Download Evaluation of the Efficacy and Safety of Botulinum Toxin

ORIGINAL ARTICLE
JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS
Volume 00, Number 00, 2012
ª Mary Ann Liebert, Inc.
DOI: 10.1089/jop.2012.0119
Evaluation of the Efficacy and Safety of Botulinum Toxin
Type A to Induce Temporary Ptosis in Dogs
Maura Krähembühl Wanderley Bittencourt,1 José Paulo Cabral de Vasconcellos,1
Matheus Domingues Bittencourt,2 Rodolfo Malagó,3 and Marianna Bacellar 4
Abstract
Purpose: To verify the safety and efficacy of botulinum toxin type A (BoNT/A) to promote protective ptosis in
dogs.
Methods: In this prospective interventional study, a total of 10 dogs underwent transcutaneous anterior chemodenervation of levator palpebral superioris with 15 U of BoNT/A. The systemic changes, ocular mobility,
visual function, intraocular pressure (IOP), tear production, and the onset, degree, and duration of ptosis were
evaluated on a daily basis during the first 7 days and on days 14, 21, and 28 after application.
Results: The onset of the clinical effect was observed between 2 and 3 days after application of the toxin; the time
taken for maximum ptosis to develop varied from 4 to 7 days (mean 5 days) and the average duration of the
toxin effect was 21 days. The mean percentage reduction in palpebral fissure height was 42,859% (SD – 35,714%–
59,821%). There was not a statistically significant difference in IOP before and after the BoNT/A application
(P = 0.974), or lacrimal production evaluation (P = 0.276). There was no change in ocular mobility and no other
adverse effect was observed in association with the administration of the study drug.
Conclusion: The application of BoNT/A into the levator palpebral superioris muscle in dogs was effective and
safe to promote protective ptosis with a temporary covering of the cornea.
efficacy, and safety related to the local use of BoNT/A, even
in cases of prolonged use.8
The therapeutic use of BoNT/A in ophthalmology is
mainly for the treatment of essential blepharospasm, strabismus correction, chronic dry eye syndrome, congenital
nystagmus, facial paralysis, lacrimal hypersecretion syndromes, eyelid retraction, entropion, and for the production
of protective ptosis.1,2,9 In this last case, BoNT/A when applied into the levator palpebral superioris muscle promotes
ptosis with a temporary covering of the cornea, thus producing the same protective effect of some routine surgical
procedures such as tarsorrhaphy.10–14
The only published study describing the use of BoNT/A
in veterinary ophthalmology refers to a case in which the
toxin was successfully used during 3 years for the treatment
of a possible essential blepharospasm in a dog.15
Therefore, the objective of this work was to verify the
ability of BoNT/A to promote protective ptosis in dogs.
Qualitative and quantitative evaluations of ptosis were obtained after applying the drug into the levator palpebral
Introduction
B
otulinum toxin, the most potent biological toxin, is an
exotoxin produced by the sporulating anaerobic Grampositive organism Clostridium botulinum.1,2 This toxin can
lead to a high rate of mortality when large doses are ingested
through contaminated food.1,3,4 In 1817, the German physician Justinus Kerner published the pioneering report on
botulinum toxin, suggesting its potential use for therapeutic
purposes.2,5,6 However, the first publication demonstrating
the clinical use of botulinum toxin type A (BoNT/A) occurred in 1980 by Alan Scott, who described its use in eye
muscles to correct strabismus.5,7 Since this work, the research
on the therapeutic use of BoNT/A has expanded rapidly,
and currently several disorders characterized by excessive,
abnormal, or inappropriate muscle contraction are being
treated with BoNT/A in different areas of medicine, such as
gastroenterology, orthopedics, otolaryngology, dermatology,
and ophthalmology.1,2 According to Naumann et al., results
of the clinical studies and trials had shown excellent tolerance,
1
Department
Department
Department
4
Department
2
3
of
of
of
of
Ophthalmology, School of Medicine, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
Ophthalmology, Beneficência Portuguesa Hospital, São Paulo, Brazil.
