(Investigative Ophthalmology and Visual Science. 2000;41:3399-3401.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Histologic Effect of Mitomycin C on Strabismus Surgery in the Rabbit
Nilza Minguini1,
Keila M. Monteiro de Carvalho1,
Patrícia M. S. Akaishi1 and
Iara M. S. De Luca2
1 From the Department of Ophthalmology, Faculty of Medical Sciences, and the
2 Institute of Biology, State University of Campinas, Brazil.
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Abstract
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PURPOSE. To evaluate the efficiency of mitomycin C (MMC) in limiting the
postoperative inflammatory response and scarring after strabismus
surgery.
METHODS. A prospective, two-stage, masked, controlled trial was conducted. In
the first stage, the inflammatory response at the extraocular muscle
reattachment site was increased after inferior rectus recession in
eight rabbits. In the second stage, MMC (0.4 mg/ml) was applied during
surgery to the eyes of 22 rabbits with inferior rectus recession. As a
control, contralateral eyes were treated with saline solution. Seven
weeks later, exenteration was performed, and the sites of muscle
reattachment were processed for histologic examinations. The sums of
the areas of the granulomas in the extraocular muscle reattachment
sites of control and treated eyes were compared.
RESULTS. There was no significant inhibitory effect of MMC on the inflammatory
response of treated eyes compared with that of control eyes.
CONCLUSIONS. The intraoperative use of MMC (0.4 mg/ml) was not effective in
controlling the postoperative inflammatory response in rabbit eyes
after extraocular muscle surgery. These data do not support the
hypothesis that MMC reduces postoperative adhesions after strabismus
surgery.
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Introduction
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Mechanical contributions to the success of strabismus surgery
include the absence of excessive fibrous tissue.1
Recently, the usefulness of mitomycin C (MMC), an antimetabolic agent,
has been tested experimentally2
3
4
5
to reduce
postoperative adhesions in strabismus surgery. Cruz3
reported reduced formation of adhesions and postoperative scarring when
MMC was used during surgery. In contrast, Brooks et al.4
found no significant reduction in scarring and restriction of motility
with MMC. Similarly, Ohtsuki et al.5
found MMC to be
ineffective in preventing muscle migration, a supposed result of
vigorous healing response.
The objective of this study was to further examine the influence of MMC
on postoperative inflammatory response and scarring in strabismus
surgery. This was done by performing a semiquantitative histologic
analysis of the effect of MMC after inferior rectus (IR) recession in
rabbits.
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Materials and Methods
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Experimental Design
This was a prospective, two-stage, masked, controlled animal
trial. In the first stage, inflammatory response surrounding the muscle
insertion site was enhanced to allow a better evaluation of the
supposed inhibitory effect of MMC on fibrosis, and in the second stage
the effect of MMC was examined.
Animals
Thirty New Zealand White rabbits (2–3 kg) were used. Each rabbit
was anesthetized with an intramuscular injection of ketamine HCl (40
mg/kg, Ketalar; Parke–Davis, Guarulhos, Brazil) and xylazine (4 mg/kg,
Rompum; Bayer, São Paulo, Brazil) before surgery and orbit
exenteration, which were followed by euthanasia. The procedures
followed the recommendations of the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research and the policies in the
Guidelines for the Care and Use of Laboratory Animals (National
Institutes of Health publication No. 85-23, rev. 1985).
First Stage: Increasing Inflammatory Response
Eight rabbits had IR muscle of both eyes recessed 2 mm, as
described elsewhere.6
In one randomly chosen eye of each
pair (treated eye), the procedure included cauterization of a
1-cm2 area of the muscle’s inferior surface and
the underlying sclera; the other eye served as a control. Seven weeks
later, the orbits were exenterated with care, to ensure that the IR
junction was not disrupted, and the eyes were then processed for
histologic analysis. The extent of the inflammatory response was
accessed histologically followed by comparison of the treated and
control eyes.
Second Stage: MMC Testing
Twenty-two rabbits underwent 2-mm recession with cauterization in
both eyes as described for the treated eyes in the first stage. After
cauterization, a sponge soaked in MMC (0.4 mg/ml, Mitocin; B-MS,
São Paulo, Brazil) was placed for 3 minutes between the muscle
and the sclera in a randomly chosen eye in each pair. The area was then
irrigated with 200 ml normal saline. A sponge soaked in balanced salt
solution was placed on the contralateral eye as a control. The orbits
were exenterated 7 weeks later, as described for the first stage.
