The effectiveness of colchicine combined with mitomycin C to prolong bleb function in trabeculectomy in rabbits
The effectiveness of colchicine combined with mitomycin C to prolong bleb function in trabeculectomy in rabbits
Taiki KokubunID 0 2
Kotaro Yamamoto 0 2
Kota Sato 0 1 2
Takahiro Akaishi 2
Atsushi Shimazaki 2
Masatsugu Nakamura 2
Yukihiro Shiga 0 2
Satoru Tsuda 0 2
Kazuko Omodaka 0 2
Toru Nakazawa 0 1 2
0 Department of Ophthalmology, Tohoku University Graduate School of Medicine , Sendai, Miyagi , Japan
1 Collaborative Program for Ophthalmic Drug Discovery, Tohoku University Graduate School of Medicine , Sendai, Miyagi , Japan , 3 Research and Development Division, Santen Pharmaceutical Co. Ltd. , Ikoma, Nara , Japan , 4 Department of Retinal Disease Control, Ophthalmology, Tohoku University Graduate School of Medicine , Sendai, Miyagi , Japan , 5 Department of Advanced Ophthalmic Medicine, Tohoku University Graduate School of Medicine , Sendai, Miyagi , Japan , 6 Department of Ophthalmic imaging and information analytics, Tohoku University Graduate School of Medicine , Sendai, Miyagi , Japan
2 Editor: Demetrios G. Vavvas, Massachusetts Eye & Ear Infirmary, Harvard Medical School , UNITED STATES
Experiments with a rabbit model of trabeculectomy showed that 0; 04% MMC plus 0; 01%
Colchicine may be a promising adjuvant for strengthening the effect of MMC and improving
the survival of the filtering bleb in trabeculectomy.
To investigate the potential of colchicine to improve bleb function after trabeculectomy.
To find the maximum usable colchicine concentration, an ocular irritation study was
performed with the Draize test at concentrations of 0.001%, 0.01% and 0.1%. Additionally, the
synergistic effect of topical colchicine instillation and MMC application to surgical site was
evaluated in a rabbit model by measuring changes after trabeculectomy in intraocular
pressure (IOP) and bleb morphology score at 3, 7, 14, 21, 28, 35, 42, and 49 days.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: The funder (Santen Co. Ltd.) provided
support in the form of salaries for authors TA, AS,
and MN, but did not have any additional role in the
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. The
specific roles of these authors are articulated in the
?author contributions? section.
Trabeculectomy is one of the most common types of filtration surgery to reduce intraocular
pressure (IOP) in glaucoma[
]. This technique involves creating a filtering bleb, through
which aqueous humor flows from the anterior chamber into the sub-Tenon?s space and is
absorbed into the bleb wall (which consists of Tenon?s tissue). The success of trabeculectomy
depends on the postoperative wound healing process in the filtering bleb. During healing, the
excessive proliferation of fibroblasts or fibrosis in the subconjunctiva causes tissue scarring,
and this scarred tissue cannot adequately absorb the aqueous humor, causing bleb failure[
Clinically, to prevent fibrosis after trabeculectomy, adjuvants such as mitomycin C (MMC)
are used to counteract the proliferative activity of cells[
]. Currently, adjuvant MMC is the
?gold standard? against which other potential anti-fibrotic therapeutics are compared.
However, although the success rate is improved with the use of MMC, bleb failure still occurs in
some cases due to strong fibrosis. Increased IOP can then re-occur, necessitating additional
]. In addition, MMC has nonspecific cytotoxic effects that are associated
with severe complications, such as blebitis, keratitis, bleb leakage, chronic hypotony and
endophthalmitis caused by the un-targeted, generally destructive cellular toxicity of MMC[
]. Hence, it is important to identify safer and more broadly targeted anti-fibrotic agents that
can maintain bleb function for a longer period and can be used postoperatively in addition to
MMC, to help preserve the filtering bleb when it shows signs of failure.
Previous reports have shown that colchicine can suppress migration and proliferation of
fibroblasts, in addition to its well-known ability to act as an anti-inflammatory[
Furthermore, the administration of colchicine can prolong the maintenance of a functional bleb after
glaucoma filtration surgery in human subjects by suppressing inflammatory fibrosis[
However, there are no detailed reports comparing the effectiveness of MMC and colchicine
and investigating the effectiveness of colchicine combined with MMC for preserving the
In the present study, we evaluated the effect of topical colchicine administration in a rabbit
model of trabeculectomy. We determined the most appropriate concentration of colchicine to
ensure both effectiveness and safety, and investigated the potential of colchicine eye drops as a
supplementary postoperative adjuvant in trabeculectomy.
