Journal of Food and Nutrition Research, 2017, Vol. 5, No. 5, 320-330
Available online at http://pubs.sciepub.com/jfnr/5/5/6
©Science and Education Publishing
Fermented Cordyceps cicadae Mycelia Extracts
Ameliorate Dry Eye Symptoms through Reduction of
Cornea Epithelial Cell Apoptosis and Maintenance of
Conjunctival Goblet Cells in a Mouse Dry Eye Model
Tung-Yu Lin1,#, Han-Hsin Chang2,#, Yu-Jun Tang3, Chin-Chu Chen4,5, Li-Ya Lee6, David Pei-Cheng Lin1,7,*
1Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung City, Taiwan
2Department of Nutrition, Chung Shan Medical University, Taichung City, Taiwan
3Institute of Biochemistry, Microbiology, and Immunology, Chung Shan Medical University, Taichung City, Taiwan
4Institute of Food Science and Technology, National Taiwan University, Taiwan
5Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University, Taiwan
6Department of Veterinary Medicine, National Chung Hsin University, Taiwan
7Department of Ophthalmology, Chung Shan Medical University Hospital, Taichung City, Taiwan
#These are first authors.
*Corresponding author: firstname.lastname@example.org
Abstract Cordyceps cicadae (Cc), a traditional Chinese medicine, has been shown to possess immunomodulatory
and anti-inflammatory activities, and is regarded as having effects in vision improvement, but with no reported
evidence. This study investigated the effects of Cordyceps cicadae fermented mycelia extracts (Cc extracts) in a
benzalkonium chloride (BAC)-induced mouse dry eye model. Female ICR mice aged 6 weeks were randomly
divided into four groups: blank, BAC-damaged without Cc extracts, BAC-damaged with 10 mg/kg bodyweight of
Cc extracts, BAC-damaged with 100 mg/kg bodyweight of Cc extracts. The results showed that tear volume, tear
film breakup time, and cornea surface indexes, including smoothness, opacity, topography, and the extent of
lissamine green staining, were all improved with intake of Cc extracts intake, when compared to the status of the
BAC-damaged group without Cc extracts. Immunohistochemical assays showed moderate change of Ki-67+ and
Np63+ epithelial cell populations, while apoptotic epithelial cells, as detected by TUNEL assay, were decreased.
PAS stain showed that the conjunctival goblet cell number and total cell area were decreased in the BAC-damaged
group with Cc extracts at 10 mg/kg bodyweight. This study demonstrated that Cc extracts effectively ameliorate
BAC-induced dry eye symptoms through enhancement of cornea resilience against BAC-induced damages and
maintenance of conjunctival goblet cells.
Keywords: Cordyceps cicadae, mycelia extracts, amelioration, dry eye symptoms, cornea epithelial cell apoptosis,
conjunctival goblet cells
Cite This Article: Tung-Yu Lin, Han-Hsin Chang, Yu-Jun Tang, Chin-Chu Chen, Li-Ya Lee, and David
Pei-Cheng Lin, “Fermented Cordyceps cicadae Mycelia Extracts Ameliorate Dry Eye Symptoms through
Reduction of Cornea Epithelial Cell Apoptosis and Maintenance of Conjunctival Goblet Cells in a Mouse Dry
Eye Model.” Journal of Food and Nutrition Research, vol. 5, no. 5 (2017): 320-330. doi: 10.12691/jfnr-5-5-6.
Dry eye disease (DED) prevails between 7.4% and
33.7% among human populations  and it was estimated
that 25% of patients who visited ophthalmic clinics due to
symptoms of dry eye . The risk factors of DED include
age, gender, autoimmune disease, long-term contact lens
wearing, refractive laser surgery, excessive visual task
performance, intake of certain systemic medications,
smoking, and low humidity environments [2,3]. These risk
factors characterize DED as a chronic ocular surface
disorder that affects significantly quality of life through
enduring discomfort and visual disturbances. Due to such
extended impact, DED has attracted much attention and
many efforts have been exerted in finding the ways to
alleviate its symptoms.
