·Basic
Research· ·Current Issue· ·Achieve· ·Search Articles· ·Online
Submission· ·About
IJO·
Cytotoxicity
of pilocarpine to human corneal stromal cells and its underlying cytotoxic
mechanisms
Xiao-Long Yuan, Qian Wen, Meng-Yu Zhang, Ting-Jun Fan
Laboratory for Corneal Tissue Engineering, College of
Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong
Province, China
Correspondence
to: Ting-Jun
Fan. Laboratory for Corneal Tissue
Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
tjfan@ouc.edu.cn
Received: 2015-03-18 Accepted:
2015-09-29
Abstract
AIM: To examine the cytotoxic effect of pilocarpine, an
anti-glaucoma drug, on human corneal stromal (HCS) cells and its underlying
cytotoxic mechanisms using an in vitro model of non-transfected HCS
cells.
METHODS: After HCS cells were treated with pilocarpine at a
concentration from 0.15625 g/L to 20.0 g/L, their morphology and viability were
detected by light microscopy and MTT assay. The membrane permeability, DNA
fragmentation and ultrastructure were examined by acridine orange (AO)/ethidium
bromide (EB) double-staining. DNA electrophoresis and transmission
electron microscopy (TEM), cell cycle, phosphatidylserine (PS) orientation and
mitochondrial transmembrane potential (MTP) were assayed by flow cytometry
(FCM). And the activation of caspases was checked by ELISA.
RESULTS: Morphology observations and viability assay showed that pilocarpine at concentrations above 0.625 g/L induced dose- and
time-dependent morphological abnormality and viability decline of HCS cells.
AO/EB double-staining, DNA electrophoresis and TEM noted that pilocarpine at concentrations above 0.625 g/L induced dose- and/or
time-dependent membrane permeability elevation, DNA fragmentation, and
apoptotic body formation of the cells. Moreover, FCM and ELISA assays revealed
that 2.5 g/L pilocarpine also induced S phase arrest, PS externalization, MTP disruption, and
caspase-8, -9 and -3 activation of the cells.
CONCLUSION: Pilocarpine
at concentrations above 0.625 g/L (1/32 of its clinical therapeutic dosage) has
a dose- and time-dependent cytotoxicity to HCS cells by inducing apoptosis in
these cells, which is most probably regulated by a death receptor-mediated
mitochondrion-dependent signaling pathway.
KEYWORDS: pilocarpine; cytotoxicity; human
corneal stromal cells; apoptosis; mitochondrion
Citation: Yuan XL, Wen Q, Zhang MY, Fan TJ. Cytotoxicity
of pilocarpine to human corneal stromal cells and its underlying cytotoxic
mechanisms. Int J Ophthalmol 2016;9(4):505-511
Human corneal stroma
(HCS) is composed of highly organized extracellular matrix (ECM) and
mitotically quiescent HCS cells[1]. The
specialized HCS cells play important roles in maintaining corneal
transparency and wound healing by synthesizing ECM
components[2-4]. Damages to corneal
epithelium and/or stroma from trauma and drugs often induce
apoptosis of HCS cells[5], and excessive loss of HCS cells
usually results in serious complications[6]. Therefore, it will be of great significance to
characterize the cytotoxicity of topical drugs to the
cornea and the underlying cytotoxic mechanisms, especially to HCS.
Pilocarpine, a cholinergic anti-glaucoma drug, is widely and chronically used in eye
clinic for glaucoma treatment[7-9]. Unfortunately, it has been reported that repeated and
prolonged medication of pilocarpine often results in adverse effects, such as seizures, posterior
synechiae, iris cysts, and so on[10-13]. However, the cytotoxicity of pilocarpine
to HCS remains unkown due to the lack of an in vitro model of HCS cells that can be used for cytotoxicity
investigations[14]. Recently,
a
non-transfected HCS cell line was successfully established in our laboratory[15], and it makes it possible to study
intensively the cytotoxicity of pilocarpine to HCS cells and the underlying
mechanisms in vitro. The present
study was intended to investigate the cytotoxicity of Pilocarpine to HCS and
the underlying cellular and molecular mechanisms using an in vitro model of HCS cells.