Materials & methods
Colchicine was purchased from Nacalai Tesque, Inc (Kyoto, Japan). Mitomycin C (MMC;
2-mg injection) was purchased from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). Ketamine
hydrochloride (Ketalar for intramuscular injection; 500 mg) was purchased from Daiichi
Sankyo Co., Ltd. (Tokyo, Japan). Xylazine hydrochloride (Selactar for injection; 2%) was
purchased from Bayer Yakuhin Ltd. (Osaka, Japan). A 0.01% ophthalmic solution of
betamethasone sodium phosphate was purchased from Shionogi Co., Ltd. (Osaka, Japan). A 0.5%
ophthalmic solution of levofloxacin hydrate and a 0.4% ophthalmic solution of oxybuprocaine
were supplied by Santen Pharmaceutical Co., Ltd. (Osaka, Japan).
Male Japanese white rabbits, weighing 1.7 to 2.9 kg, were supplied by Kitayama Labes Co., Ltd.
(Nagano, Japan). The animals were housed under a 12-hour light/dark cycle and had free
access to water and a standard laboratory diet. All animal care and experimental procedures
were performed in accordance with the ARVO Statement for the Use of Animals in
2 / 12
Ophthalmic and Vision Research and were approved and monitored by the Animal Care
and Use Committee of Santen Pharmaceutical Co., Ltd (Approval number: DR120505 and
DR120520). After the study was completed, animals were euthanized with an intravenous
injection of pentobarbital.
To find the maximum safety colchicine concentration, an ocular irritation study was
performed with the Draize test[
]. Twelve male albino rabbits with no existing ocular irritation
were used to evaluate each test material; the animals were assigned to one of four groups (n = 3
in each group) in each experiment.
A 50 ?L test preparation (containing 0.001%, 0.01% or 0.1% colchicine dissolved in saline)
or saline was instilled in one eye. This was repeated every 30 min for 4.5 h (10 times). The
ocular response was then scored at 0.5, 1, 2 and 4, and 24 h after the last instillation. Irritation
was scored using the Draize method[
]. This method involves weighting and summing six
directly observable changes in the eye?s anterior segment: density (A; score: 1?4), area of
corneal opacification (B; score: 1?4), severity of iritis (C; score: 1?2), conjunctival redness (D;
score: 1?3), edema (E; score: 1?4), and discharge (F; score: 1?3). The mean total score was
calculated as (A?B?5)+(C?5)+(D+E+F)?2, ranging from 0 to 110 at each measured time point.
After the induction of general anesthesia, which was performed with an intramuscular
injection of ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (4 mg/kg),
trabeculectomy was performed with previously reported methods[
]. Briefly, a limbus-based
conjunctival flap was prepared, a scleral flap was created, and Medical Quick Absorber (MQA)
(Inami Co, Ltd. Tokyo, Japan) saturated with 0.04% MMC solution or saline was placed under
the conjunctiva and over the scleral flap site for 5 minutes. The area was irrigated with 120 ml
of 0.9% sodium chloride solution (Otsuka Pharmaceutical Factory Inc., Tokushima, Japan).
Sclerotomy was performed under the scleral flap; a Kelly Descemet?s membrane punch (M.E.
Technica, Tokyo Japan) was used to form a scleral tunnel opening into the aqueous chamber.
The scleral flap was not sutured, but the conjunctiva was sutured with 10?0 sutures. Topical
0.01% betamethasone sodium phosphate and 0.5% levofloxacin hydrate ophthalmic solution
were administered three or four times daily (according to specific experimental protocols) for
the first five days after surgery. The right eye underwent this procedure in all rabbits, while the
other eye was left untreated as a control. During this study, no perioperative complications,
such as inadvertent penetration to the anterior chamber or aqueous drainage, were observed
in any of the eyes.
To compare colchicine and MMC, experiment A was performed, which included 8 rabbits.
The animals received either 0.04% MMC or 0.01% colchicine (each n = 4). To compare
colchicine combined with MMC vs. MMC alone, experiment B was performed, which included 20
rabbits assigned to receive saline (n = 4), 0.04% MMC (n = 8), or 0.04% MMC plus 0.01%
colchicine (n = 8). IOP values were confirmed to be approximately the same in all groups before
In experiments A and B, the 0.04% MMC groups were treated with MQA saturated with
0.04% MMC, applied under the conjunctiva and over the scleral flap site for 5 minutes during
glaucoma filtration surgery. Saline (50 ?L) was then instilled 4 times daily in experiment A and
3 times daily in experiment B. In experiment A, the 0.01% colchicine group received saline
3 / 12
under the conjunctiva and over the scleral flap site during glaucoma filtration surgery. After
surgery, colchicine (0.01%) eye drops were applied 4 times daily. In experiment B, the control
group was treated with MQA saturated with saline, applied under the conjunctiva and over the
scleral flap site for 5 minutes during glaucoma filtration surgery, and saline was instilled 3
times daily. The 0.04% MMC plus 0.01% colchicine group was treated with MQA saturated
with 0.04% MMC, applied under the conjunctiva and over the scleral flap site for 5 minutes
during glaucoma filtration surgery. After surgery, 0.01% colchicine dissolved in saline (50 ?L)
was instilled 3 times daily.