DED is a multifactorial disease that is manifested in
many aspects. Previous studies indicated that inflammation,
such as seen in systemic autoimmune diseases including
Sjogren’s syndrome and systemic lupus erythematosus, is
a common manifestation of dry eye symptoms [4,5]. Some
researchers have thus proposed to use topical or systemic
immunomodulatory therapy in severe dry eye conditions
[6,7]. Increase of pro-inflammatory markers with
excessive oxidants in the tear is also a hallmark of DED
[8,9]. Thus, anti-inflammation by use of anti-oxidants,
Journal of Food and Nutrition Research 321
such as the use of dietary alpha-lipoic acid or topic
administration of CoQ10, has been experimentally applied
for dry eye symptom alleviation [10,11,12].
Cordyceps cicadae (Cc), a traditional Chinese health
food, was shown to possess immunomodulatory activities
 and anti-inflammatory effects . These effects, if
exerted in the eye, may help to relieve dry eye symptoms.
Nevertheless, whether intake of Cc extracts may
ameliorate dry eye symptoms is not known. This study
investigated this potential effect in benzalkonium chloride
(BAC)-induced mouse dry eye model .
2.1. Dietary Cc Extracts Ameliorate Ocular
Surface Damages Induced by
The ocular surface damages were assessed by semi-
quantitative grading systems according to smoothness,
topography, opacity, and the extent of corneal staining by
lissamine green. The results showed that dietary intake of
Cc extracts helped to alleviate ocular surface damages in
all of the four grading systems, except for opacity, while
those without intake of Cc extracts showed significant
damages on the ocular surface (Figure 1). The effective
dose was 100 mg/kg of body weight and this effective
dose was consistent for mitigation of the smoothness,
topography, and corneal staining grading.
2.2. Tear Production under the Influence of
Dietary intake of Cc extracts helped to increase the
aqueous tear production after ocular surface damage by
BAC (Figure 2). At day 7 after the BAC-induced damage,
Cc extracts given at 100 mg/Kg of body weight lead to
significant increase of tear production as compared to that
of the BAC-damaged group. At day 10 after the damage,
both groups given at 10 mg/Kg and 100 mg/Kg of body
weight of Cc extracts showed significant increase of tear
production. Notably, starting from day 4, with intake of
Cc extracts at 10 mg/Kg or 100 mg/Kg, the tear
production was higher than either the control or the BAC-
In A, less irregularity of reflective light was observed with intake of intake of Cc extracts. Negative images were also shown for the extent of lissamine
green staining. In B, semi-quantitative analysis showed that the effective dose was at 100 g/Kg of bodyweight. BAC: benzalkonium chloride-damaged
group without intake of CC extracts. BAC-[C] 10 mg/kg and BAC-[C] 100 mg/kg: benzalkonium chloride-damaged groups with intake of CC extracts
at 10 mg/kg and 100 mg/Kg, respectively.
Figure 1. Ocular surface grading for corneal smoothness, opacity, topography, and extent of lissamine green staining at day 10 of the experiment
322 Journal of Food and Nutrition Research
BAC: benzalkonium chloride-damaged group without intake of CC extracts. 10 mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with
intake of Cordyceps cicadae mycelia fermented extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 2. Aqueous tear production (in millimeter) assessed with a 1-mm-width strip cut off from a pH test paper at days 1, 4, 7, and 10 of the
BAC: benzalkonium chloride-damaged group without intake of Cc extracts. 10 mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with
intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 3. Tear film breakup time (in seconds) assessed after fluorescein staining and recording at days 1, 4, 7, and 10 of the experiment
Journal of Food and Nutrition Research 323
2.3. Increased Tear Film Breakup Time with
Since the tear volume was increased with intake of Cc
extracts, we further assessed the tear quality as reflected
by tear film breakup time (TBUT). The results showed that
BAC caused significant reduction of TBUT at day 1 after
the damage (Figure 3) and this detrimental effect aggravated
more severely at days 4, 7, and further to day 10. TBUT was
significantly increased at day 4 with intake of Cc extracts
in both 10 mg/Kg and 100 mg/Kg dose groups, as compared
to that of the BAC-damaged group. However, the significance
of increase did not persist to day 7. At day 10, the TBUT
increase regained its significance. Furthermore, the TBUTs
of 100 mg/Kg dose group was significantly longer than
that of the 10 mg/Kg dose group at both day 4 and day 10.
