal microscope z-scan feature. It was evident that fluorescein had entered the cell as demonstrated by representative images (Fig. 1A) and the fluorescent intensity profile across the single HCLE cells (Fig. 1B). Similar results were observed when untreated ex vivo corneal epithelial cells were imaged using the z-scanning feature of the confocal microscope; following staining with fluorescein (data not presented). 3.2. Identification of apoptotic and dead cells using PI and AN-V-Cy3 staining Confocal microscopy provides information on a particular focused plane. When focus changes from one plane to other a different configuration of the cell at that particular plane becomes visible resulting in different appearance of cell e.g. at one plane of focus the nuclei of cell might not be visible however with slightest change tip of nuclei may appear, resulting in different appearance of the cell. Fig. 2 demonstrates the appearance of the dead, apoptotic and live cells after staining with PI (nuclei-red), AN-V-Cy3 (membrane-red) and HO (nuclei-blue) when the cell nucleolus was in focus (Fig. 2A–C) or out of focus (Fig. 2D–F). It can be seen from Fig. 2 that dead cells can be identified by positive PI staining with intense red fluorescence in the nuclear region along with AN-V-Cy3 positive staining on the cell membrane (Fig. 2A and D). Apoptotic cells can be identified by positive AN-V-Cy3 staining seen as faint red fluorescence on cell membranes but with no PI staining in the nuclear region (Fig. 2B and E). Live cells were identified as cells with positive staining to HO and negative staining to PI, i.e. no red fluorescence in the nucleus (Fig. 2C), and no staining to AN-V-Cy3 on the cell membrane (Fig. 2F). 3.3. Association of fluorescein staining of HCLE cells with their physiological health status under stress conditions Fig. 3 shows a monolayer of HCLE cells after exposure to hypertonic stress and subsequently stained with fluorescein, PI, HO and AN-V-Cy3. Normal, healthy cells stained positive to HO (blue) but negative to both PI and AN-V-Cy3 (no red) and showed a moderate level of fluorescein intensity (a representative such cell is circled). Also evident was a population of apoptotic cells stained positive to both HO (blue) and AN-V-Cy3 (faint red) but negative to PI (no bright red), which exhibited a very high level of fluorescein intensity (a representative group of such cells is outlined by dotted lines). Early necrotic or late apoptotic, dead cells positive to HO (blue), and to AN-V-Cy3 and PI (bright red) showed a low level of fluorescein staining (indicated with rectangles). In
addition, there is also a population of cells which display very low levels of fluorescein staining but stained positive to HO (blue) and AN-V-Cy3 (faint red), but negative to PI (no bright red) (examples are indicated by arrows). These could be the cells in later stage of apoptosis or early stages of necrosis. We made similar observations for fluorescein and HO/AN-V-Cy3/PI staining of HCLE cells stressed by other stimuli including exposure to MilliQ water (hypotonic condition), or culture medium containing PHMB at 0.0001% PHMB, or stressing cells using scratch or alkali wounding (Fig. 6). 3.4. Effect of simultaneous use of HO, PI and AN-V-Cy3 mixture dyes on fluorescein staining properties There appeared to be no difference in the microscopic fluorescein staining appearance of the cells between staining only with fluorescein (Fig. 4A–C) and staining with fluorescein plus a mixture of PI, HO and AN-V dyes (Fig. 4D–L), indicating there was no visible interference of fluorescein stain in the presence of the other dyes. 3.5. Fluorescein staining of rabbit corneal epithelium under stress conditions Fig. 5 shows representative images of the superficial corneal epithelial cells of an ex vivo rabbit eye stained with fluorescein, PI, HO and AN-V-Cy3, after exposure to hypertonic solution. Similar to the observations with cultured HCLE cells previously described, the fluorescence intensity of dead cells (PI +ve, HO +ve, indicated by rectangle) was very low whereas that of normal/healthy cells (PI −ve, HO +ve, circle) was moderate. Cells exhibiting high fluorescence intensity showed apoptotic characteristics (dotted lines). 3.6. Correlation of low, moderate and hyper-fluorescence with cellular physiological health status Average grey scale intensity values for healthy and dead cells as well as those damaged by the various stress stimuli are shown in Fig. 6. Dead cells, identified as PI +ve, had a mean fluorescence intensity of 23 ± 7, which was not significantly different from the background intensity of 20 ± 11 (p > 0.05). This level of fluorescence was categorized as low. Normal, healthy cells (PI −ve, AN-V-Cy3 −ve), showed a significantly higher level of fluorescence (62 ± 24, p < 0.05), and this was designated as moderate. Apoptotic cells (ANV-Cy3 +ve) had the highest fluorescence intensity values with a mean of 138 ± 38. This was significantly higher than all other groups (p < 0.05) and was thus termed as “hyper-fluorescence”. Grey scale intensities in the range 0–40 were classified as low, those in 41–100 were as moderate, and in 101–256 as high. There was good consistency among the various days on which measurements were performed as well as between the various methods of inducing stress. Based on data obtained from the alternative analysis performed on the selected cell fields, the majority of normal live cells (78–89%) exhibited moderate levels of fluorescence, whereas the majority of apoptotic cells (79–85%) were hyper-fluorescent, and the majority of dead cells (76–95%) showed low levels of fluorescence
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Fig. 1. (A) Representative images of fluorescein staining across a single healthy HCLE cell. The cell was imaged over 1 m depth intervals across a cell using z-scanning feature of the confocal microscope at magnification 600×. The depth interval between the representative image planes was 5 M. (B) Fluorescent intensity profile across the cell diameter.
(Table 3). This suggests that the physiological status of the majority of cells (live, apoptotic or dead) can be indicated by the observed fluorescent intensity, independent of the type of stress stimuli applied.
3.7. Effect of stress stimuli on morphology of cultured cells Fig. 7 shows light microscopic images of the HCLE cell under each of the stress condition. Changes in cell morphology became visible
Fig. 2. Confocal microscopic images of dead, apoptotic and live cells after staining with PI (nuclei-red), AN-V-Cy3 (membrane-red) and HO (nuclei-blue) when the cell nucleolus was in focus (A–C) or out of focus (D–F). Dead cells stained positive to PI with intense red fluorescence in the nuclear region (A) along with AN-V-Cy3 positive staining on the cell membrane (A and D). Apoptotic cells stained positive to AN-V-Cy3 with faint red fluorescence on cell membranes but with no PI staining (B) in the nuclear region (B and E). Live cells stained positive to HO but negative to PI with no red fluorescence in the nucleus (C) and negative to AN-V-Cy3 staining on the cell membrane (F). Cropped images from original image obtained at 200× original magnification.