Instillation of all types of eye drops was performed on days 1 to 14 in experiment A and on
days 6 to 14 in experiment B.
IOP was measured with a pneumatonograph (Model 30 Classic Pneumatonometer; Reichert
Technologies, Depew, NY, USA) after general anesthesia was induced with the intramuscular
injection of a mixture of ketamine (40 mg/kg body weight) and xylazine (4 mg/kg body
weight). Binocular IOP was measured within 7 min of the injection of the anesthetic. For
corneal anesthesia, an oxybuprocaine solution (Benoxil ophthalmic solution 0.4%; Santen
Pharmaceutical Co., Ltd.) was applied topically prior to IOP measurement. IOP measurement was
performed between 3 pm and 7 pm. IOP was measured in experiment A 7, 14, 21, and 28 days
after surgery. IOP was measured in experiment B 3, 7, 14, 21, 28, 35, 42, and 49 days after the
The blebs were examined via slit lamp and were graded with a slightly modified version of the
method described by Perkins et al[
]. This method uses a qualitative scale of 1+ to 4+,
reflecting increasing bleb height and size as follows: 1+, minimal height, conjunctiva thickening, no
microcysts; 2+, microcysts present covering less than 75? of the eye; 3+, elevated bleb covering
75 to 135? of the eye; and 4+, greatly elevated bleb covering more than 135? of the eye. A score
of 0 indicated no observable bleb. In experiment A, bleb evaluation was performed 7, 14, 21,
and 28 days after surgery. In experiment B, bleb evaluation was performed 3, 7, 14, 21, 28, 35,
42, and 49 days after the surgery.
We compared the IOP data in the treated eyes in all groups separately (two groups in
experiment A: 0.04% MMC and 0.01% colchicine; and three groups in experiment B: saline, 0.04%
MMC, and 0.04% MMC plus 0.01% colchicine). Values were compared with the unpaired
Student?s t-test in experiment A and with the Tukey-Kramer test for multiple comparisons in
experiment B (P < 0.05 was considered to be statistically significant). We also confirmed
changes in slit lamp appearance and compared bleb score in these same groups with the
Wilcoxon-Mann-Whitney test in experiment A and with the Steel-Dwass test for multiple
comparisons in experiment B (P < 0.05 was considered to be statistically significant).
Ocular irritation caused by colchicine
To determine the maximum safe concentration of colchicine for topical administration, the
Draize test was performed. Table 1 shows the main results for total score; a detailed profile of
the test results is shown in S1 Table. Eyes treated with 0.001% colchicine showed no obvious
4 / 12
Data represent the mean score + S.E.M. of 3 eyes for the saline group and colchicine groups (0.1%, 0.01%, and 0.001%).
ocular irritation compared to the eyes treated with saline. The colchicine eyes had total
scores of 0.0 + 0.0 at all observed time points. The eyes treated with 0.01% colchicine also
showed very little ocular irritation, with total scores of 0.0 + 0.0, 0.0 + 0.0, 0.0 + 0.0, 0.0 + 0.0,
0.7 + 0.7 and 0.7 + 0.7 (n = 3, mean + S.E.M) at each observed time point (Table 1). On the
other hand, the eyes treated with 0.1% colchicine showed redness in the palpebral
conjunctiva and chemosis of the conjunctiva after treatment (S1 Table), with total scores of 0.0 + 0.0,
0.0 + 0.0, 0.7 + 0.7, 5.3 + 2.7, 8.0 + 2.1 and 1.3 + 0.7 (n = 3, mean + S.E.M) at each time point
These results suggest that there is a relationship between colchicine concentration and a
number of key indices of ocular irritation. Minimal irritation effects were observed at a
concentration of colchicine of 0.01% or less.
Effect of colchicine and MMC on bleb preservation in a rabbit model
Firstly, we evaluated the bleb preservation effect of uncombined colchicine. We found that
topical 0.01% colchicine instillation alone was less effective than the intraoperative application
of 0.04% MMC in preserving reduced IOP and bleb score (S1 Fig).
Secondly, we evaluated whether combined treatment with colchicine and MMC had a
synergistic effect on bleb preservation. Fig 1 shows IOP at each measured time point in each
group: control, 0.04% MMC, or 0.04% MMC plus 0.01% colchicine. There was no statistically
significant difference in mean initial IOP in the groups (range: 21.4 mmHg to 21.7 mmHg).