MATERIALS AND METHODS
Materials Pilocarpine
(C11H16N2O2) was purchased from
Alfa Aesar (Tianjin, China). HCS cells, from the nontransfected HCS cell line (utHCSC01) established previously in
our laboratory[15], were maintained and cultured in
10% bovine calf serum (BCS, Invitrogen,
Carlsbad, CA, USA)-containing Dulbecco’s modified Eagle medium: Ham’s nutrient
mixture F-12 medium (DMEM/F12, 1:1) (Invitrogen, Carlsbad, CA, USA) at 37oC in 25 cm2 culture flasks (Nunc,
Copenhagen, Denmark) as described previously.
Experimental
design After cultured
logarithmic HCS cells were treated with
pilocarpine at
concentrations from 0.3125 g/L
to 20.0 g/L, and the morphology, viability, and cell cycle were
checked by light microscopy, MTT assay, and flow cytometry (FCM) using propidium iodide (PI)
staining for
cytotoxicity evaluation. The
membrane permeability, phosphatidylserine
(PS) orientation, DNA integrality, and ultrastructure were examined by acridine
orange (AO)/ethidium bromide (EB) double-staining, FCM using Annexin-V/PI staining,
DNA electrophoresis, and transmission electron microscopy (TEM) for apoptosis characterization. And the caspase activation and mitochondrial
transmembrane potential (MTP) was assayed by ELISA and FCM using JC-1 staining for apoptosis signaling pathway investigation. HCS cells cultured in the
same medium without any Pilocarpine
addition at the same time point were used as controls in all experiments.
Methods
Morphological observation Cell morphology was observed by light
microscopy. HCS cells were harvested from
culture flasks by trypsin digestion and centrifugation as described previously[15],
and inoculated into a 24-well culture plate (Nunc) at 6.0×104
cells/well and grown at 37oC in a 5% CO2 incubator. When
the cells reached to logarithmic phase, their culture medium was replaced
entirely with 0.3125-20.0 g/L pilocarpine-containing 10% BCS-DMEM/F12 medium. The morphology and growth status of the cells were monitored successively
with a TS-100 light microscope (Nikon, Tokyo, Japan) at a 4h interval.
Cell
viability assay Cell
viability was determined by
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay
as described previously[16]. Briefly, HCS cells were inoculated into a 96-well culture plate (Nunc) at a density of 1×104
cells/100 μL/well,
and were cultured and treated as described above. At a 4h interval, the pilocarpine-containing
medium was replaced entirely with 100 μL serum-free DMEM/F12 medium containing 1.0 g/L MTT (Sigma-Aldrich, St.
Louis, MO, USA), and the cells were incubated at 37 oC in
the dark for 4h. After the MTT-containing medium was discarded with caution, 150 μL dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to dissolve the produced formazan crystals
at 37oC in the dark for 15min, and the absorbance at 490 nm was
measured with a Multiskan GO microplate reader (Thermo Scientific, MA, USA).
Plasma membrane permeability detection Plasma membrane permeability of HCS cells was measured by
AO/EB double-staining as described previously[16]. In brief, HCS
cells in a 24-well culture plate (Nunc) were cultured, treated
and harvested as described above. After stained with 100 μg/mL AO/EB (Sigma-Aldrich)
(1:1) solution for
1min, the cell suspension was observed under a Ti-S fluorescent microscope
(Nikon, Tokyo, Japan). HCS cells with red or orange nuclei were designated as apoptotic cells
while those with green nuclei as non-apoptotic cells, and at least 300 cells
from several random fields were counted in each group. The apoptotic ratio was
calculated according to the formula of “Apoptotic rate (%)=Apoptotic
cells/(Apoptotic cells+non-apoptotic cells)×100%”.
DNA electrophoresis DNA fragmentation was detected by agarose gel
electrophoresis according to the method reported previously[17]. Briefly, HCS cells in 25 cm2 flasks (Nunc) were cultured, treated and
harvested as described above. The DNA from 1.4×105 cells was isolated with a Quick Tissue/Culture Cells
Genomic DNA Extraction Kit (Dongsheng
Biotech, Guangzhou, Guangdong Province, China). The DNA extracts from each group was
electrophoresed on 1% agarose gel (15 V, 4h), stained with 0.5 mg/L ethidium bromide for
10min, and observed with an EC3 imaging system (UVP, LLC Upland, CA, USA).