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Fig. 3. Confocal microscopic images of fluorescein, HO, PI and AN-V-Cy3 stained cultured HCLE cells under hypertonic stress. Live cells (HO +ve/PI −ve/AN-V-Cy3 −ve) showed moderate fluorescence (example indicated by circles); dead cells showed low fluorescence (HO +ve/PI +ve/AN-VCy3 +ve, example indicated by rectangle) and apoptotic cells showed hyper-florescence (HO +ve/PI −ve/AN-V-Cy3 +ve, example indicated by dotted margins). Some of the cells in the field show lo fluorescein intensity but do not stain with PI (example indicated by arrows); these may be the cells in later stage of apoptosis. Bar indicates 30 m.
within 10 s of exposure but were most apparent after 1–3 min. Compared with PBS treated controls (Fig. 7A) the hypotonic environment resulted in cell swelling (Fig. 7B) while hypertonicity (Fig. 7C) caused cell shrinkage. Incubation with PHMB (Fig. 7D) caused visible cell membrane damage. When wounded with either a scratch (Fig. 7E) or alkaline patch (Fig. 7F) the cells in the immediate area of the injury were washed off during rinsing, creating a cell free, clear zone (Fig. 7E and F, indicated with a rectangle). Cells
Table 3 Fluorescence intensity of live, apoptotic or dead cells grouped according to the type of stress stimuli the cells were exposed to. Values obtained from measurement of all cells in 5 randomly selected images for each stress model. Observed level of fluorescence
Live (%)
Apoptotic (%)
Dead (%)
Control
Low Moderate Hyper
5 82 12
0 5 79
95 13 9
Hypotonic
Low Moderate Hyper
8 89 4
7 6 84
85 5 12
Hypertonic
Low Moderate Hyper
6 84 6
5 9 85
89 7 9
PHMB
Low Moderate Hyper
2 88 5
3 11 89
95 1 6
Scratch wound
Low Moderate Hyper
13 78 7
5 9 85
82 13 8
Alkaline wound
Low Moderate Hyper
10 86 5
14 10 83
76 4 12
Stimulus
Bold values signify the highest percentage of cells identified in that particular category.
at the wound edges could be seen to have distorted membranes (Fig. 7E indicated with arrow, Fig. 7F indicated with circle). Slit-lamp bio-microscopic appearance of fluorescein staining of cultured corneal epithelial cells under healthy and stressed conditions: To determine the meaning of the differences in cell morphology seen in Fig. 7 in terms of fluorescein staining of these cells, we observed similarly treated test and control cultures using a clinical slit-lamp bio-microscope following staining with fluorescein. Fig. 8A shows that controls have minimal micropunctate staining whereas all the test stimuli-treated HCLE cells exhibited an increased staining response. Exposure to hypotonicity (Fig. 8B), hypertonicity (Fig. 8C) and ophthalmic preservative (Fig. 8D) resulted in diffuse staining whereas the scratch (Fig. 8E) and alkaline (Fig. 8F) wounds showed bright fluorescent dots along the wound edges. 4. Discussion There are three findings in this study that potentially change our understanding of micropunctate fluorescein staining of the cornea. The first of these is the observation that fluorescein dye enters normal, healthy, epithelial cells. Such cells, characterized by lack of staining with either PI or AN-V-Cy3, demonstrated fluorescein staining with an intensity that was significantly higher than that of the background and self-exhibited auto-fluorescence. The second relates to the observation of a population of cells that were hyperfluorescent relative to normal. This phenomenon appears similar to that reported by both Feenstra [2] and Wilson [13] but based on the present findings, it now appears possible to ascribe this behaviour to apoptotic changes taking place within these particular cells. Thus, irrespective of the means used to induce physiological stress, almost 85% of cells that hyper-fluoresced when stained with fluorescein were also positive to AN-V-Cy3 staining and thus apoptotic. Third is the evidence that fluorescein does not interact with cells that are dead. PI +ve necrotic dead cells showed a marked absence of fluorescein staining in both cell and organ culture
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Fig. 4. Representative images showing confocal microscopic appearance of HCLE cells in response to hypertonicity under various staining conditions. Panels A–C: cells stained only with fluorescein; imaged using fluorescein filter. Panels D–L: cells stained simultaneously with fluorescein, HO, AN-V-Cy3 and PI. Panels D–F: images captured using fluorescein filter only. Panels G and H: images captured with recommended filter for PI, HO and AN-V-Cy3. Panels J–L: overlay of images captured with recommended filter for all dye. Note similarity in appearance of panels A–C and D–F.
models and the fluorescence intensity levels of such cells were similar to the background. Taken together these findings have considerable impact on the clinical interpretation of fluorescein when applied as a corneal stain. Conventional wisdom has been that normal epithelial health guarantees the exclusion of fluorescein and thus staining will be absent from a corneal surface comprised entirely of healthy cells. The data presented here indicate that this is probably not correct. When a clinician observes a normal corneal surface after instilling fluorescein and records “no staining”, they are in fact, unlikely to be seeing an absence of “staining,” but rather a homogenous field of moderately intense fluorescence, emanating from all the cells in the area. In general, and consistent with the findings of Wilson [13], fluorescein will have permeated throughout each cell (Fig. 1A) and this will contribute to the regularity of the overall appearance. Despite the fact that all healthy cells are actually glowing more brightly than the background, the lack of a reference to provide contrast leads to the perception of a uniformly “dark” field. Observation of fluorescence of the healthy cells could also be affected by limitation of the slit-lamp bio-microscope to observe lower level of fluorescence.
When a cell does become visible, it is primarily because it is displaying hyper-fluorescence and therefore, is now brighter than its neighbours. It seems reasonable to suggest that clinicians can interpret such brightly fluorescent regions as indicating that some sufficiently stressful or damaging stimulus has caused the component cells to enter an apoptotic pathway. Thus, what we call “fluorescein staining” in reality represents hyper-fluorescence relative to a moderately fluorescent background and can be taken to indicate initiation of apoptosis in the affected cells. The presence of low level, micropunctate staining in the normal, healthy, non-contact lens wearing population [10,30,31] can also be understood in these terms as evidence of epithelial apoptosis occurring as part of regular cell turnover [32]. Hence, staining at sites of normal desquamation before exposure to stress stimuli, might also contribute to the overall appearance of fluorescein staining following exposure. This contribution is expected to be minimal however. When cells eventually die, they lose all fluorescence. Thus, if dead cells are visible at all after fluorescein instillation, it will be because they appear darker (i.e. less fluorescent) than the surrounding normal cells. This may be one explanation for the phenomenon
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Fig. 5. Fluorescence confocal microscopic images of live, dead, apoptotic and fluorescein staining of ex vivo rabbit corneal epithelium after treatment with hypertonic solution. Live cells showed moderate fluorescence (circle); dead cells showed low fluorescence (rectangle) and apoptotic cells showed hyper-florescence (dotted margin). Bar indicates 40 m.
of “negative staining”, i.e. relatively dark areas, that may sometimes be seen in the fluorescein pattern [33]. The argument that the PHMB related corneal staining is an artefact was put forward by Barrett et al. [17] and has recently received further support by Bright et al. [34]. Based on studies using liposomal mimics of cell membranes, Bright et al. [34] have suggested that the corneal staining associated with the use of PHMB based MPS is created due to the interaction of PHMB and fluorescein and
does not indicate true involvement of corneal epithelial cells. While nothing in the current report suggests that such mechanisms do not occur, the data presented here support the view that the observation of hyper-fluorescence indicates that distinct physiological changes are occurring in the epithelial cells. How fluorescein enters corneal epithelial cells is not clear. Active transport systems have been documented in other epithelial tissues such as the intestine [35] and across the blood–retinal barrier
PI -ve AN-V +ve
Damaged
High
160 140
PI -ve AN-V -ve
Moderate
120 100 Day 1
80 60
PI +ve AN-V +ve
Day 2 Day 3
40 Low
Gray scale fluorescence intensity value
200 180
20 0
Fig. 6. Average fluorescence of dead, healthy and damaged cells in terms of grey scale pixel intensity values (Image J analysis software). Error bars represents standard deviations (n = 3200 cells, n = 320 fields for background). Measurements were repeated on three separate days. Dead cells showed low levels of fluorescence, compared to healthy cells (p < 0.05). Damaged cells showed significantly higher levels of fluorescence compared with healthy cells (p < 0.05). No significant differences were found between results obtained on separate observation days (p > 0.05). No statistically significant differences were found between the levels of hyper-fluorescence seen for the various stress stimuli (p > 0.05).