2.4. Cc Extracts Help to Maintain Central
Cornea Epithelium Thickness after BAC-
Histological analysis showed that BAC administration
generally caused cornea damage on the surface (Figure 4A),
but did not reduce the total cornea thickness (Figure 4B).
On the contrary, BAC caused significant increase of total
cornea thickness in the peripheral cornea area (Figure 4B).
The main reduction of thickness caused by BAC was on
the central cornea epithelium (Figure 4C). This reduction,
however, did not extend to the peripheral cornea. There
was even a slight increase of epithelial thickness in the
peripheral cornea (Figure 4C).
BAC administration caused increase of cornea stroma
thickness in the central and peripheral cornea, but not in
the limbus when compared with that of the control group
(Figure 4D). With intake of Cc extracts, the thickness of
stroma in the central cornea and peripheral cornea was
even more increased, particularly in the central cornea
where dietary intake of Cc extracts at 100 mg/Kg dose
lead to the most significant increase. On the contrary, in
the limbus, intake of Cc extracts did not increase stromal
thickness (Figure 4D). At 10 mg/Kg dose, the stroma
thickness in the limbus was even significantly reduced as
compared to that of the BAC-damaged group.
2.5. Moderate Mobilization of Cornea Ki-67+
and Np63+ Epithelial Cells with Cc
To further characterize the damages caused by BAC
administration, we performed immunohistochemistry for
Ki-67+ and Np63+ cells and calculated the cell numbers in
the cornea central, peripheral, and limbus regions at day
10 of the experiment. The results showed that Ki-67+ cells
in all of the 3 cornea regions were fewer in the blank
group than in the BAC-damaged group in all of the three
cornea regions (Figure 5A). Interestingly, dietary intake of
Cc extracts did not appear to promote further increase of
Ki-67+ cells, adding to the mobilizing effect occurred after
BAC damage. Instead, the number of Ki-67+ cells became
less as compared to that of the BAC-damaged group,
particularly in the peripheral cornea and limbus areas
when 100 mg/Kg dose was administered (Figure 5B).
CC: central cornea. PC: peripheral cornea. L: limbus. BAC: benzalkonium chloride-damaged group without intake of Cc extracts. 10
mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 4. Histological analysis and thickness measurement of total cornea, cornea epithelium, and cornea stroma at day 10 of the experiment after
324 Journal of Food and Nutrition Research
CC: central cornea. PC: peripheral cornea. L: limbus. BAC: benzalkonium chloride-damaged group without intake of CC extracts. 10 mg/kg
and 100 mg/kg: benzalkonium chloride-damaged groups with intake of Cordyceps cicadae mycelia fermented extracts at 10 mg/kg and 100
Figure 5. Immunohistochemical staining and quantitative analysis of Ki-67+ cells at day 10 of the experiment
Immunohistochemical detection of Np63+ cells at day 10 of the experiment. CC: central cornea. PC: peripheral cornea. L: limbus. BAC:
benzalkonium chloride-damaged group without intake of Cc extracts. 10 mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with
intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively
Figure 6. Immunohistochemical detection of Np63+ cells at day 10 of the experiment
In contrast to the Ki-67+ cell population, the Np63+
cells did not appear to be mobilized to the same extent in
the peripheral cornea and limbus areas (Figure 6). When
compared with that of the BAC-damaged group, the
Np63+ cell number with dietary intake of Cc extracts
showed no significant difference in either the 10 mg/Kg
group or in the 100 mg/Kg group (data not shown).