No abnormalities in the eyes or the general health of the animals were observed.
Compared to the saline group, there was a significant IOP reduction in the 0.04% MMC
group at each time point between 7?35 days (P < 0.05 at all time points). The 0.04% MMC
plus 0.01% colchicine group showed a significant reduction in comparison with the saline
group at days 7?49 (P < 0.01 at all time points) and in comparison with the 0.04% MMC
group at day 49 (P < 0.05).
We scored the morphology of the bleb based on its appearance and size, as previously
described by Perkins TW[
]. Representative slit-lamp photographs of blebs with each score
are shown in Fig 2A. Representative slit-lamp photographs of the control, 0.04% MMC, and
0.04% MMC plus 0.01% colchicine groups are shown in Fig 2B. The average bleb score of the
three groups at each measured time point is shown in Fig 2C.
Compared to the saline group, the 0.04% MMC group showed a significantly higher bleb
score at days 7?35 (P < 0.05 at all time points) and the 0.04% MMC plus 0.01% colchicine
group showed a significantly higher bleb score at days 7?21, and 35?49 (P < 0.05 at all time
points). The 0.04% MMC plus 0.01% colchicine group showed no significant difference in
comparison with the 0.04% MMC group at any time point.
5 / 12
Fig 1. Changes in intraocular pressure (IOP) after treatment with MMC and MMC plus colchicine in a rabbit model of trabeculectomy.
The white circles, black squares and gray triangles indicate controls (saline), 0.04% MMC and 0.04% MMC plus 0.01% colchicine, respectively
(n = 4, 8 and 8, respectively). Error bar = SEM. The bars indicate periods with a significant difference between groups. a: 0.04% MMC plus
0.01% colchicine vs. control. b: 0.04% MMC vs. control. c: 0.04% MMC plus 0.01% colchicine vs. 0.04% MMC.
This study found that the combination of 0.04% MMC and 0.01% colchicine prolonged the
low-IOP period after surgery more effectively than a control treatment. Furthermore, this
period was longer than the effective low-IOP period after treatment with uncombined 0.04%
MMC, in comparison to a control treatment. We also found that IOP in the 0.04% MMC
group tended to increase starting at 28 days, even though the difference in IOP between the
0.04% MMC and 0.04% MMC plus 0.01% colchicine groups at 28?42 days was not statistically
significant, and a statistically significant difference was only present on day 49. Similarly,
combined 0.04% MMC and 0.01% colchicine preserved the subjectively-observed appearance of
the bleb more effectively than in the control group. This period was longer than the similar
period in the uncombined 0.04% MMC group compared to controls. The 0.04% MMC group
also showed a tendency towards a worse bleb score starting at 35 days, even though there was
not a statistically significant difference in bleb score between the 0.04% MMC and 0.04%
MMC plus 0.01% colchicine groups at any time point. Thus, the combination of colchicine
instillation and MMC application might improve the outcome of trabeculectomy by helping to
maintain low IOP and a functional bleb.
MMC is used in a variety of medical settings to prevent cellular proliferation; it acts by
halting the synthesis of new DNA and protein[
]. However, strong fibrosis can occur in the
6 / 12
Fig 2. Bleb scoring via slit lamp observation. (A) Representative bleb scores (0?4) according to the method described by Perkins et al.
Briefly, with increasing score, the bleb becomes more elevated and covers a wider area. (B) Representative photographs of blebs in
control and treated rabbit eyes. Top to bottom rows: blebs after treatment with saline as a control, 0.04% MMC, or 0.04% MMC plus
0.01% colchicine. Left to right: images taken 14, 28 and 49 days after surgery. The blebs are visible in the upper right of each image. (C)
Changes in bleb score after surgery with saline-treated controls, 0.04% MMC, and 0.04% MMC plus 0.01% colchicine. The white circles,
black squares and gray triangles indicate controls, 0.04% MMC and 0.04% MMC plus 0.01% colchicine, respectively (n = 4, 8 and 8 in
each group). The error bar indicates SEM. The bars indicate time periods with a significant difference between groups. d: 0.04% MMC
plus 0.01% colchicine vs. control. e: 0.04% MMC vs. control.
7 / 12
filtering bleb even with intraoperative MMC, causing bleb failure in some cases[
]. This may
be because MMC-treated fibroblasts continue to express transforming growth factor-?