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Fig. 7. Morphological changes in HCLE cells after exposure to control and test stimuli. (A) PBS treated control showing normal healthy cells. (B) Hypotonicity: cells are swollen and rounded. (C) Hypertonicity: cell shrinkage is visible. (D) Ophthalmic preservative (0.0001% PHMB): shape irregularities, rounding and detachment can be seen. (E) Scratch wound: ruptured cells are visible at the wound edge (arrows). (F) Alkaline wound: cell damage can be seen at wound edge (circle). The dotted rectangles show the cell free zones created due to detachment of cells severely damaged by the stress stimuli. Original magnification 400×.
[36–39] but whether such mechanisms are also active within the corneal epithelium remains to be determined. Whatever the route fluorescein takes to enter normal, ocular, epithelial cells, one interpretation of the observed increase in fluorescent intensity is that the onset of apoptosis enhances the process, so that a substantially higher intra-cellular fluorescein concentration results. Cell death then causes a breakdown of membrane integrity releasing the contents and eliminating the fluorescence. As an alternative, it is also conceivable that normal cells take up fluorescein in amounts that are sufficiently high to cause the
intensity of the emitted light to be reduced due to the phenomenon of quenching. This is the property of fluorescent molecules where the initially linear increase of emitted intensity with concentration reaches a peak at some critical concentration and then falls away at higher values [5]. Thus, cells will appear dimmer than expected if they contain a sufficiently high internal fluorescein concentration and actually brighten as this concentration decreases. This might occur if, as seems reasonably likely, the onset of apoptosis were accompanied by a reduction in metabolic activity and/or a loss of membrane
Fig. 8. Slit lamp bio-microscopic appearance of fluorescein-stained HCLE cells after exposure to various stimuli: (A) control PBS, (B) Hypotonicity; (C) Hypertonicity; (D) Ophthalmic preservative (0.0001% PHMB); (E) Scratch wound; (F) Alkaline wound. Arrows indicate hyper-fluorescent cells, circles indicate areas with dead cells and rectangles indicate areas with no cells due to detachment of cells severely damaged by the stress stimuli. Original magnification 10×.
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integrity. Further concentration reduction would then return the emission characteristics to the linear region of the curve, resulting in progressively dimmer fluorescence and finally “darkness” as the cell dies. We note in passing that such a mechanism would produce an initial increase in cell brightness immediately after fluorescein instillation, followed by dimming as the critical quenching concentration was reached. This is not typically what is observed in practice. Evidently this is an area requiring further research. One criticism that could rise is that, optically the red fluorescein can often be detected by using excitation/emission wavelength sets for green fluorescence. This common phenomenon is referred as bleed-through effect, typically a result of crossover of emission spectrum. While in our experiments for simultaneous imaging with multiple dyes the optimization for each excitation and emission wavelength was performed, one can never be sure that the bleedthrough effect has been eliminated completely. Based on this it could be argued that the faint red fluorescence of AN-V-Cy 3 of hyper-fluorescent apoptotic cells is an artefact. However to find out if such an effect had biased our observations, we induced apoptosis in monolayer of HCLE cells using a known apoptotic agent i.e. Digitonin. Following exposure of 0.3 mg/ml (w/v in culture medium) for 5 min the cells were rinsed and stained as described earlier with HO, PI and AN-V i.e. in absence of fluorescein. Another set of similarly treated cells were stained with HO, PI and AN-V in presence of fluorescein. It was observed that apoptotic cells showed similar level of red-fluorescence with AN-V-Cy-3 in presence or absence of fluorescein (data not shown). Suggesting that the results were minimally affected by crossover of emission spectrum. Another criticism that might be raised in respect of this analysis is that epithelial cells in live, human corneas may behave differently than they do in culture. This is of course entirely possible but pending the development of methods that permit in vivo human confirmation, some reassurance can be gained from the ex vivo observations which, for all the stress stimuli investigated, entirely replicated the cell culture work. While this study suggests that micropunctate staining of the corneal epithelium corresponds to the hyper-fluorescence of apoptotic cells, by no means does it preclude the possibility that fluorescein passes through intercellular spaces. However we found no evidence that the micropunctate appearance was due to dye accumulation in intercellular spaces. It can be seen from Figs. 4, 6 and 7 that the fluorescein stained cells within the boundaries of their membranes. 5. Conclusion Based on an in vitro cell culture model and ex vivo animal model, the well known clinical phenomenon of micropunctate fluorescein staining appears to indicate individual cells that are undergoing apoptosis have internalized fluorescein and are hyper-fluorescent relative to their neighbours. Normal, healthy cells also take up fluorescein, but fluoresce less brightly than apoptotic cells. Dead cells do not fluoresce appreciably at all. Sodium fluorescein can thus be considered as a clinical probe, capable of indicating cells in the corneal epithelium that are undergoing apoptosis. References [1] Pfluger. Zur Ernahrung der Cornea. Klin Monbl Augenheilkd Augenarztl Fortbild 1882;20:13. [2] Feenstra RP, Tseng SC. Comparison of fluorescein and rose bengal staining. Ophthalmology 1992;99:605–17. [3] Norn MS. Lissamine green. Vital staining of cornea and conjunctiva. Acta Ophthalmol (Copenh) 1973;51:483–91. [4] Norn MS. Fluorescein vital staining of the cornea and conjunctiva. Studied by triple staining with fluorescein, rose bengal, and alcian blue. Acta Ophthalmol (Copenh) 1964;42:1038–45.
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Fluorescein staining and physiological state of corneal epithelial cells Kalika L. Bandamwar a,b,∗ , Eric B. Papas a,b,c , Qian Garrett a,b a
Brien Holden Vision Institute, Sydney, Australia School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia c Vision Cooperative Research Centre, Sydney, Australia b
a r t i c l e
i n f o
Article history: Received 7 April 2013 Received in revised form 1 September 2013 Accepted 20 November 2013 Keywords: Fluorescein Micropunctate staining Corneal epithelium Apoptosis Live-dead cells
a b s t r a c t Purpose: To evaluate the physiological status of corneal epithelial cells exhibiting fluorescein staining. Methods: Fluorescein staining properties of corneal epithelial cells under normal and stressed conditions were studied using cell-culture (human corneal limbal epithelial cells – HCLE) and organ-culture (rabbit) models. Stress stimuli comprised exposure to hypotonicity, hypertonicity, preservatives, scratch, and alkaline wounding. In addition to fluorescein, cells were stained with Hoechst-33342 (HO), Propidiumiodide (PI), and Annexin-V (AN-V) to identify live, dead and apoptotic cells. Clinical-slit-lamp and fluorescence confocal-microscopic (FCM) observations were performed. FCM images were quantified for fluorescence intensity using Image-J software. Results: Healthy HCLE cells uniformly took up fluorescein to a moderate degree with a mean grey value of 62 ± 24 (mean ± SD) on a scale of 0–256 (no unit). Fluorescence levels similar to those observed prior to stress were associated with healthy cells. Apoptotic cells showed the highest fluorescence (138 ± 38). Dead cells showed minimal fluorescence (23 ± 7) that was similar to the background (20 ± 11, p > 0.05). Observations in whole rabbit eyes were in general agreement with these cell culture findings. Conclusions: The clinical observation of corneal staining with fluorescein suggests the presence of epithelial cells that are undergoing apoptosis but does not indicate dead cells. Under in vitro or ex vivo conditions, healthy cells took up fluorescein at levels that were lower than those of apoptotic cells and thus, are not likely to be perceived as exhibiting staining during clinical observation. Sodium fluorescein may be considered as a probe for apoptotic epithelial cells. © 2013 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.