2.6. Cc Extracts Helps to Reduce
BAC-induced Apoptosis in
the Cornea Epithelium
Since our data indicated that dietary intake of Cc
extracts can effectively ameliorate ocular surface damages
induced by BAC administration, the cornea epithelium
Journal of Food and Nutrition Research 325
must either be protected from the damages or they will die
and be replaced though mobilization of limbal stem cells
or basal progenitor cells . Therefore, we performed
TUNEL assay to examine the apoptotic status of the corneal
epithelial cell populations. The results showed extensive
apoptotic cells in all 3 cornea areas following BAC
administration (Figure 7A). Quantitatively, the peripheral
cornea area contained more apoptotic epithelial cells than
the central cornea and the limbus areas following the
damages induced by BAC (Figure 7B). With dietary
intake of Cc extracts, the numbers of apoptotic cornea
epithelial cells were generally reduced in both 10 mg/Kg
and 100 mg/Kg study groups and the 100 mg/Kg study
group apparently reduced more apoptotic epithelial cells.
2.7. Maintenance of Conjunctival Goblet Cell
with Cc Extracts Intake
Since the tear quality was improved with intake of Cc
extracts, as indicated by increased tear film breakup
time, we performed PAS stain to assess the conjunctival
goblet cell status. The results showed that PAS+
conjunctival goblet cells became fewer after the damage
caused by BAC treatment (Figure 8A). With intake of
CC extracts at 10 mg/Kg, the number of PAS+
conjunctival goblet cells was higher than that in the
damaged group (Figure 8B). However, less conjunctival
goblet cells were observed with intake of Cc extracts at
100 mg/Kg dose.
In A, the upper panel showed an example of combination of PI (in red) and FITC (in green) images to yield cells with yellow signals (indicated by
arrows) that were regarded as positive for TUNEL assay. In B, quantitative analysis showed evident reduction of apoptotic cornea epithelial cells with
intake of Cc extracts. CC: central cornea. PC: peripheral cornea. L: limbus. BAC: benzalkonium chloride-damaged group without intake of CC extracts.
10 mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 7. In situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay at day 10 of the experiment
326 Journal of Food and Nutrition Research
In A, the morphology and number of goblet cells were shown in the inferior conjunctiva. In B, the PAS+ conjunctival
goblet cells were quantified. The results showed that more goblet cells were maintained with intake of Cc extracts.
BAC: benzalkonium chloride-damaged group without intake of CC extracts. 10 mg/kg and 100 mg/kg: benzalkonium
chloride-damaged groups with intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 8. Periodic acid Schiff (PAS) staining at day 10 of the experiment
Dry eye disease (DED) is a multifactorial disorder of
the tear film and ocular surface [3,5]. For many years,
researchers have been trying to unveil the core mechanism
of DED, which is reflected by the number of publications
directly related to dry eye syndromes. A recent review
paper  was published by using the key words “dry eye,
inflammation” on the PubMed and Web of Science
databases for scientific articles published in English
between January 1, 1900 and August 30, 2013. Based on
the literature survey, the authors clearly demonstrated that
inflammation is the core mechanism and plays a key role
in the pathogenesis of DED. The authors concluded that
immune dysregulation leads to a cycle of continued
inflammation and eventually leads to DED. Thus,
functional foods that possess both anti-inflammatory and
immunomodulatory activities may be used as candidates
for alleviation of dry eye symptoms.
Cordyceps cicadae (Cc), a caterpillar-shaped medicinal
mushroom that derives its nutrients from larvae of Cicada
flammata Dist., has been used as a dietary supplement in
Chinese for hundreds of years. Previous studies have
shown that the ergosterol peroxide from C. cicadae
inhibits the activation and proliferation signals in
primary human T lymphocytes  and ameliorates
TGF-β1-induced activation of kidney fibroblasts .