(TGF?), as well as pro-inflammatory cytokines such as interleukin-8 (IL-8) and monocyte
chemoattractant protein-1 (MCP-1)[
]. These cytokines promote scarring during the remodeling
phase of the wound healing cascade[
], and they have been observed to have mildly
elevated levels in aqueous humor samples for a prolonged period after intraocular surgery[
Colchicine is considered to be an anti-mitotic drug, and acts via a very different mechanism
than MMC. It is an alkaloid that binds to tubulin molecules and inhibits their polymerization
into microtubules, resulting in disruption of the mitotic spindle[
], causing widespread cell
death and apoptosis[
]. In addition, some studies have found evidence that colchicine
attenuates inflammatory cytokines, such as TGF-? [
], connective-tissue growth factor
(CTGF), and MCP-1 [
], and suppresses extracellular matrix accumulation[
work has shown that colchicine can directly inhibit the release of fibronectin and collagen into
the extracellular space by fibroblasts, reduce collagen-processing enzymes, and stimulate tissue
]. Thus, colchicine has a broad range of anti-fibrotic functions that
could complement the mechanisms underlying the ability of MMC to control scarring
formation after trabeculectomy. Combined treatment with colchicine and MMC might therefore
have a synergistic effect and lengthen the period of IOP reduction after trabeculectomy, in
comparison to uncombined MMC.
Past studies have reported on the limitations of MMC treatment. Intraocular irrigation
by the aqueous humor significantly reduces the tissue concentration of MMC, causing it to
disappear rapidly in the subconjunctival tissue[
]. This may another reason that intraoperative
administration of MMC alone is insufficient to keep bleb function at long postoperative
period. Furthermore, inflammatory cytokines can persist for several months after surgery and
counteract the anti-scarring effects of MMC[
]. These past findings suggested that new
drugs might reduce scarring by suppressing this prolonged increase of inflammatory
cytokines, especially if they could be applied postoperatively. Thus, previous studies investigated
whether targeting specific cytokines could modulate scarring after glaucoma filtration surgery.
Research in animal models showed that postoperative TGF-? inhibition with monoclonal
antibodies inhibited subconjunctival scarring and prevented the failure of experimental glaucoma
]. However, clinical trials in patients undergoing primary trabeculectomy failed to
reproduce this effect[
]. We speculated that this therapy might have failed because the
monoclonal antibodies targeted only a single cytokine, TGF-?. Therefore, we hypothesized
that colchicine might be more effective in modulating fibrosis after trabeculectomy, because it
can suppress a variety of cytokines related to fibrosis[
], and can also directly suppress
fibrosis by acting on fibroblasts[
Despite our finding that colchicine was effective when combined with MMC, we found
that uncombined colchicine instillation was less effective than MMC in preserving bleb score
and reducing IOP. This may be because a colchicine concentration of 0.01% was insufficient.
Past studies of anti-metabolites as topical medicines after ocular surgery have found that
these drugs must have a lower concentration when they are applied topically, rather than
intraoperatively, to avoid ocular surface complications such as corneal and conjunctival
]. Here, we found that in a Draize study, a 0.1% concentration of colchicine caused
ocular irritation, so we used a 0.01% concentration, applied topically four times daily.
However, interestingly, one previous study reported that the topical administration of 0.25%
colchicine only once daily significantly decreased IOP up to 4 weeks after filtration surgery.
Thus, the possibility remains that if we adjust the number of times per day the treatment is
applied, we might be able to safely increase the concentration of colchicine and improve its
8 / 12
In conclusion, this study suggests that combining topical colchicine instillation and MMC
application as surgical adjuvants after trabeculectomy can increase the postoperative period
during which IOP is reduced and bleb morphology is preserved. Thus, colchicine might be a
promising novel adjunct to trabeculectomy, strengthening the suppression of postoperative
fibrosis when used as a complement to MMC. We anticipate that this combined therapy will
be explored further in future studies.
S1 Table. Detailed profile of Draize ocular irritation test.
S1 Fig. Intraocular pressure (IOP) and bleb score with MMC or uncombined colchicine.
The black squares and gray triangles indicate 0.04% MMC and uncombined 0.01% colchicine,
respectively (all n = 4). Error bars = SEM. A: IOP; B: bleb score. The bars indicate time periods
with a significant difference between groups; a: 0.04% MMC vs. uncombined 0.01% colchicine;
b: 0.04% MMC vs. uncombined 0.01% colchicine.
S1 File. Data underlying Table 1.
S2 File. Data underlying Fig 1.
S3 File. Data underlying Fig 2.
S4 File. Data underlying S1 Table.
S5 File. Data underlying S1A Fig.
S6 File. Data underlying S1B Fig.
We thank Mr. Tim Hilts for reviewing and editing the language of the manuscript and Dr.
Kazuichi Maruyama for providing valuable comments. This work was supported by Santen
Pharmaceutical Co., Ltd., the Platform for Drug Discovery, Informatics, and Structural Life
Science of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Conceptualization: Takahiro Akaishi, Atsushi Shimazaki, Masatsugu Nakamura, Toru
Data curation: Taiki Kokubun, Kotaro Yamamoto, Takahiro Akaishi.