Ultrastructural observation Ultrastructure of HCS cells was
checked by
transmission electron microscopy (TEM) as described previously[17]. In brief, HCS cells in 25 cm2 flasks were cultured, treated with 2.5 g/L pilocarpine and harvested as described
above. After fixed successively with 4% glutaraldehyde and 10 g/L osmium
tetroxide, the cells were dehydrated and embedded in epoxy resin. Ultrathin
sections were stained with 20 g/L uranyl acetate-lead citrate and observed with
an H700 TEM (Hitachi, Tokyo, Japan).
Flow cytometry analyses The cell cycle status, PS orientation
and MTP were detected and analyzed by FCM as reported previously[16].
Briefly, HCS cells in 6-well plates (Nunc) were cultured, treated with 2.5 g/L pilocarpine and harvested as described above. After washed
twice with 1 mL phosphate-buffered saline (PBS), the cells were fixed with 70%
alcohol at 4°C overnight. Then the cells were stained with PI (BD Biosciences, San Jose,
CA, USA) for cell cycle assay,
stained with Annexin-V/PI using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) for PS externalization assay, and stained with 10
μg/mL 5,5’,6,6’-tetrachloro-1,1’,3,3’- tetraethybenzimida (JC-1; Sigma-Aldrich)
for MTP assay, respectively. The stained cells, 3.6×105 cells at a minimum, were detected by a FACScan flow cytometer and analyzed with CXP
analysis software (BD Biosciences).
Caspase activation assay Caspase activation was measured by enzyme-linked
immunosorbent assay (ELISA) as described previously[16]. Briefly, HCS cells in 6-well plates (Nunc) were cultured, treated with
2.5 g/L pilocarpine and harvested as described above. Whole-cell
protein extracts from 5.0×105 cells were prepared by utilizing a
RIPA lysis kit (Biotime, Beijing, China) according to the manufacturer’s
instructions. About 100 μL supernatant was coated into high-binding 96-well
microtiter plates (Nunc) overnight at 4°C. After blocked with 5% non-fat milk (BD Biosciences), the wells were incubated with
rabbit anti-human caspase-3, -8, and -9 (active form) antibodies (1:500) (Biosynthesis Biotechnology, Beijing, China),
and the HRP-conjugated goat anti-rabbit secondary antibody (1:3000) (cwBiotech, Nanjing, China),
respectively, at 37°C for 2h. A colorimetric reaction was induced by 1%
tetramethylbenzidine (TMB) for 25min in the dark at room temperature. Color
development was stopped with 50 μL H2SO4
(0.5 mol/L), and the 490 nm absorbance of each well was measured using a
Multiskan GO microplate reader (Thermo Scientific).
Statistical Analysis Each experiment was
repeated 3 times independently. Data are shown as mean±standard deviation (SD).
Group comparisons were conducted using one-way analysis of variance (ANOVA) using SPSS 21.0 software (Chicago, IL, USA). Differences
to controls were considered statistically significant when P<0.05.
RESULTS
Cytotoxic Effect of Pilocarpine on Human Corneal Stromal Cells To evaluate the cytotoxicity of pilocarpine,
the morphology and viability of HCS cells were examined by light microscopy and
MTT assay, respectively. Morphological observations showed that HCS cells
exposed to pilocarpine at a concentration from
0.625 to 20 g/L exhibited dose- and time-dependent proliferation retardation
and morphological abnormality such as cellular shrinkage, cytoplasmic
vacuolation, detachment from culture matrix, and eventually death,
while no obvious difference was observed between those exposed to pilocarpine
below the concentration of 0.625 g/L and controls (Figure 1A). Results of MTT
assay revealed that the cell viability of HCS cells decreased with time and
concentration after exposing to pilocarpine above the
concentration of 0.625 g/L (P<0.01 or 0.05), while that of HCS cells
treated with pilocarpine below the concentration of
0.625 g/L showed no significant difference to controls (Figure 1B).
Figure 1 Dose and time dependent cytotoxicity of pilocarpine to HCS
cells Cultured HCS cells were treated with the
indicated concentration and exposure time of pilocarpine. A: Light microscopy.
One representative photograph from three independent experiments was shown.
Scale bar=50 µm. B: MTT assay. The cell viability of pilocarpine-treated HCS
cells in each group was expressed as percentage (mean±SD) of 490 nm absorbance
compared to its corresponding control (n=3).
aP<0.05, bP<0.01 versus control.