Another earlier study  indicated that C. cicadae
extracts possess immunomodulatory functions. We
therefore hypothesized that C. cicadae may help to relieve
dry eye symptoms and evaluated its fermented mycelia
extracts in a benzalkonium chloride (BAC)-induced
mouse dry eye model. The results demonstrated that
dietary intake of Cc extracts, particularly at 100 mg/Kg
dose, helped to ameliorate BAC-induced dry eye
symptoms through multiple aspects, including improvement
of ocular surface grading of smoothness, topography, and
lissamine green staining, increasing tear volume and
TBUT period, prevention against the extensive epithelial
layer apoptosis in the central, peripheral, and limbal areas
of the cornea, and maintenance of conjunctival goblet cell
number and area at 10 mg/Kg dose.
Multi-fold mechanisms may be unveiled underlying the
efficacy after intake of Cc extracts. The amelioration of
BAC-induced damages to the ocular surface may be
through accelerated repair by mobilization of limbal stem
cells or corneal progenitor cells or both [17,20].
Alternatively, intake of Cc extracts may render the corneal
epithelial cells more resilient to the damages caused by
BAC. We conducted immunohistochemistry of Ki-67 and
Np63 cellular proliferation markers to investigate the
changes of corneal epithelial cells following damages
induced by BAC and compare the status with or without
intake of Cc extracts. Ki-67 is a marker for proliferating
but poorly differentiated corneal epithelial cells  and
unspecified ΔNp63 isoforms are detectable in the basal
layer of the corneal epithelium at all times . The
results demonstrated more Ki-67+ cells in all of the three
cornea areas following BAC damage, in contrast to the
steady status in the blank control group. Evidently, BAC
Journal of Food and Nutrition Research 327
damage caused more mobilization of proliferative cornea
epithelial cells, either from the limbus or form the local
If dietary intake of Cc extracts ameliorated the damages
caused by BAC through accelerated cell mobilization, it
would have promoted even more increase of Ki-67+ cells
as compared to that of the BAC damage group without Cc
extracts intake. Instead, Ki-67+ cell number became
significantly less (p<0.05 at 100 mg/Kg dose) as compared to
that of the BAC-damaged group, particularly in the
peripheral cornea and limbus areas. The result suggests
that intake of Cc extracts did not help to ameliorate
BAC-induced dry eye symptoms mainly through promotion
for faster corneal epithelium replacement. Instead, a moderate
condition of mobilization was more likely to occur. The
result that Np63+ cells did not appear to be evidently
mobilized in the peripheral cornea and limbus areas also
indicates this moderate condition of mobilization.
If the corneal stem cells or progenitor cells were only
moderately mobilized under the effects of BAC damage,
ocular surface improvement with intake of Cc extracts
would be resulted from more resilience to the damages.
The results of TUNEL assay confirmed that fewer
apoptotic cells were found as Cc extracts were given,
particularly in the peripheral and limbus areas when the
Cc extracts were given at 100 mg/Kg dose.
Notably, as Cc extracts were given to the mice, the
effective dose at 100 mg/Kg correlates in almost all
aspects of dry eye symptom amelioration, except for the
cell number and area of conjunctival goblet cells.
Significant amelioration was observed at 100 mg/Kg dose
for 3 out of the 4 ocular surface grading criteria. The
results of tear production and TBUT were also better with
100 mg/Kg dose. Another result that is also noteworthy is
the significant increase of stromal thickness in the central
cornea, in contrast to the non-significance in the
peripheral cornea and the limbus. This data is conversely
correlated with the non-significant change of Ki-67+ cell
number in the central cornea and the significant change in
the peripheral cornea and in the limbus. Interestingly,
fewer apoptotic cornea epithelial cells were detected in the
central cornea in the BAC-damaged group as well as in
the groups given Cc extracts. The reduction of central
cornea epithelial apoptotic activity is likely due to the
more increase of stromal thickness in the central cornea
where differential corneal epithelial-keratocyte cell
interactions may occur in this area, leading to differential
release of protective factors from the keratocytes. For
example it has been known that paracrine mediators such
as hepatocyte growth factor (HGF) and keratinocyte
growth factor (KGF) are produced by the keratocytes to
regulate proliferation, motility, differentiation, and
possibly other functions, of epithelial cells [22,23]. If the
differential epithelial-keratocyte cell interaction occurred,
since the epithelial cells in the central cornea were more
protected, apoptotic activities were relatively low and the
loss of central cornea epithelial cells would be less. The
demand for repair would be less, as reflected by the
non-significant change of Ki-67+ cell number in the
central cornea. Arguably, however, increase of the central
stroma thickness may be an indication of edema caused by
inflammatory activities therein. Besides, the effects to
maintain conjunctival goblet cell were better with Cc
extracts at 10 mg/Kg than at 100 mg/Kg dose.