1. Introduction The use of dyes to aid in evaluation of the ocular surface is common clinical practice. Several examples have been used over the last 130 years [1], including rose bengal [2], lissamine green [3] and most commonly, sodium fluorescein, which is also known as “fluorescein” [4]. The application of fluorescein during ocular examination was first reported by Pfluger [1] for staining epithelial abrasions on rabbit corneas. Since then, its use has been widely reported in the ophthalmic literature and it is now an important part of ocular surface assessment. Despite its well accepted clinical utility, the exact interpretation of corneal staining (we use the term “staining” to refer to superficial micropunctate fluorescein staining unless stated otherwise) is not
∗ Corresponding author at: Brien Holden Vision Institute Research Centre, Level 4, B V Raju Bhavan, Banjara Hills, Road No. 2, Hyderabad 500034, India. Tel.: +91 814 250 32 11. E-mail addresses: [email protected], [email protected] (K.L. Bandamwar).
well understood and has been the topic of continuing debate [5–9]. Early reports proposed that staining was the result of fluorescein accumulation in voids on the ocular surface or intercellular spaces [10] while others [2,11] suggested that normal desquamation of the corneal epithelium might create cavities on the ocular surface in which fluorescein could accumulate to cause a punctuate appearance. Recently however, these “pooling” based theories have been challenged by the observation that repeated saline rinsing does not substantially alter the appearance of corneas exhibiting superficial micro-punctate staining [12]. It is also unlikely that this specific clinical picture could be produced by the rapid stromal diffusion of fluorescein through disturbed cell–cell junctions [2], as has also been suggested. The hypothesis that epithelial cells might actually take up fluorescein was confirmed by Wilson et al. [13] who proposed that fluorescein uptake was probably secondary to cell damage, as had earlier been suggested by Tabery [14–16]. However, correlation of fluorescein staining with cell damage was not demonstrated. More recently, it has been suggested that certain types of staining, in particular those associated with contact lens multipurpose solution (MPS) (i.e. SICS – solution induced corneal staining), is not indicative of ocular surface damage but rather due to an interaction
1367-0484/$ – see front matter © 2013 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.clae.2013.11.003
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between fluorescein molecules, certain components of the lens care solution and the ocular surface [17]. This suggestion proposes that the appearance of clinically visible staining under these circumstances is an artefact produced by the interaction of fluorescein with certain compounds in the solution, specifically ophthalmic preservative molecules. These complexes then adhere to the ocular surface giving the appearance of punctuate staining. Thus, evaluation of the physiological state of cultured corneal epithelial cells or ex vivo corneal cells induced by various stress stimuli (mechanical or chemical) and confirmed using apoptotic or necrotic specific stains would help to indicate the association between the observed clinical appearance and the underlying status of the cells. Based on these observations, it is evident that there is a range of potential mechanisms by which fluorescein could interact with the ocular surface. Which of these theories accounts for the observed clinical appearance might vary depending on the particular circumstances. A corneal abrasion caused by mechanical injury might fluoresce in the presence of fluorescein due to a different mechanism than, for example, that associated with the use of a multipurpose disinfecting solution [12]. Understanding which of these responses describes the response of epithelial cells in any situation is important to correctly interpret the clinical picture, identify potential etiological factors, and determine appropriate remedial actions [7,18]. The purpose of the present study was to investigate the association between fluorescein staining and the physiological state of the cells. We used in vitro cell cultures and ex vivo organ culture models to study how various stress stimuli affect the response of corneal epithelial cells to fluorescein. By using both in vitro cell culture and ex vivo organ culture models, together with a series of well characterized alternative stains for detecting cellular apoptosis and necrosis, we aimed to establish how physiological alteration of epithelial cells affects the associated fluorescein staining characteristics. 2. Materials and methods 2.1. Reagents Sodium Fluorescein, Propidium iodide (PI) and Hoechst-33342 (HO) were obtained from Sigma–Aldrich, MO, USA. AnnexinV-Cy3 was purchased from Biovision (Mountain View, CA, USA). Eagle’s minimum essential medium (EMEM), foetal bovine serum (FBS), Keratinocyte serum free medium (K-SFM), antibiotics (Penicillin–Streptomycin) and rat tendon fibre (RTF) were obtained from Invitrogen-GIBCO, Carlsbad, CA, USA. Epithelial growth factor (EGF), trypsin–EDTA and bovine pituitary extract (BPE) were obtained from Invitrogen, Mount Waverley, VIC, Australia. Sodium chloride, calcium chloride and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were also obtained from Sigma. Polyhexamethylene biguanide (PHMB) (Cosmocil-CQ® ) was obtained from Snow Drift, South Tucson, AZ. Organ culture medium was prepared by adding 10% FBS (w/v), 1% RTF (v/v) and 0.1% antibiotic (v/v) in EMEM. Binding buffer was prepared in Milli-Q water and contained 140 mmol/l NaCl, 10 mmol/l HEPES and 2.5 mmol/l CaCl2 . 2.2. Cell culture Immortalized human corneal limbal epithelial (HCLE) cells were cultured using previously described methods [19–21]. Briefly, HCLE cells were maintained at 2 × 104 cells/cm2 in a K-SFM supplemented with 25 g/ml of BPE, 0.2 ng/ml of EGF and 0.4 mM CaCl2 and grown at 37 ◦ C in a 5% carbon dioxide humidified atmosphere. Once 80% confluence was reached, the cells were detached using
trypsin–EDTA and transferred to grow on glass cover slips, maintained in the same culture medium. Cells were then incubated overnight to allow attachment. 2.3. Organ culture Freshly excised eyes (n = 20) of New Zealand white rabbits were obtained within 1 h post-mortem. These rabbits were obtained from other research that did not require ocular tissue. All the procedures were conducted in accordance with ARVO statement for the use of animals in ophthalmic and vision research. Only clear corneas with no visible oedema or pre-existing staining were used. Two surgical needles were passed through the lateral or posterior conjunctiva of the eye globe to provide support and to aid handling. During imaging, these needles were temporarily inserted into a Styrofoam block, creating a stable fixture for the globes which could then be attached to the bio-microscope chin rest during imaging. In this way direct contact with the corneal surface was avoided. 