Cell Cycle Arrest of Human
Corneal Stromal Cells To
verify the proliferation retarding mechanisms of pilocarpine, the cell cycle status of HCS cells were examined
by FCM using PI
staining. Results showed that the number of 2.5 g/L pilocarpine-treated HCS cells in S phase
increased from 21.6% of control to 25.7% (P<0.05)
at 4h, 30.7% (P<0.01) at 8h, and
56.6% (P<0.01) at 12h,
respectively, while that in G1 phase and G2/M phase decreased with time (P<0.01
or 0.05), respectively,
when compared
with controls
(Figure 2).
Figure 2 Pilocarpine induced cell cycle arrest of HCS cells Cultured HCS cells were treated with the
indicated concentration and exposure time of pilocarpine,
and their cell cycle status was checked by FCM with PI staining. One
representative photograph from three independent experiments was shown.
Plasma Membrane Abnormality
of Human
Corneal Stromal Cells To examine whether the cytotoxicity of
pilocarpine was achieved by inducing apoptosis, plasma membrane permeability
and PS orientation of pilocarpine-treated HCS cells were detected by AO/EB
double-staining and FCM using Annexin V/PI staining, respectively. Our results
of AO/EB double-staining displayed that pilocarpine at concentrations above
0.625 g/L could elevate the plasma membrane permeability of HCS cells in a dose- and time-dependent manner (P<0.01
or 0.05), while that of HCS cells treated with pilocarpine below 0.625 g/L
showed no significant difference to controls (Figure 3A). Results of FCM using
Annexin V/PI staining showed that HCS cells treated with 2.5 g/L pilocarpine
exhibited time-dependent increase of PS externalization when compared with its
corresponding control (Figure 3B). The number of early and late apoptotic cells
(PS externalized cells) increased from 1.77% of control to 56.38% (P<0.01) at 4h, 95.46% (P<0.01) at 8h, and 91.87% (P<0.01) at 12h, respectively.
Figure 3 Pilocarpine induced plasma membrane
abnormality of HCS cells Cultured HCS
cells treated with the indicated concentration and exposure time of pilocarpine.
A: AO/EB double staining. The apoptotic ratio was calculated as percentage
(mean±SD) of the total number of cells based on the permeability elevation of
plasma membrane of HCS cells (n=3).
Data are presented as mean±SD. aP<0.05,
bP<0.01 versus control.
B: FCM using Annexin V/PI staining. The
cell number of HCS cells in each group was expressed as percentage of the total
number of cells. One representative photograph from three independent
experiments was shown. K1: Necrosis; K2: Late apoptosis; K3: Normal
cell; K4: Early apoptosis. The cells in K2 and K4 phase exhibit PS
externalization.
DNA Fragmentation and
Ultrastructural Abnormality of Human Corneal Stromal Cells To verify
the apoptosis inducing effect of pilocarpine, DNA fragmentation and
ultrastructure of pilocarpine-treated HCS cells was characterized by DNA electrophoresis and TEM, respectively.
Results of DNA electrophoresis indicated that the genomic DNA extracted
from pilocarpine-treated HCS cells was fragmented into a highly dispersed state,
and typical DNA ladders were found in 0.625-2.5 g/L pilocarpine-treated cells,
while no DNA ladder was found in those of controls (Figure 4A). Moreover,
TEM observations postulated that 2.5 g/L pilocarpine-treated HCS
cells
exhibited ultrastructural disorganizations, including cytoplasmic
vacuolation, mitochondrion swelling, chromatin condensation, and apoptotic body
formation (Figure 4B).
Figure 4 Pilocarpine induced DNA
fragmentation and ultrastructural abnormality of HCS cells Cultured HCS cells were treated with the
indicated concentration and exposure time of pilocarpine. A: DNA
electrophoresis. DNA isolated from HCS cells was electrophoresed in 1% agarose
gel, and dispersed DNA ladders were shown. B: TEM images. One representative
photograph from three independent experiments was shown. N: Nucleus; v: Vacuoles;
m: Swollen mitochondrion; a: Apoptotic body.