Complicated local factors may be involved, resulting in
impedance of goblet cell generation with Cc extracts given
at higher dose. Obviously, this entire scenario demands
The underlying mechanisms between maintenance of
healthy cornea epithelial layers and improvement of tear
production and quality are multifold. Particularly, since
Cc extracts possess both immunomodulatory and
anti-inflammatory activities, the ameliorating effects
found in the present study are of complexity and warrant
further investigation. Also, the clinical significance
between protecting corneal epithelium from extensive
apoptosis rather than acceleration of stem cell
mobilization and replacement of damaged cells is different.
To the aged corneas or corneas with limbal stem cell
deficiency, as their cornea stemness capacity is diminished,
stimulation to accelerate cornea repair is less likely to
ameliorate dry eye symptoms. Under these conditions,
dietary Cc extracts are potentially more helpful.
4. Materials and methods
4.1. Mice and Study Groups
A total of 24 six-week-old female ICR mice were
purchased from BioLASCO Taiwan Co., Ltd, Taipei,
Taiwan. The mice were fed ad libitum and kept under
standard conditions with a 12-h light/dark cycle. The mice
were acclimatized and habituated to the laboratory for at
least one week before experiments. All mice were
examined with a slit lamp (Model 99 BQ; Haag-Streit,
Bern, Switzerland). Only mice without anomalies of the
anterior segment of the eye (cornea, anterior chamber, iris,
or lens) were included in the experiments. The mice were
randomly split into 4 groups: blank with 0.9 % NaCl as
vehicle control, BAC-damaged without CC extracts,
BAC-damaged with 10 mg/kg bodyweight of Cc extracts,
BAC-damaged with 100 mg/kg bodyweight of Cc extracts.
Each group contained 6 mice. All experiment protocols
were reviewed and approved by the Animal Care and Use
Committee of Chung Shan Medical University, Taichung,
Taiwan and were performed in agreement with the
Association for Research in Vision and Ophthalmology
(ARVO) Resolution on the Use of Animals in Research.
4.2. Cornea Surface Damage by
The BAC damages were performed from day 1 to day
10, with the mice anaesthetized and their ocular surfaces
exposed to 5 μL of 0.2% BAC administered topically
twice daily (9 AM and 9 PM). Care was taken to ensure
that BAC covered the entire mouse ocular surface for at
least 1 minute during each BAC administration.
4.3. Preparation and Feeding of Cc Extracts
Cordyceps cicadae was harvested from mountain areas
of New Taipei City, Taiwan. The mycelium was isolated
and cultured on potato dextrose agar and genotyped to
328 Journal of Food and Nutrition Research
confirmed its identity. The colony has been stored (stock
number MU30106) in Bioresource Collection and
Research Centre (BCRC), Hsinchu, Taiwan. The
mycelium was primarily cultured at 25°C in 2% glucose,
1% yeast extract, 1% soy bean powder at pH 6.0, followed
by expanded culture and fermentation. The crude
fermented fluids were concentrated, cold-dried into
powder, and stored at 4°C before feeding. The Cc extracts
were given by daily tube feeding at 10 AM starting from 3
days prior to the BAC damage and continuing until day 10.
All mice were sacrificed on day 10 for tissue collection.