Formal analysis: Taiki Kokubun, Kotaro Yamamoto, Takahiro Akaishi.
Investigation: Kotaro Yamamoto.
Methodology: Kota Sato.
9 / 12
Supervision: Kota Sato, Atsushi Shimazaki, Masatsugu Nakamura.
Writing ? original draft: Taiki Kokubun.
Writing ? review & editing: Kota Sato, Takahiro Akaishi, Atsushi Shimazaki, Masatsugu
Nakamura, Yukihiro Shiga, Satoru Tsuda, Kazuko Omodaka, Toru Nakazawa.
10 / 12
11 / 12
1. Cairns JE . Symposium: microsurgery of the outflow channels . Trabeculectomy. Trans Am Acad Ophthalmol Otolaryngol . 1972 ; 76 : 384 - 8 . PMID: 4582682
2. Costa VP , Spaeth GL , Eiferman RA , Orengo-Nania S . Wound healing modulation in glaucoma filtration surgery . Ophthalmic Surg . 1993 ; 24 : 152 - 70 . PMID: 8483566
3. Addicks EM , Quigley HA , Green WR , Robin AL . Histologic characteristics of filtering blebs in glaucomatous eyes . Arch Ophthalmol . 1983 ; 101 : 795 - 8 . PMID: 6847472
4. Mietz H , Arnold G , Kirchhof B , Diestelhorst M , Krieglstein GK . Histopathology of episcleral fibrosis after trabeculectomy with and without mitomycin C . Graefes Arch Clin Exp Ophthalmol . 1996 ; 234 : 364 - 8 . PMID: 8738702
5. Kitazawa Y , Kawase K , Matsushita H , Minobe M. Trabeculectomy with mitomycin. A comparative study with fluorouracil . Arch Ophthalmol . 1991 ; 109 : 1693 - 8 . PMID: 1841578
6. Kitazawa Y , Yamamoto T , Sawada A , Hagiwara Y . Surgery for refractory glaucoma . Aust N Z J Ophthalmol . 1996 ; 24 : 327 - 32 . PMID: 8985544
7. Kokubun T , Tsuda S , Kunikata H , Himori N , Yokoyama Y , Kunimatsu-Sanuki S , et al. Anterior-Segment Optical Coherence Tomography for Predicting Postoperative Outcomes After Trabeculectomy . Curr Eye Res . 2018 ; 1 - 9 .
8. Fontana H , Nouri-Mahdavi K , Lumba J , Ralli M , Caprioli J . Trabeculectomy with mitomycin C: outcomes and risk factors for failure in phakic open-angle glaucoma . Ophthalmology . 2006 ; 113 : 930 - 6 . https:// doi.org/10.1016/j.ophtha. 2006 . 01 .062 PMID: 16647135
9. Jampel HD , Pasquale LR , Dibernardo C . Hypotony maculopathy following trabeculectomy with mitomycin C. Arch Ophthalmol . 1992 ; 110 : 1049 - 50 .
10. Greenfield DS , Su?er IJ , Miller MP , Kangas TA , Palmberg PF , Flynn HW . Endophthalmitis after filtering surgery with mitomycin . Arch Ophthalmol . 1996 ; 114 : 943 - 9 . PMID: 8694729
11. Khaw PT , Doyle JW , Sherwood MB , Grierson I , Schultz G , McGorray S . Prolonged localized tissue effects from 5-minute exposures to fluorouracil and mitomycin C. Arch Ophthalmol . 1993 ; 111 : 263 - 7 . PMID: 8431167
12. Entzian P , Schlaak M , Seitzer U , Bufe A , Acil Y , Zabel P. Antiinflammatory and antifibrotic properties of colchicine: implications for idiopathic pulmonary fibrosis . Lung . 1997 ; 175 : 41 - 51 . PMID: 8959672
13. Lemor M , de Bustros S , Glaser BM . Low-dose colchicine inhibits astrocyte, fibroblast, and retinal pigment epithelial cell migration and proliferation . Arch Ophthalmol . 1986 ; 104 : 1223 - 5 . PMID: 3741255
14. Joseph JP , Grierson I , Hitchings RA . Taxol, cytochalasin B and colchicine effects on fibroblast migration and contraction: a role in glaucoma filtration surgery? Curr Eye Res . 1989 ; 8 : 203 - 15 . PMID: 2565797
15. Fuller JR , Bevin TH , Molteno ACB , Vote BJT , Herbison P . Anti-inflammatory fibrosis suppression in threatened trabeculectomy bleb failure produces good long term control of intraocular pressure without risk of sight threatening complications . Br J Ophthalmol . 2002 ; 86 : 1352 - 4 . PMID: 12446362
16. Wilhelmus KR . The Draize eye test . Surv Ophthalmol . 2001 ; 45 : 493 - 515 . PMID: 11425356