Caspase Activation and
Mitochondrial Transmembrane Potential Disruption of Human Corneal Stromal Cells To postulate the possible triggering
pathways involved in pilocarpine-induced apoptosis, caspase activation and MTP
disruption was further measured by ELISA using antibodies to the active form of
caspase-3, -8 and -9 and FCM using JC-1 staining, respectively. Results of
ELISA revealed that 2.5 g/L pilocarpine could activate caspase-8, -9, and -3 in
HCS cells successively (P<0.01 or
0.05), of which caspase-8 was first activated to its peak value at 2h (P<0.01), caspase-9 activated to its
peak value at 4h (P<0.01), while
caspase-3 was activated continually during the monitoring period (P<0.01 or 0.05) (Figure 5A).
Moreover, FCM using JC-1 staining indicated that 2.5 g/L pilocarpine could
induce MTP disruption in HCS cells, and the number of JC-1 positive cells
(MTP-disrupted cells) increased from 0.97% of control to 26.30% (P<0.01) at 4h, 51.42% (P<0.01) at 8h, and 82.55% (P<0.01) at 12h, respectively (Figure 5B).
Figure 5 Pilocarpine induced caspase
activation and MTP disruption of HCS cells Cultured HCS cells were treated with the
indicated exposure time of 2.5 g/L pilocarpine. A: ELISA. Caspase activation induced by pilocarpine was assayed using the
active form of caspase-3, -8 and -9 antibodies. The activation ratio of each
group was expressed as percentage (mean±SD) compared to its
corresponding control based on 490 nm absorbance (n=3). aP<0.05, bP<0.01 versus control. B: FCM images of JC-1 staining. JC-1 in
mitochondria with MTP depolarized maintains monomers in green fluorescence,
while that in mitochondria with normal MTP incorporates into aggregates in red
fluorescence. One representative photograph from three independent experiments
was shown. The cell number of
MTP disrupted HCS cells in each group was expressed as percentage of the total
number of cells.
DISCUSSION
Anti-glaucoma drugs, such as latanoprost, have been
reported to have toxic effects on HCS and result in reduction in central
corneal thickness and even keratoconus progression[18-19]. However, the effect of pilocarpine,
a widely used
anti-glaucoma drug in eye clinic, on HCS remains unclear. To provide cytotoxic references for prospective
therapeutic interventions of anti-glaucoma drugs, the present study was intended to
investigate the cytotoxicity of pilocarpine to HCS and its possible
cellular and molecular mechanisms using an in
vitro model of non-transfected HCS cells[15].
To evaluate the cytotoxicity of pilocarpine, the
morphology, viability and cell cycle status of pilocarpine-treated HCS cells
were examined at first in this study. We found that pilocarpine at
concentrations above 0.625 g/L (1/32 of its clinical therapeutic dosage) could
induce dose- and time-dependent morphological abnormality, proliferation
retardation, and viability decline of HCS cells. Moreover, 0.625 g/L pilocarpine
could also induce S phase arrest of HCS cells. Our findings suggest that pilocarpine
above 1/32 of its clinical therapeutic dosage has significant cytotoxicity to
HCS cells in vitro, and S phase
arrest is involved in the retarded proliferation of HCS cells induced by pilocarpine.
These findings are supported by previously reported cytotoxicity of ophthalmic
drugs and anesthetics to human corneal cells[16,20-24].
Since cell viability decline and cell cycle arrest are often related with
apoptosis triggered by chemotherapeutic agents[22-25],
it can be deduced that the cytotoxic effect of pilocarpine on HCS cells might
also be related with apoptosis.
As well
postulated previously, plasma membrane permeability elevation, PS
externalization, DNA fragmentation and apoptotic body formation are the
hallmark features of apoptosis[26-29]. To
verify whether the cytotoxicity of pilocarpine was achieved by inducing
cell apoptosis, we then investigated the plasma membrane permeability, PS
externalization, DNA fragmentation, and apoptotic body formation of HCS cells
using AO/EB double staining, FCM using Annexin V/PI staining, DNA
electrophoresis, and TEM, respectively. Our results showed that pilocarpine above concentrations of 0.625 g/L could induce dose-
and time-dependent membrane permeability elevation of HCS cells, and 2.5 g/L pilocarpine could also induce PS externalization, DNA
fragmentation and apoptotic body formation. As well defined, the key difference between
apoptosis and necrosis is the formation of apoptotic bodies with externalized
PS in their plasma membrane, which is a vital feature for clearing away the
apoptotic cells by phagocytosis[27,29].