2.4. Stress stimuli Near confluent monolayers of HCLE cells and freshly excised eyes were rinsed once with the respective culture media and then exposed to a single stress stimulus. Stimuli were chosen based on conditions which could reasonably be expected to mimic clinically observable corneal insults in a real life situation. Chosen stimuli included exposure to hypotonicity, hypertonicity, ophthalmic preservative, and scratch or alkaline wounding. All experiments were replicated at least three times. The time course for development of corneal staining arising from moderate hyperosmolar environment in dry eye or exposure to preservative in solution induced corneal staining (SICS) [22] ranged from minutes to hours. However, in ex vivo animal eyes it is important to reproduce a similar environment more rapidly, to avoid post-mortem changes. Hence stimulus strengths were increased above those typical of human, in vivo situations such as hyperosmolarity in dry eyes and preservative exposure in SICS. Milli-Q® water was introduced to create a hypotonic environment [23]. Likewise, hyper-osmolar solution (5% w/v NaCl in culture medium, >2000 mOsm) was used to produce hypertonicity [24]. A solution of 0.001% PHMB (w/v in phosphate buffered saline, PBS) (concentration ∼10 times higher than used in ophthalmic preparations) represented a commonly used ophthalmic preservative [25]. For all the above stimuli, cells were incubated with 1 ml of the stimulus solution for a period of 1 min PBS was used as a control. For rabbit corneas, 1 ml of test solution was administered, dropwise over period of 1 min. Confluent monolayer cultures were wounded by scratching with a blunt ended 200 l pipette tip, producing a wound of approximately 1 mm diameter [26]. Alkaline wounds were induced by placing a 2.6 mm circular disc of filter paper that had been freshly soaked for 20 s in 2 l of 0.2 N NaOH, on the centre of the cell sheet for 20 s [20,21]. Following exposure to the various stress stimuli, cultured cells and eye balls were rinsed with culture media prior to staining. As the depth of the injury could not be accurately limited to the superficial cornea epithelium, scratch and alkaline wounds were not performed in ex vivo rabbit eyes. 2.5. In vitro confocal microscopic evaluation of cultured cells under normal and stressed conditions Fluorescein staining of the cells in response to various stress stimuli and its association with cell physiological health status were evaluated using a confocal microscope (FluoView FV1000 Confocal
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Table 1 Criteria to distinguish live, apoptotic and dead cells based on staining with Propidium iodide (PI), Hoechst 33342 (HO) and Annexin-V-Cy3 (AN-V-Cy3) dyes. Dye
Fluorescence
Specific location
Normal live
Early apoptotic
Late apoptotic or early necrotic dead cells
PI HO AN-V-Cy3
Red Blue Red
Nucleus Nucleus Cell membrane
−ve +ve −ve
−ve +ve +ve
+ve +ve +ve
Microscope, Olympus, Japan) in conjunction with the use of specific dyes, Hoechst-3342 (HO), Propidium iodide (PI), and Annexin-V (AN-V) for live, dead or apoptotic cell identification. HO is a bright blue nucleic acid dye which fluoresces in contact with nucleic acid. PI is also a nucleic acid dye and fluoresces more brightly when it comes in contact with nucleic acids after the cell membrane it is permeabilized. This property of PI is used to identify permeable cells such as late apoptotic to late necrotic i.e. dead cells. Annexin-V (AN-V) is a probe to identify apoptotic cell membrane changes. It has calcium dependent specific affinity towards phosphotidylserine (PS), a cell membrane protein that flips from inside to outside of the membrane of cells which are undergoing early to late apoptosis [27–29]. Hence, Annexin-V stains early apoptotic to late stage apoptotic cells. It should be noted that AN-V is commercially available with various conjugated fluorophores, such as AN-V-FITC (bright green), AN-V-Cy5 (week blue-violet florescence) and AN-V-Cy3 (red), in the present study, we used the AN-V conjugated with Cy3 (red) in order to distinguish from the fluorescein staining (green). Although PI and AN-V-Cy3 both display red fluorescence, they have distinct locations on cells and hence were considered suitable for simultaneous use. The criteria for defining normal (live), apoptotic, and necrotic dead cells in the present study are described in Table 1. Optimal conditions for fluorescein staining of the cells were determined after a series of experiments were performed using varying concentrations of the fluorescein dye (in the range of 0.001–1.0%) and incubation times with cells (from 15 s to 10 min), as well as the number of rinses (effect of rinsing up to 6 times was tested). Table 2 summarize the resulting optimal microscopic settings which were then used throughout the experiments for acquiring images. (1) Concentration of fluorescein: 1% (w/v in culture medium). (2) Incubation time: 1 min. (3) Number of rinses: 3 (with 2 ml volume of culture medium). Following the treatment with or without stress, both stressed and control (unstressed/health) HCLE cells were stained with 1 ml fluorescein (1% w/v in culture medium) for 1 min followed by 3 rinses with culture medium. Cells were then stained with a mixture of PI, HO and AN-V-Cy3 (as per manufacturer’s recommendation). Briefly, the cells were incubated with PI (0.02 ng/ml w/v in culture medium, Sigma–Aldrich, MO, USA), HO (0.01 ng/ml w/v in culture medium, Sigma–Aldrich, MO, USA) and AN-V-Cy3 (20 l/ml v/v in culture medium) Biovision (Mountain View, CA) for 5 min at room temperature followed by one rinse with culture medium prior to imaging. To evaluate the effect of mixture of other dyes (PI, HO, AN-V) on fluorescein staining of the cells, the stressed cells were divided in two groups. One group was stained with fluorescein only and the other was stained with a mixture of fluorescein, PI, HO and AN-V. Quantification of low, moderate and hyper-fluoresce intensity: The fluorescence intensity of cells stained with fluorescein was obtained by extracting the green component (8 bits) from digitally acquired RGB, then using Image-J software to determine the relevant grey scale intensity values. All images were obtained at
200× magnification using constant viewing conditions. For each test condition and control, 3 separate samples were prepared and multiple images were obtained at various points across the cell field (10–15 images/sample). This was repeated on three separate days for repeatability. Of these, at least 10 pictures for each test and control condition were randomly selected. Using the HO/AN-V-Cy3/PI staining characteristics (Table 1) as a guide for cell physiological status, 10 cells for each of the dead, healthy and apoptotic states respectively were identified from each image and grey scale fluorescein intensity values measured. Although auto-fluorescence of HCLE cells was observed at high laser intensities, when the optimized confocal settings were employed, auto-fluorescent intensity did not differ appreciably from the background level of the culture surface that was devoid of cells. Hence, cover slips with no cells were measured on each day to establish the background fluorescence intensity. To eliminate the possibility of bias introduced as a consequence of the manual selection of cells for this analysis, an alternative means of establishing fluorescence characteristics was also conducted. All cells in the field of view of five randomly selected images from each test and control stimulus were analyzed for their grey scale fluorescein intensity values followed by classification of each cell as either live, apoptotic or dead based on their response to HO, AN-V and PI. Three distinct levels of fluorescence intensities were observed. Grey scale values in the range 0–40 were classified as low, those between 41 and 100 as moderate and 101 and 256 as high. In vitro light microscopic evaluation of the effect of stress stimuli on morphology of cultured cells and clinical slit-lamp microscopic evaluation of fluorescein staining of cultured corneal epithelial cells under healthy and stressed conditions: The stressed HCLE cells induced by various stimuli as described previously were further evaluated under light microscope for cell morphology. The fluorescein-staining of these HCLE cells was further assessed using a clinical slit-lamp bio-microscope (Telaval-31, Carl ZEISS, Germany) equipped with cobalt blue light, Kodak Wratten-12 filter and a camera attachment (Canon Power Shot A620). Images were obtained using slit-lamp standard settings as described elsewhere [22]. 2.6. Evaluation of fluorescein staining of the ex vivo corneas under stress Several images were captured from various focal planes to determine intensities of live, dead and apoptotic cells. No statistical analysis was performed for the ex vivo observations which were conducted primarily to provide visual confirmation of the in vitro findings. Prior to the in vitro confocal microscopic evaluations, the fluorescein stained corneas were evaluated and imaged using a clinical slit-lamp bio-microscope. Soon after slit-lamp bio-microscopic observation of fluorescein staining, corneas were incubated with 1 ml of culture medium containing 0.1 ng/ml of HO, 0.2 ng/ml of PI, and 10 l of AN-V (Annexin-V) reagent and incubated for 5 min in the dark. Corneas were then evaluated under the in vitro laboratory fluorescent confocal microscope (FluoView FV1000, OLYMPUS, Japan) at various magnifications.