4.4. Grading of Corneal Surface Damages
Corneal surface damages were graded according to
smoothness, topography, opacity, and the extent of
lissamine green staining, following a previous publication
. Briefly, one eye of each mouse was randomly
selected for assessment of corneal smoothness and
topography. The other eye was then assessed for corneal
opacity. Images of the cornea surface were taken with a
stereoscopic zoom microscope equipped with ring
illuminator (SMZ 1500; Nikon). The digital images were
then used to score corneal smoothness on a 5-point scale
according to the number of distorted quadrants in the
reflected ring: 0, no distortion; 1, distortion in one
quadrant of the ring (3 clock hours); 2, distortion in two
quadrants (6 clock hours); 3, distortion in three quadrants
(9 clock hours); 4, distortion in all four quadrants (12
clock hours); and 5, severe distortion, in which no ring
could be recognized. The scoring of corneal topography
was the same as that of corneal smoothness, but covered
much more area of the corneal surface. The other set of
images on the other eye was used for corneal opacity
scoring (0, normal cornea; 0.5, mild haze seen only under
dissection microscope; 1, mild haze; 2, moderate haze
with visible iris; 3, severe haze with invisible iris; 4,
severe haze with corneal ulceration). For lissamine green
staining, both corneas from each mouse were stained with
2% lissamine green B (Sigma-Aldrich, cat. no. 199583) in
0.9% NaCl. Images were taken and scored according to
the areas of stain. Briefly, the total area of punctuate
staining was designated as grade 0; grade 1, less than 25%
of cornea stained with scattered punctuate staining; grade
2, 25% to 50% of cornea stained with diffuse punctate
staining; grade 3, 50% to 75% of cornea stained with
punctuate staining and apparent epithelial defects; grade 4,
more than 75% of cornea stained with abundant punctuate
staining and large epithelial defects. All scorings were
performed by two observers without prior knowledge of
the experiment and study groups.
4.5. Tear Production and Tear Film Breakup
Aqueous tear production was assessed with phenol
red-impregnated cotton threads (Zone-Quick; Oasis,
Glendora, CA, USA) at days 1, 4, 7, and 10. The animals
were anesthetized and rest for a fixed period of 20 seconds.
A 1-mm-width strip was held with forceps under a
dissection microscope and placed in the lateral cantus of
the conjunctival fornix of the eye for 20 seconds. The tear
distance (in millimeters) was read under a microscope.
Tear film breakup time was assessed by in vivo staining
with 0.1% liquid sodium fluorescein [10,16]. The mice
were anesthetized with intraperitoneal tribromoethanol
injection (0.3 ml at 20 mg/ml). One or two microliter of
fluorescein stain was dropped into the conjunctival sac.
After 3 blinks, tear film fluorescein signals were recorded
under an Olympus BX51 fluorescence microscope
(Olympus, Tokyo, Japan). The tear film breakup time was
read in seconds by two observers without prior knowledge
of the experiment and study groups.
4.6. Histology and Immunohistochemistry
After mouse sacrifice by cervical dislocation, the
eyes were extracted, processed, and stained with
Hematoxylin-Eosin for histopathologic analysis following
conventional procedures as previously described [10,16].
For immunohistochemistry, the tissue sections were boiled
in citrate buffer (pH 6.0) for 20 minutes for antigen
retrieval and then incubated, respectively, with either
mouse anti-Np63 (1/50, cat. no. sc-8431; Santa Cruz
Biotechnology, Santa Cruz, CA) or rabbit anti-Ki-67
antibody (1/100, cat. no. NB110–89719; Novus Biologicals),
followed by incubation with a horseradish peroxidase–
conjugated secondary antibody (1/200), either anti-mouse
or anti-rabbit IgG (Jackson ImmunoResearch Laboratories,
Inc., West Grove, PA), and followed by washes and incubation
in diaminobenzidine tetrahydrochloride solution for color
detection, and counterstained with hematoxylin.