Therefore, our findings confirmed that pilocarpine could induce apoptosis of
HCS cells in vitro. The pilocarpine-induced
HCS cell apoptosis is consistent with our previous reports of topical
anesthetic- and ophthalmic drug-induced apoptosis in human corneal cells[16,20-24].
As we know, apoptosis is a
normal physiological process regulated orderly by intricate pathways, such as
the death receptor-mediated extrinsic pathway and the mitochondrion-dependent
intrinsic pathway[30-31], which
will result in the activation of initiator caspases (caspase-2, -8, -10, and
-9) and subsequent activation of executioner caspases (caspase-3, -6, and -7)[32-33]. Meanwhile, MTP disruption is a
prerequisite for triggering release of mitochondrion sequestered apoptotic
proteins, such as cytochrome c, an indispensable activator of caspase 9,
apoptosis inducing factor, and Smac/Diablo[31,34-36]. To
postulate the possible apoptosis-triggering pathways of pilocarpine, the
activation pattern of caspase-3, -8, and -9 and MTP disruption was further
studied by ELISA using antibodies of their active forms and FCM using JC-1
staining, respectively. We found that 2.5 g/L pilocarpine could activate
caspase-8, -9, and -3 successively, and induce MTP disruption in a
time-dependent manner in HCS cells. Since caspase-8 is an important mediator of
tumor necrosis factor receptor 1 (TNFR1)-mediated extrinsic pathway while
caspase-9 is a key mediator of the mitochondrion-dependent intrinsic pathway[33,37], our results of the activation
of caspase-8, -9, and -3 in pilocarpine-treated HCS cells, combined with MTP
disruption, suggest that the pilocarpine-induced HCS cell apoptosis might be
triggered via both a death
receptor-mediated pathway and a mitochondrion-dependent pathway. The
involvement of both an extrinsic and an intrinsic signaling pathway has also
been reported in other chemical induced apoptosis[16,21-22,38-39].
In conclusion, pilocarpine
above 1/32 of its clinical therapeutic concentration has a dose- and
time-dependent cytotoxicity to HCS cells in
vitro, and its cytotoxicity is achieved by inducing death receptor-mediated
mitochondrion-dependent apoptosis. By now, this is the first attempt of
investigating the cytotoxicity of pilocarpine to HCS cells and its toxic
mechanisms at cellular and molecular levels
in vitro. Even these findings do not allow predicting clinical inferences in vivo directly without further
investigations, they provide new insights into the acute cytotoxicity and
apoptosis-inducing effect of pilocarpine on HCS cells.
ACKNOWLEDGEMENTS
Foundation: Supported by National High Technology Research and
Development Program ("863" Program) of China (No. 2006AA02A132).
Conflicts
of Interest: Yuan XL, None; Wen Q, None; Zhang MY, None; Fan TJ,
None
REFERENCES [Top]
1 DelMonte
DW, Kim T. Anatomy and physiology of the cornea. J Cataract Refr Surg 2011;37(3):588-598. [CrossRef]
[PubMed]
2 Jester
JV, Barry PA, Lind GJ, Petroll WM, Garana R, Cavanagh HD. Corneal keratocytes:
in situ and in vitro organization of cytoskeletal contractile proteins. Invest Ophth Vis Sci 1994;35(2):730-743.
[PubMed]
3 Shtein
RM, Kelley KH, Musch DC, Sugar A, Mian SI. In vivo confocal microscopic
evaluation of corneal wound healing after femtosecond laser-assisted
keratoplasty. Ophthalmic Surg Lasers
Imaging 2012;43(3):205-213. [CrossRef]
[PubMed] [PMC free article]
4 Chien
Y, Liao YW, Liu DM, Lin HL, Chen SJ, Chen HL, Peng CH, Liang CM, Mou CY, Chiou
SH. Corneal repair by human corneal keratocyte-reprogrammed iPSCs and
amphiphatic carboxymethyl-hexanoyl chitosan hydrogel. Biomaterials 2012;33(32):8003-8016. [CrossRef]
[PubMed]
5 Lee
KS, Ko DA, Kim ES, Kim MJ, Tchah H, Kim JY. Bevacizumab and rapamycin can
decrease corneal opacity and apoptotic keratocyte number following
photorefractive keratectomy. Invest Ophth
Vis Sci 2012;53(12):7645-7653. [CrossRef]
[PubMed]
6 Wilson
SE, Chaurasia SS, Medeiros FW. Apoptosis in the initiation, modulation and
termination of the corneal wound healing response. Exp Eye Res 2007;85(3):305-311. [CrossRef]
[PubMed] [PMC free article]
7 Yung RL, Francis S. Sjögren’s Syndrome
in the elderly. Geriatric Rheumatology Springer; 2011:287-291.