Histologic Preparation and Quantification of Inflammatory Response
The eyes were fixed in 10% phosphate-buffered formalin and
embedded in paraffin. Three consecutive sagittal sections for each eye
were cut perpendicularly to the line of the postsurgical muscle
insertion. The sections were stained with Masson trichrome, and the
intensity of the inflammatory response was semiquantified by summing
the area of each granuloma. The area (A) of the granuloma
was estimated using the formula for the area of an ellipse:
A = 
(a · b), where
a is major diameter and b is minor diameter.
The diameters of the granulomas were measured using a light optical
system supplied with a graduated eyepiece micrometer and x10 or x40
objective (for final magnification of 100 or 400).
Statistical Analysis
Group differences in total granuloma area were compared using the
Mann–Whitney test with P < 0.05 indicating
significance.
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Results
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First Stage
Histologic analysis revealed a significant difference
(P = 0.01) in the total areas of granulomas
between the control eyes and those treated with cauterization
(Table 1)
. The data for rabbit 6 were not considered, because the
animal developed an ocular infection.
Second Stage
Table 2
shows the total area of the granulomas for 19 rabbits. Although a few
MMC-treated eyes unexpectedly showed a more prominent inflammatory
response than the control eyes (Fig. 1)
, there was no significant difference (P = 0.23) in
areas of granulomas. Data from two rabbits that died during this stage
and one that developed an ocular infection were excluded from the final
analysis.
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Figure 1. Histologic appearance of the IR muscle in a control eye (A)
and in an eye treated with 0.4 mg/ml MMC (B). Compared with
the control, the treated eye shows a more prominent inflammatory
response, with more fibrous tissue around the suture materials
(asterisks). Masson trichrome; original magnification,
x64.
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Discussion
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This study was designed to examine further the tissue effects of
MMC in rabbits after extraocular muscle surgery, particularly with
regard to the effect of this compound on scarring. We have created a
new way to quantify the inflammatory response of the
muscle-tendon–scleral interface. We showed that cautery to the muscle
and sclera increases the area of the subsequent granuloma at the site
of muscle reattachment. This provides a new animal model for measuring
that inflammation.
Previous studies using similar animal models have emphasized the
clinical effects of this antimetabolic,2
3
7
which were
evaluated indirectly based on tensile strength or directly by
dissection exploration of postoperative adhesions. The general
histologic characteristics described for the muscle insertion sites
agreed with the clinical findings in these investigations, although the
histologic analysis was only qualitative with no statistical
evaluation.
In the present study, an initial qualitative histologic analysis 7
weeks after surgery unexpectedly revealed a more prominent
granulomatous inflammation in the MMC-treated eyes of a few animals,
compared with the controls (Fig. 1)
. However, there was no significant
difference between areas of granulomas in treated and control eyes.
This finding highlights the importance of using a sensitive histologic
method for evaluating the tissue reaction.
Although there were no prominent signals of fibrosis present in the
examined sections, fibrosis is a common and important complication in
granulomatous inflammation.8
This suggests that the amount
of resultant fibrosis would be proportional to the severity of the
previous inflammation.
Thus, contrary to other reports,2
3
7
and according to
this new animal model for measuring granulomatous inflammation, our
data do not support the hypothesis that MMC would be effective in
reducing postoperative scarring after strabismus procedure in rabbits.
However, this study could not offer conclusions about other
inflammatory interactions. The scar tissue of strabismus surgery
involves conjunctiva, Tenon’s fascia, muscle-tendon, sclera, orbital
fat, and probably other factors.
Clearly, more animal studies are needed to investigate other aspects of
tissue adherence and to establish or refute the usefulness of MMC in
extraocular muscle surgery.
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Acknowledgements
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The authors thank everyone in the Faculty of Medical Science,
particularly from the departments of Experimental Surgery, Anatomic
Pathology, and Statistics, for their contributions.
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Footnotes
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Supported by Grant 95/6987-1 from Fundação de Amparo a Pesquisa do Estado de São Paulo (MdeC).
Submitted for publication May 26, 1999; revised February 22, 2000; accepted March 8, 2000.
Commercial relationships policy: N.
Corresponding author: Nilza Minguini, Departamento de Oftalmologia, FCM/UNICAMP, Cidade Universitária Zeferino Vaz, Campinas, SP, Caixa Postal 6111, 13084-970, Campinas, SP, Brazil. minguini{at}uol.com.br
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References
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Brooks, SE, Ribeiro, GB, Archer, SM, Elner, VM, Del Monte, MA (1996) Fat adherence syndrome treated with intraoperative mitomycin-C: a rabbit model J Pediatr Ophthalmol Strabismus 33,21-27[Medline][Order article via Infotrieve]
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Abstract
Introduction