17. Okuda T , Higashide T , Fukuhira Y , Sumi Y , Shimomura M , Sugiyama K. A thin honeycomb-patterned film as an adhesion barrier in an animal model of glaucoma filtration surgery . J Glaucoma . 2009 ; 18 : 220 - 6 . https://doi.org/10.1097/IJG.0b013e31817eee32 PMID: 19295377
18. Bergstrom TJ , Wilkinson WS , Skuta GL , Watnick RL , Elner VM . The effects of subconjunctival mitomycin-C on glaucoma filtration surgery in rabbits . Arch Ophthalmol . 1991 ; 109 : 1725 - 30 . PMID: 1841585
19. Perkins TW , Faha B , Ni M , Kiland JA , Poulsen GL , Antelman D , et al. Adenovirus-mediated gene therapy using human p21WAF-1/Cip-1 to prevent wound healing in a rabbit model of glaucoma filtration surgery . Arch Ophthalmol . 2002 ; 120 : 941 - 9 . PMID: 12096965
20. Bass PD , Gubler DA , Judd TC , Williams RM . Mitomycinoid alkaloids: mechanism of action, biosynthesis, total syntheses, and synthetic approaches . Chem Rev . 2013 ; 113 : 6816 - 63 . https://doi.org/10. 1021/cr3001059 PMID: 23654296
21. Occleston NL , Daniels JT , Tarnuzzer RW , Sethi KK , Alexander RA , Bhattacharya SS , et al. Single exposures to antiproliferatives: long-term effects on ocular fibroblast wound-healing behavior . Invest Ophthalmol Vis Sci . 1997 ; 38 : 1998 - 2007 . PMID: 9331263
22. Seet L-F , Su R , Toh LZ , Wong TT . In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C . J Cell Mol Med . 2012 ; 16 : 1245 - 59 . https:// doi.org/10.1111/j.1582- 4934 . 2011 . 01400 . x PMID : 21801304
23. Leask A , Abraham DJ . TGF-? signaling and the fibrotic response . FASEB J . 2004 ; 18 : 816 - 27 . https:// doi.org/10.1096/fj.03-1273rev PMID: 15117886
24. Yuan H , Li X , Yang B , Shao Z , Yan L . Expression of connective tissue growth factor after trabeculectomy in rabbits . Zhonghua Yan Ke Za Zhi . 2009 ; 45 : 168 - 74 . PMID: 19573340
25. Inoue T , Kawaji T , Tanihara H . Monocyte chemotactic protein-1 level in the aqueous humour as a prognostic factor for the outcome of trabeculectomy . Clin Exp Ophthalmol . 2014 ; 42 : 334 - 41 . https://doi.org/ 10.1111/ceo.12204 PMID: 24025148
26. Akiyama H , Kachi S , Silva RLE , Umeda N , Hackett SF , McCauley D , et al. Intraocular injection of an aptamer that binds PDGF-B: a potential treatment for proliferative retinopathies . J Cell Physiol . 2006 ; 207 : 407 - 12 . https://doi.org/10.1002/jcp.20583 PMID: 16419035
27. Pan LH , Ohtani H , Yamauchi K , Nagura H. Co-expression of TNF alpha and IL-1 beta in human acute pulmonary fibrotic diseases: an immunohistochemical analysis . Pathol Int . 1996 ; 46 : 91 - 9 . PMID: 10846556
28. Kawai M , Inoue T , Inatani M , Tsuboi N , Shobayashi K , Matsukawa A , et al. Elevated levels of monocyte chemoattractant protein-1 in the aqueous humor after phacoemulsification . Invest Ophthalmol Vis Sci . 2012 ; 53 : 7951 - 60 . https://doi.org/10.1167/iovs.12-10231 PMID: 23132797
29. Ng GF , Raihan IS , Azhany Y , Maraina CHC , Banumathi KG , Liza-Sharmini T. Conjunctival TGF-B Level in Primary Augmented Trabeculectomy. Open Ophthalmol J. 2015 ; 9 : 136 - 44 . https://doi.org/10. 2174/1874364101509010136 PMID: 26401171
30. Hastie SB . Interactions of colchicine with tubulin . Pharmacol Ther . 1991 ; 51 : 377 - 401 . PMID: 1792241
31. Yang Y , Zhu X , Chen Y , Wang X , Chen R. p38 and JNK MAPK, but not ERK1/2 MAPK, play important role in colchicine-induced cortical neurons apoptosis . Eur J Pharmacol . 2007 ; 576 : 26 - 33 . https://doi. org/10.1016/j.ejphar. 2007 . 07 .067 PMID: 17716651