8 Bowman
RJ, Dickerson M, Mwende J, Khaw PT. Outcomes of goniotomy for primary
congenital glaucoma in East Africa. Ophthalmology
2011;118(2):236-240. [CrossRef]
[PubMed]
9 Wu
LL, Huang P, Gao YX, Wang ZX, Li B. A 12-week, double-masked, parallel-group
study of the safety and efficacy of travoprost 0.004% compared with pilocarpine
1% in Chinese patients with primary angle-closure and primary angle-closure
glaucoma. J Glaucoma 2011;20(6):388-391.
[CrossRef]
[PubMed]
10 Turski L, Cavalheiro EA, Czuczwar SJ,
Turski WA, Kleinrok Z. The seizures induced by pilocarpine: behavioral,
electroencephalographic and neuropathological studies in rodents. Pol J Pharmacol 1987;39(5):545-555.
11 Phillips
CI, Clark CV, Levy AM. Posterior synechiae after glaucoma operations:
aggravation by shallow anterior chamber and pilocarpine. Br J Ophthalmol 1987;71(6):428-432. [CrossRef]
12 Kuchenbecker
J, Motschmann M, Schmitz K, Behrens-Baumann W. Laser iridocystotomy for
bilateral acute angle-closure glaucoma secondary to iris cysts. Am J Ophthalmol 2000;129(3):391-393. [CrossRef]
13 Everitt
DE, Avorn J. Systemic effects of medications used to treat glaucoma. Ann Intern Med 1990;112(2):120-125. [CrossRef]
[PubMed]
14 Kanjilal
B, Keyser BM, Andres DK, Nealley E, Benton B, Melber AA, Andres JF, Letukas VA,
Clark O, Ray R. Differentiated NSC-34 cells as an in vitro cell model for VX. Toxicol Mech Methods 2014;24(7):488-494.
[CrossRef]
[PubMed]
15 Fan
TJ, Hu XZ, Zhao J, Niu Y, Zhao WZ, Yu MM, Ge Y. Establishment of an
untransfected human corneal stromal cell line and its biocompatibility to
acellular porcine corneal stroma. Int J
Ophthalmol 2012;5(3):286-292. [PMC free article]
[PubMed]
16 Yu
HZ, Li YH, Wang RX, Zhou X, Yu MM, Ge Y, Zhao J, Fan TJ. Cytotoxicity of
lidocaine to human corneal endothelial cells in vitro. Basic Clin Pharmacol 2014;114(4):352-359. [CrossRef] [PubMed]
17 Lee
PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the
separation of DNA fragments. J Vis Exp 2012;(62):pii: 3923. [CrossRef]
18 Sen
E, Nalcacioglu P, Yazici A, Aksakal FN, Altinok A, Tuna T, Koklu G. Comparison
of the effects of latanoprost and bimatoprost on central corneal thickness. J Glaucoma 2008;17(5):398-402. [CrossRef]
[PubMed]
19 Amano
S, Nakai Y, Ko A, Inoue K, Wakakura M. A case of keratoconus progression
associated with the use of topical latanoprost. Jpn J Ophthalmol 2008;52(4):334-336. [CrossRef]
[PubMed]
20 Zhou
X, Li YH, Yu HZ, Wang RX, Fan TJ. Local anesthetic lidocaine induces apoptosis
in human corneal stromal cells in vitro. Int
J Ophthalmol 2013;6(6):766-771. [PMC free article]
[PubMed]
21 Wen
Q, Fan T, Bai S, Sui Y. Cytotoxicity of proparacaine to human corneal
endothelial cells in vitro. J Toxicol Sci
2015; 40(4):427-436. [CrossRef]
[PubMed]
22 Tian
CL, Wen Q, Fan TJ. Cytotoxicity of atropine to human corneal epithelial cells
by inducing cell cycle arrest and mitochondrion dependent apoptosis. Exp Toxicol Pathol 2015;67(10): 517-524.