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Table 2 Filter setting for visualization of PI, HO, AN-V, and fluorescein staining of HCLE cells. Dye
Excitation filter (nm)
Dichrome mirror (nm)
Barrier filter (nm)
Gain
Off set
HV
Laser power
PI HO AN-V-Cy3 Fluorescein
520–550 330–385 520–550 440–490
565 400 565 505
565–675 425–473 565–675 510–550
1 1 1 1
12 12 12 10
450 350 450 350
18 12 18 10
Fluorescent intensity values were compared between groups using analysis of variance. A p-value of less than 0.05 was considered to be statistically significant. 3. Results 3.1. Fluorescein staining of healthy HCLE cells Fluorescein staining of the healthy cells was imaged over 1 m depth intervals across a single cell using the confocal microscope z-scan feature. It was evident that fluorescein had entered the cell as demonstrated by representative images (Fig. 1A) and the fluorescent intensity profile across the single HCLE cells (Fig. 1B). Similar results were observed when untreated ex vivo corneal epithelial cells were imaged using the z-scanning feature of the confocal microscope; following staining with fluorescein (data not presented). 3.2. Identification of apoptotic and dead cells using PI and AN-V-Cy3 staining Confocal microscopy provides information on a particular focused plane. When focus changes from one plane to other a different configuration of the cell at that particular plane becomes visible resulting in different appearance of cell e.g. at one plane of focus the nuclei of cell might not be visible however with slightest change tip of nuclei may appear, resulting in different appearance of the cell. Fig. 2 demonstrates the appearance of the dead, apoptotic and live cells after staining with PI (nuclei-red), AN-V-Cy3 (membrane-red) and HO (nuclei-blue) when the cell nucleolus was in focus (Fig. 2A–C) or out of focus (Fig. 2D–F). It can be seen from Fig. 2 that dead cells can be identified by positive PI staining with intense red fluorescence in the nuclear region along with AN-V-Cy3 positive staining on the cell membrane (Fig. 2A and D). Apoptotic cells can be identified by positive AN-V-Cy3 staining seen as faint red fluorescence on cell membranes but with no PI staining in the nuclear region (Fig. 2B and E). Live cells were identified as cells with positive staining to HO and negative staining to PI, i.e. no red fluorescence in the nucleus (Fig. 2C), and no staining to AN-V-Cy3 on the cell membrane (Fig. 2F). 3.3. Association of fluorescein staining of HCLE cells with their physiological health status under stress conditions Fig. 3 shows a monolayer of HCLE cells after exposure to hypertonic stress and subsequently stained with fluorescein, PI, HO and AN-V-Cy3. Normal, healthy cells stained positive to HO (blue) but negative to both PI and AN-V-Cy3 (no red) and showed a moderate level of fluorescein intensity (a representative such cell is circled). Also evident was a population of apoptotic cells stained positive to both HO (blue) and AN-V-Cy3 (faint red) but negative to PI (no bright red), which exhibited a very high level of fluorescein intensity (a representative group of such cells is outlined by dotted lines). Early necrotic or late apoptotic, dead cells positive to HO (blue), and to AN-V-Cy3 and PI (bright red) showed a low level of fluorescein staining (indicated with rectangles). In
addition, there is also a population of cells which display very low levels of fluorescein staining but stained positive to HO (blue) and AN-V-Cy3 (faint red), but negative to PI (no bright red) (examples are indicated by arrows). These could be the cells in later stage of apoptosis or early stages of necrosis. We made similar observations for fluorescein and HO/AN-V-Cy3/PI staining of HCLE cells stressed by other stimuli including exposure to MilliQ water (hypotonic condition), or culture medium containing PHMB at 0.0001% PHMB, or stressing cells using scratch or alkali wounding (Fig. 6). 3.4. Effect of simultaneous use of HO, PI and AN-V-Cy3 mixture dyes on fluorescein staining properties There appeared to be no difference in the microscopic fluorescein staining appearance of the cells between staining only with fluorescein (Fig. 4A–C) and staining with fluorescein plus a mixture of PI, HO and AN-V dyes (Fig. 4D–L), indicating there was no visible interference of fluorescein stain in the presence of the other dyes. 3.5. Fluorescein staining of rabbit corneal epithelium under stress conditions Fig. 5 shows representative images of the superficial corneal epithelial cells of an ex vivo rabbit eye stained with fluorescein, PI, HO and AN-V-Cy3, after exposure to hypertonic solution. Similar to the observations with cultured HCLE cells previously described, the fluorescence intensity of dead cells (PI +ve, HO +ve, indicated by rectangle) was very low whereas that of normal/healthy cells (PI −ve, HO +ve, circle) was moderate. Cells exhibiting high fluorescence intensity showed apoptotic characteristics (dotted lines). 3.6. Correlation of low, moderate and hyper-fluorescence with cellular physiological health status Average grey scale intensity values for healthy and dead cells as well as those damaged by the various stress stimuli are shown in Fig. 6. Dead cells, identified as PI +ve, had a mean fluorescence intensity of 23 ± 7, which was not significantly different from the background intensity of 20 ± 11 (p > 0.05). This level of fluorescence was categorized as low. Normal, healthy cells (PI −ve, AN-V-Cy3 −ve), showed a significantly higher level of fluorescence (62 ± 24, p < 0.05), and this was designated as moderate. Apoptotic cells (ANV-Cy3 +ve) had the highest fluorescence intensity values with a mean of 138 ± 38. This was significantly higher than all other groups (p < 0.05) and was thus termed as “hyper-fluorescence”. Grey scale intensities in the range 0–40 were classified as low, those in 41–100 were as moderate, and in 101–256 as high. There was good consistency among the various days on which measurements were performed as well as between the various methods of inducing stress. Based on data obtained from the alternative analysis performed on the selected cell fields, the majority of normal live cells (78–89%) exhibited moderate levels of fluorescence, whereas the majority of apoptotic cells (79–85%) were hyper-fluorescent, and the majority of dead cells (76–95%) showed low levels of fluorescence
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Fig. 1. (A) Representative images of fluorescein staining across a single healthy HCLE cell. The cell was imaged over 1 m depth intervals across a cell using z-scanning feature of the confocal microscope at magnification 600×. The depth interval between the representative image planes was 5 M. (B) Fluorescent intensity profile across the cell diameter.
(Table 3). This suggests that the physiological status of the majority of cells (live, apoptotic or dead) can be indicated by the observed fluorescent intensity, independent of the type of stress stimuli applied.
3.7. Effect of stress stimuli on morphology of cultured cells Fig. 7 shows light microscopic images of the HCLE cell under each of the stress condition. Changes in cell morphology became visible
Fig. 2. Confocal microscopic images of dead, apoptotic and live cells after staining with PI (nuclei-red), AN-V-Cy3 (membrane-red) and HO (nuclei-blue) when the cell nucleolus was in focus (A–C) or out of focus (D–F). Dead cells stained positive to PI with intense red fluorescence in the nuclear region (A) along with AN-V-Cy3 positive staining on the cell membrane (A and D). Apoptotic cells stained positive to AN-V-Cy3 with faint red fluorescence on cell membranes but with no PI staining (B) in the nuclear region (B and E). Live cells stained positive to HO but negative to PI with no red fluorescence in the nucleus (C) and negative to AN-V-Cy3 staining on the cell membrane (F). Cropped images from original image obtained at 200× original magnification.