32. Ozmen N , Kaya-Sezginer E , Bakar-Ates F. The Cellular Uptake and Apoptotic Efficiency of Colchicine is Correlated with Down-regulation of MMP-9 mRNA Expression in SW480 Colon Cancer Cells . Anticancer Agents Med Chem . 2018 ; https://doi.org/10.2174/1871520618666180821102047 PMID: 30129419
33. Malawista SE . Colchicine: a common mechanism for its anti-inflammatory and anti-mitotic effects . Arthritis Rheum . 1968 ; 11 : 191 - 7 . PMID: 4868927
34. Disel U , Paydas S , Dogan A , Gulfiliz G , Yavuz S . Effect of colchicine on cyclosporine nephrotoxicity, reduction of TGF-beta overexpression, apoptosis, and oxidative damage: an experimental animal study . Transplant Proc . 2004 ; 36 : 1372 - 6 . https://doi.org/10.1016/j.transproceed. 2004 . 05 .078 PMID: 15251335
35. Tzortzaki EG , Antoniou KM , Zervou MI , Lambiri I , Koutsopoulos A , Tzanakis N , et al. Effects of antifibrotic agents on TGF-?1, CTGF and IFN-? expression in patients with idiopathic pulmonary fibrosis . Respir Med . 2007 ; 101 : 1821 - 9 . https://doi.org/10.1016/j.rmed. 2007 . 02 .006 PMID: 17391951
36. Li JJ , Lee SH , Kim DK , Jin R , Jung D-S , Kwak S-J , et al. Colchicine attenuates inflammatory cell infiltration and extracellular matrix accumulation in diabetic nephropathy . Am J Physiol Renal Physiol . 2009 ; 297 : 200 - 9 .
37. Dehm P , Prockop DJ . Time lag in the secretion of collagen by matrix-free tendon cells and inhibition of the secretory process by colchicine and vinblastine . Biochim Biophys Acta . 1972 ; 264 : 375 - 82 . PMID: 4337621
38. Diegelmann RF , Peterkofsky B . Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs . Proc Natl Acad Sci U S A . 1972 ; 69 : 892 - 6 . PMID: 4502941
39. Harris ED , Krane SM . Effects of colchicine on collagenase in cultures of rheumatoid synovium . Arthritis Rheum . 1971 ; 14 : 669 - 84 . PMID: 4109310
40. Kawase K , Matsushita H , Yamamoto T , Kitazawa Y. Mitomycin concentration in rabbit and human ocular tissues after topical administration . Ophthalmology . 1992 ; 99 : 203 - 7 . PMID: 1553208
41. Cordeiro MF , Reichel MB , Gay JA , D'Esposita F , Alexander RA , Khaw PT . Transforming growth factorbeta1 , - beta2 , and -beta3 in vivo: effects on normal and mitomycin C-modulated conjunctival scarring . Invest Ophthalmol Vis Sci . 1999 ; 40 : 1975 - 82 . PMID: 10440251
42. Mead AL , Wong TTL , Cordeiro MF , Anderson IK , Khaw PT . Evaluation of anti-TGF-beta2 antibody as a new postoperative anti-scarring agent in glaucoma surgery . Invest Ophthalmol Vis Sci . 2003 ; 44 : 3394 - 401 . PMID: 12882787
43. Khaw P , Grehn F , Hollo? G, Overton B , Wilson R , et al. A phase III study of subconjunctival human antitransforming growth factor beta(2) monoclonal antibody (CAT-152) to prevent scarring after first-time trabeculectomy . Ophthalmology . 2007 ; 114 : 1822 - 30 . https://doi.org/10.1016/j.ophtha. 2007 . 03 .050 PMID: 17908591
44. Siriwardena D , Khaw PT , King AJ , Donaldson ML , Overton BM , Migdal C , et al. Human antitransforming growth factor beta(2) monoclonal antibody-a new modulator of wound healing in trabeculectomy: a randomized placebo controlled clinical study . Ophthalmology . 2002 ; 109 : 427 - 31 . PMID: 11874742
45. Singh G , Wilson MR , Foster CS . Mitomycin eye drops as treatment for pterygium . Ophthalmology . 1988 ; 95 : 813 - 21 . PMID: 3211484
46. Hayasaka S , Noda S , Yamamoto Y , Setogawa T. Postoperative instillation of low-dose mitomycin C in the treatment of primary pterygium . Am J Ophthalmol . 1988 ; 106 : 715 - 8 . PMID: 3143266
47. Yu YS , Youn DH . The effect of colchicine on fibroblast proliferation after glaucoma filtering surgery . Korean J Ophthalmol . 1987 ; 1 : 59 - 71 . https://doi.org/10.3341/kjo. 1987 . 1 .2.59 PMID: 3508228