[CrossRef]
[PubMed]
23 Li
YH, Wen Q, Fan TJ, Ge Y, Yu MM, Sun LX, Zhao Y. Dose dependent cytotoxicity of
pranoprofen in cultured human corneal endothelial cells by inducing apoptosis. Drug Chem Toxicol 2015;38(1):16-21. [CrossRef]
[PubMed]
24 Miao
Y, Sun Q, Wen Q, Qiu Y, Ge Y, Yu MM, Fan TJ. Cytotoxic effects of betaxolol on
healthy corneal endothelial cells both in vitro and in vivo and in vivo. Int J Ophthalmol 2014;7(1):14-21. [PMC free article]
[PubMed]
25 Lobner D. Comparison of the LDH and MTT
assays for quantifying cell death: validity for neuronal apoptosis? J Neurosci Methods 2000;96(2):147-152. [CrossRef]
26 Ciniglia C, Pinto G, Sansone C, Pollio
A. Acridine orange/Ethidium bromide double staining test: a simple in-vitro
assay to detect apoptosis induced by phenolic compounds in plant cells. Allelopathy J 2010;26(2):301-308.
27 Vermes I, Haanen C, Steffens-Nakken H,
Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of
phosphatidylserine early apoptotic cells using fluorescein labeled expression
on Annexin V. J Immunol Methods
1995;184(1):39-51. [CrossRef]
28 Oberhammer
F, Wilson JW, Dive C, Morris ID, Hickman JA, Wakeling AE, Walker PR, Sikorska
M. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb
fragments prior to or in the absence of internucleosomal fragmentation. EMBO J 1993;12(9):3679-3684. [PMC free article]
[PubMed]
29 Brustmann
H. Apoptotic bodies as a morphological feature in serous ovarian carcinoma:
correlation with nuclear grade, Ki-67 and mitotic indices. Pathol Res Pract 2002;198(2):85-90. [CrossRef]
[PubMed]
30 Jin Z, El-Deiry WS. Overview of cell death
signaling pathways. Cancer Biol Ther 2005:4(2):139-163.
[CrossRef]
[PubMed]
31 Spierings D, McStay G, Saleh M, Bender C,
Chipuk J, Maurer U, Green DR. Connected to death: the (unexpurgated)
mitochondrial pathway of apoptosis. Science
2005;310(5745):66-67. [CrossRef]
[PubMed]
32 Kumar S, Vaux DL. Apoptosis. A cinderella
caspase takes center stage. Science
2002;297(5585):1290-1291. [CrossRef]
[PubMed]
33 Fan T, Han L, Cong RS, Liang J. Caspase
family proteases and apoptosis. Acta
Biochim Biophys Sin (Shanghai) 2005;37(11):719-727. [CrossRef]
34 Brenner C, Kroemer G. Apoptosis.
Mitochondria--the death signal integrators. Science
2000;289(5482):1150-1151. [CrossRef]
[PubMed]
35 Cai J, Yang J, Jones DP. Mitochondrial
control of apoptosis: the role of cytochrome c. Biochim Biophys Acta 1998;1366(1-2):139-149. [CrossRef]
36 Pop C, Timmer J, Sperandio S, Salvesen GS.
The apoptosome activates caspase-9 by dimerization. Mol Cell 2006;22(2):269-275. [CrossRef]
[PubMed]
37 Wajant H. The Fas signaling pathway: more
than a paradigm. Science 2002;296(5573):1635-1636. [CrossRef]
[PubMed]
38 Sun XM, MacFarlane M, Zhuang J, Wolf BB,
Green DR, Cohen GM. Distinct caspase cascades are initiated in
receptor-mediated and chemical-induced apoptosis. J Biol Chem 1999;274(8):5053-5060. [CrossRef]
39 Zhang FT, Ding Y, Shah Z, Xing D, Gao Y,
Liu DM, Ding MX. TNF/TNFR1 pathway and endoplasmic reticulum stress are
involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes. Toxicol Appl Pharmacol 2014;276(2):121-128.
[CrossRef]
[PubMed]
[Top]