4.7. Demarcation of Central, Peripheral, and
Limbal Cornea Areas and Thickness
To describe differential cell distribution and corneal
thickness in different areas, each cornea was demarcated
into central, peripheral, and limbal areas. The eye was
sectioned on sagittal plane and the cornea length, spanning
from the upper to the lower limbus, was split into 3 equal
parts. The upper one third and the lower one third were
regarded as the peripheral corneas, while the middle one
third as the central cornea. Following Hematoxylin-Eosin
stain, the tissue sections with the longest corneal length
were selected and measured for total, epithelial, and
stromal thickness under a Nikon E 100 microscope (Nikon,
Tokyo, Japan), equipped with a digital camera linked to a
desktop computer. Each thickness measurement was
determined by 2 observers without prior information of
the experiments and study groups. All measurements were
performed on ImageJ2 (National Institutes of Health,
Bethesda, USA) software program.
4.8. In Situ Terminal Deoxynucleotidyl
Transferase dUTP Nick End
Labeling (TUNEL) Assay
In situ TUNEL labeling was performed using the
DeadEnd™ Fluorometric TUNEL System (G3250;
Promega, Madison, WI) according to manufacturer's
instructions. Tissue sections were fixed in acetone at 4°C,
rinsed with phosphate-buffer saline (PBS), permeabilized
Journal of Food and Nutrition Research 329
by 0.2% Triton X100, and incubated in equilibration
buffer for 10 min. The sections were then incubated with
TdT reaction mix for 60 min, followed by immersion in
standard saline citrate to stop reaction. For positive
controls, tissue sections were incubated in DNase I prior
to addition of equilibration buffer. For negative controls,
DDW was added instead of TdT reaction mix. All
preparations were rinsed with PBS several times,
counterstained with propidium iodide to locate the cells
(P3566; ThermoFisher), and mounted for photography.
The photo images were taken with an Olympus BX51
fluorescence microscope (Olympus, Tokyo, Japan),
followed by digital combination of the fluorescein
isothiocyanate (FITC) (green) with the propidium iodide
(red) color to produce yellow signals. Only cells with
yellow signals were regarded as positive for TUNEL assay.
4.9. PAS Stain for Conjunctival Goblet Cell
Number and Total Cell Areas
Periodic acid Schiff (PAS) staining was performed to
evaluate conjunctival epithelial morphology, and the
number of goblet cells and total cell areas in the inferior
conjunctiva was counted under a microscope (ECLIPSE
E100; Nikon, Melville, NY) with a ×20 objective and
ImageJ2 software program. For each eye, 5 different
sections were randomly selected for counting, and an average
was calculated. All scorings were performed by two
observers without prior knowledge of the study groups.
All data were obtained from repeats (n > 6). The data
are presented as the means ± standard error of the means
(SEMs) and were compared among groups. The corneal
smoothness, opacity, and fluorescein staining scores were
compared with the Kruskal–Wallis test. The corneal total,
epithelial, and stromal thickness and the number of cells
positive in immunohistochemistry and TUNEL assay were
analyzed with the Mann–Whitney test. All statistical
analyses were performed by using the SPSS program
(SPSS, Inc., Chicago, IL).
We demonstrated that C. cicadae fermented mycelia
extracts help to ameliorate dry eye symptoms after ocular
surface damages caused by benzalkonium chloride
administration. The efficacy was shown in multiple aspects,
including tear quality and quantity as well as ocular surface
assessments. Furthermore, we elucidated that C. cicadae
fermented mycelia extracts help to ameliorate the dry eye
symptoms through enhancement of cornea resilience and
maintenance of conjunctival goblet cells after benzalkonium
chloride administration. Results of this study support that
C. cicadae fermented mycelia extracts may be used as a
functional food ingredient for vision protection.
Conflicts of Interests
The authors declare no conflict of interest.
D.P.-C.L. and H.-H.C conceived and designed
experiments and wrote the paper. T.-Y.L. and Y.-J.T.
performed experiments. C. –C.C. and L.-Y.L. provided
materials and reagents and analyzed the data.
This study was supported by research grants: MOST
102-2320-B-040-013 from Ministry of Science and
Technology, Taiwan; COA 104-20-26 and COA 105-
A005-C from Council of Agriculture, Taiwan.
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