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Fig. 3. Confocal microscopic images of fluorescein, HO, PI and AN-V-Cy3 stained cultured HCLE cells under hypertonic stress. Live cells (HO +ve/PI −ve/AN-V-Cy3 −ve) showed moderate fluorescence (example indicated by circles); dead cells showed low fluorescence (HO +ve/PI +ve/AN-VCy3 +ve, example indicated by rectangle) and apoptotic cells showed hyper-florescence (HO +ve/PI −ve/AN-V-Cy3 +ve, example indicated by dotted margins). Some of the cells in the field show lo fluorescein intensity but do not stain with PI (example indicated by arrows); these may be the cells in later stage of apoptosis. Bar indicates 30 m.
within 10 s of exposure but were most apparent after 1–3 min. Compared with PBS treated controls (Fig. 7A) the hypotonic environment resulted in cell swelling (Fig. 7B) while hypertonicity (Fig. 7C) caused cell shrinkage. Incubation with PHMB (Fig. 7D) caused visible cell membrane damage. When wounded with either a scratch (Fig. 7E) or alkaline patch (Fig. 7F) the cells in the immediate area of the injury were washed off during rinsing, creating a cell free, clear zone (Fig. 7E and F, indicated with a rectangle). Cells
Table 3 Fluorescence intensity of live, apoptotic or dead cells grouped according to the type of stress stimuli the cells were exposed to. Values obtained from measurement of all cells in 5 randomly selected images for each stress model. Observed level of fluorescence
Live (%)
Apoptotic (%)
Dead (%)
Control
Low Moderate Hyper
5 82 12
0 5 79
95 13 9
Hypotonic
Low Moderate Hyper
8 89 4
7 6 84
85 5 12
Hypertonic
Low Moderate Hyper
6 84 6
5 9 85
89 7 9
PHMB
Low Moderate Hyper
2 88 5
3 11 89
95 1 6
Scratch wound
Low Moderate Hyper
13 78 7
5 9 85
82 13 8
Alkaline wound
Low Moderate Hyper
10 86 5
14 10 83
76 4 12
Stimulus
Bold values signify the highest percentage of cells identified in that particular category.
at the wound edges could be seen to have distorted membranes (Fig. 7E indicated with arrow, Fig. 7F indicated with circle). Slit-lamp bio-microscopic appearance of fluorescein staining of cultured corneal epithelial cells under healthy and stressed conditions: To determine the meaning of the differences in cell morphology seen in Fig. 7 in terms of fluorescein staining of these cells, we observed similarly treated test and control cultures using a clinical slit-lamp bio-microscope following staining with fluorescein. Fig. 8A shows that controls have minimal micropunctate staining whereas all the test stimuli-treated HCLE cells exhibited an increased staining response. Exposure to hypotonicity (Fig. 8B), hypertonicity (Fig. 8C) and ophthalmic preservative (Fig. 8D) resulted in diffuse staining whereas the scratch (Fig. 8E) and alkaline (Fig. 8F) wounds showed bright fluorescent dots along the wound edges. 4. Discussion There are three findings in this study that potentially change our understanding of micropunctate fluorescein staining of the cornea. The first of these is the observation that fluorescein dye enters normal, healthy, epithelial cells. Such cells, characterized by lack of staining with either PI or AN-V-Cy3, demonstrated fluorescein staining with an intensity that was significantly higher than that of the background and self-exhibited auto-fluorescence. The second relates to the observation of a population of cells that were hyperfluorescent relative to normal. This phenomenon appears similar to that reported by both Feenstra [2] and Wilson [13] but based on the present findings, it now appears possible to ascribe this behaviour to apoptotic changes taking place within these particular cells. Thus, irrespective of the means used to induce physiological stress, almost 85% of cells that hyper-fluoresced when stained with fluorescein were also positive to AN-V-Cy3 staining and thus apoptotic. Third is the evidence that fluorescein does not interact with cells that are dead. PI +ve necrotic dead cells showed a marked absence of fluorescein staining in both cell and organ culture
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Fig. 4. Representative images showing confocal microscopic appearance of HCLE cells in response to hypertonicity under various staining conditions. Panels A–C: cells stained only with fluorescein; imaged using fluorescein filter. Panels D–L: cells stained simultaneously with fluorescein, HO, AN-V-Cy3 and PI. Panels D–F: images captured using fluorescein filter only. Panels G and H: images captured with recommended filter for PI, HO and AN-V-Cy3. Panels J–L: overlay of images captured with recommended filter for all dye. Note similarity in appearance of panels A–C and D–F.
models and the fluorescence intensity levels of such cells were similar to the background. Taken together these findings have considerable impact on the clinical interpretation of fluorescein when applied as a corneal stain. Conventional wisdom has been that normal epithelial health guarantees the exclusion of fluorescein and thus staining will be absent from a corneal surface comprised entirely of healthy cells. The data presented here indicate that this is probably not correct. When a clinician observes a normal corneal surface after instilling fluorescein and records “no staining”, they are in fact, unlikely to be seeing an absence of “staining,” but rather a homogenous field of moderately intense fluorescence, emanating from all the cells in the area. In general, and consistent with the findings of Wilson [13], fluorescein will have permeated throughout each cell (Fig. 1A) and this will contribute to the regularity of the overall appearance. Despite the fact that all healthy cells are actually glowing more brightly than the background, the lack of a reference to provide contrast leads to the perception of a uniformly “dark” field. Observation of fluorescence of the healthy cells could also be affected by limitation of the slit-lamp bio-microscope to observe lower level of fluorescence.
When a cell does become visible, it is primarily because it is displaying hyper-fluorescence and therefore, is now brighter than its neighbours. It seems reasonable to suggest that clinicians can interpret such brightly fluorescent regions as indicating that some sufficiently stressful or damaging stimulus has caused the component cells to enter an apoptotic pathway. Thus, what we call “fluorescein staining” in reality represents hyper-fluorescence relative to a moderately fluorescent background and can be taken to indicate initiation of apoptosis in the affected cells. The presence of low level, micropunctate staining in the normal, healthy, non-contact lens wearing population [10,30,31] can also be understood in these terms as evidence of epithelial apoptosis occurring as part of regular cell turnover [32]. Hence, staining at sites of normal desquamation before exposure to stress stimuli, might also contribute to the overall appearance of fluorescein staining following exposure. This contribution is expected to be minimal however. When cells eventually die, they lose all fluorescence. Thus, if dead cells are visible at all after fluorescein instillation, it will be because they appear darker (i.e. less fluorescent) than the surrounding normal cells. This may be one explanation for the phenomenon
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Fig. 5. Fluorescence confocal microscopic images of live, dead, apoptotic and fluorescein staining of ex vivo rabbit corneal epithelium after treatment with hypertonic solution. Live cells showed moderate fluorescence (circle); dead cells showed low fluorescence (rectangle) and apoptotic cells showed hyper-florescence (dotted margin). Bar indicates 40 m.
of “negative staining”, i.e. relatively dark areas, that may sometimes be seen in the fluorescein pattern [33]. The argument that the PHMB related corneal staining is an artefact was put forward by Barrett et al. [17] and has recently received further support by Bright et al. [34]. Based on studies using liposomal mimics of cell membranes, Bright et al. [34] have suggested that the corneal staining associated with the use of PHMB based MPS is created due to the interaction of PHMB and fluorescein and
does not indicate true involvement of corneal epithelial cells. While nothing in the current report suggests that such mechanisms do not occur, the data presented here support the view that the observation of hyper-fluorescence indicates that distinct physiological changes are occurring in the epithelial cells. How fluorescein enters corneal epithelial cells is not clear. Active transport systems have been documented in other epithelial tissues such as the intestine [35] and across the blood–retinal barrier
PI -ve AN-V +ve
Damaged
High
160 140
PI -ve AN-V -ve
Moderate
120 100 Day 1
80 60
PI +ve AN-V +ve
Day 2 Day 3
40 Low
Gray scale fluorescence intensity value
200 180
20 0
Fig. 6. Average fluorescence of dead, healthy and damaged cells in terms of grey scale pixel intensity values (Image J analysis software). Error bars represents standard deviations (n = 3200 cells, n = 320 fields for background). Measurements were repeated on three separate days. Dead cells showed low levels of fluorescence, compared to healthy cells (p < 0.05). Damaged cells showed significantly higher levels of fluorescence compared with healthy cells (p < 0.05). No significant differences were found between results obtained on separate observation days (p > 0.05). No statistically significant differences were found between the levels of hyper-fluorescence seen for the various stress stimuli (p > 0.05).
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Fig. 7. Morphological changes in HCLE cells after exposure to control and test stimuli. (A) PBS treated control showing normal healthy cells. (B) Hypotonicity: cells are swollen and rounded. (C) Hypertonicity: cell shrinkage is visible. (D) Ophthalmic preservative (0.0001% PHMB): shape irregularities, rounding and detachment can be seen. (E) Scratch wound: ruptured cells are visible at the wound edge (arrows). (F) Alkaline wound: cell damage can be seen at wound edge (circle). The dotted rectangles show the cell free zones created due to detachment of cells severely damaged by the stress stimuli. Original magnification 400×.
[36–39] but whether such mechanisms are also active within the corneal epithelium remains to be determined. Whatever the route fluorescein takes to enter normal, ocular, epithelial cells, one interpretation of the observed increase in fluorescent intensity is that the onset of apoptosis enhances the process, so that a substantially higher intra-cellular fluorescein concentration results. Cell death then causes a breakdown of membrane integrity releasing the contents and eliminating the fluorescence. As an alternative, it is also conceivable that normal cells take up fluorescein in amounts that are sufficiently high to cause the
intensity of the emitted light to be reduced due to the phenomenon of quenching. This is the property of fluorescent molecules where the initially linear increase of emitted intensity with concentration reaches a peak at some critical concentration and then falls away at higher values [5]. Thus, cells will appear dimmer than expected if they contain a sufficiently high internal fluorescein concentration and actually brighten as this concentration decreases. This might occur if, as seems reasonably likely, the onset of apoptosis were accompanied by a reduction in metabolic activity and/or a loss of membrane
Fig. 8. Slit lamp bio-microscopic appearance of fluorescein-stained HCLE cells after exposure to various stimuli: (A) control PBS, (B) Hypotonicity; (C) Hypertonicity; (D) Ophthalmic preservative (0.0001% PHMB); (E) Scratch wound; (F) Alkaline wound. Arrows indicate hyper-fluorescent cells, circles indicate areas with dead cells and rectangles indicate areas with no cells due to detachment of cells severely damaged by the stress stimuli. Original magnification 10×.
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integrity. Further concentration reduction would then return the emission characteristics to the linear region of the curve, resulting in progressively dimmer fluorescence and finally “darkness” as the cell dies. We note in passing that such a mechanism would produce an initial increase in cell brightness immediately after fluorescein instillation, followed by dimming as the critical quenching concentration was reached. This is not typically what is observed in practice. Evidently this is an area requiring further research. One criticism that could rise is that, optically the red fluorescein can often be detected by using excitation/emission wavelength sets for green fluorescence. This common phenomenon is referred as bleed-through effect, typically a result of crossover of emission spectrum. While in our experiments for simultaneous imaging with multiple dyes the optimization for each excitation and emission wavelength was performed, one can never be sure that the bleedthrough effect has been eliminated completely. Based on this it could be argued that the faint red fluorescence of AN-V-Cy 3 of hyper-fluorescent apoptotic cells is an artefact. However to find out if such an effect had biased our observations, we induced apoptosis in monolayer of HCLE cells using a known apoptotic agent i.e. Digitonin. Following exposure of 0.3 mg/ml (w/v in culture medium) for 5 min the cells were rinsed and stained as described earlier with HO, PI and AN-V i.e. in absence of fluorescein. Another set of similarly treated cells were stained with HO, PI and AN-V in presence of fluorescein. It was observed that apoptotic cells showed similar level of red-fluorescence with AN-V-Cy-3 in presence or absence of fluorescein (data not shown). Suggesting that the results were minimally affected by crossover of emission spectrum. Another criticism that might be raised in respect of this analysis is that epithelial cells in live, human corneas may behave differently than they do in culture. This is of course entirely possible but pending the development of methods that permit in vivo human confirmation, some reassurance can be gained from the ex vivo observations which, for all the stress stimuli investigated, entirely replicated the cell culture work. While this study suggests that micropunctate staining of the corneal epithelium corresponds to the hyper-fluorescence of apoptotic cells, by no means does it preclude the possibility that fluorescein passes through intercellular spaces. However we found no evidence that the micropunctate appearance was due to dye accumulation in intercellular spaces. It can be seen from Figs. 4, 6 and 7 that the fluorescein stained cells within the boundaries of their membranes. 5. Conclusion Based on an in vitro cell culture model and ex vivo animal model, the well known clinical phenomenon of micropunctate fluorescein staining appears to indicate individual cells that are undergoing apoptosis have internalized fluorescein and are hyper-fluorescent relative to their neighbours. Normal, healthy cells also take up fluorescein, but fluoresce less brightly than apoptotic cells. Dead cells do not fluoresce appreciably at all. Sodium fluorescein can thus be considered as a clinical probe, capable of indicating cells in the corneal epithelium that are undergoing apoptosis. References [1] Pfluger. Zur Ernahrung der Cornea. Klin Monbl Augenheilkd Augenarztl Fortbild 1882;20:13. [2] Feenstra RP, Tseng SC. Comparison of fluorescein and rose bengal staining. Ophthalmology 1992;99:605–17. [3] Norn MS. Lissamine green. Vital staining of cornea and conjunctiva. Acta Ophthalmol (Copenh) 1973;51:483–91. [4] Norn MS. Fluorescein vital staining of the cornea and conjunctiva. Studied by triple staining with fluorescein, rose bengal, and alcian blue. Acta Ophthalmol (Copenh) 1964;42:1038–45.
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Please cite this article in press as: Bandamwar KL, et al. Fluorescein staining and physiological state of corneal epithelial cells. Contact Lens Anterior Eye (2013), http://dx.doi.org/10.1016/j.clae.2013.11.003
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Please cite this article in press as: Bandamwar KL, et al. Fluorescein staining and physiological state of corneal epithelial cells. Contact Lens Anterior Eye (2013), http://dx.doi.org/10.1016/j.clae.2013.11.003