Veterinary Clinical Medicine, Itajubá Veterinary College (FEPI), Itajubá, Minas Gerais, Brazil.
Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan.
1
2
BITTENCOURT ET AL.
superioris muscle through the eyelid. Clinical parameters
that were analyzed included the drug’s initial action, level,
and time of ptosis. Toxin influences on both intraocular
pressure (IOP) and tear production, and occurrence of side
effects were also evaluated.
Materials and Methods
This prospective interventional study was approved by
the Ethics Research Committee from the Biology Institute at
University of Campinas. The experiments were performed at
the Veterinary College Hospital in Itajubá (FEPI) according
to the Association for Research in Vision and Ophthalmology’s statement for the use of animals in ophthalmic and
vision research. The study involved 10 dogs, 5 males and 5
females, which met the eligibility criteria below.
Inclusion criteria were predefined as follows: Adult dogs,
aged from 4 to 8 years, weighing between 8 and 25 kg, males
and/or females, without clinical or ophthalmic disease. The
exclusion criteria were as follows: pregnant females, dogs
with a history of anaphylactic reactions, current bleeding
disorders, systemic and/or ocular diseases, such as entropion
or ectropion of the upper eyelid, the presence of infection or
inflammation at the proposed injection site, and scarring in
the region of the levator palpebral superioris muscle. Animals
that were receiving any medication such as aminoglycosides,
calcium channel blocking drugs, anticholinergic muscle
relaxants, or were having a known hypersensitivity to any
ingredient in the formulation were also excluded. It was decided to terminate the study if any serious adverse reactions
occurred that could compromise the visual function permanently and/or endanger the animal’s life.
A comprehensive physical and ophthalmologic evaluation
and photographic documentation was performed before and
after the implementation of BoNT/A. All examinations and
data acquisition were executed by the same researcher. The
ocular parameters evaluated included the degree of upper
eyelid ptosis, the amount of tear production (Schirmer tear
test Schering Plough Animal Health, Union, NJ), the mean of
3 readings of IOP (Tonopen Vet, Reicher, Inc., Depew, NY),
and ocular mobility evaluation. Visual function, systemic
changes, and photographic documentation of all the animals
were carefully evaluated on a daily basis during the first
7 days and on days 14, 21, and 28 after BoNT/A application.
The lyophilized BoNT/A Prosigne (Lanzhou Institute of
Biological products, Lanzhou-Gansu, China, Lot 20100902),
in a 100 U vial, maintained at a refrigeration temperature
between 2 and 8 degrees, had been diluted prior to use with
2 mL of sterile 0.9% saline solution according to the manufacturer’s recommendations to obtain a 5-unit concentration
in a volume of 0.1 mL.
The dogs received, after disinfecting the site of application
with iodine solution, a dose of 15 UI diluted in a total volume
of 0.3 mL, which was applied in the region of the levator
palpebral superioris muscle through the external face of the
left upper eyelid. The application was made with a 1 mL
sterile syringe with a 27 G needle, placed near the anterior
orbital roof slightly behind the superior orbital rim in the
mid-pupillary plane (Fig. 1). No anesthetic was required, and
the animals were not subjected to any restriction of movement after application.
Palpebral fissure length (AB) (Fig. 2) was defined as the
measure of the horizontal distance between the medial and
FIG. 1.
point.
An example of botulinum toxin type A injection
temporal canthus, the measure of the eyelid opening (CD)
(Fig. 2) was considered as a major distance between the
upper and lower lid edge in the central region as if a line was
drawn perpendicular to the 2 farthest edges from the center
of the upper and lower eyelids.
The photographic documentation of all the animals was
performed by digital photos that were taken in macro function with the Sony H50 camera at a distance of *5 cm from
the eye of each animal (Fig. 3). The photographs were processed through the public domain NIH program 1.55 (written
by Wayne Rasband at the U.S. National Institutes of Health
and available on the Internet by anonymous ftp from zippy
.nimh.nih.gov). After contrast enhancement, points were
placed along the upper and lower eyelid contours, delineating the palpebral fissure area and setting the eyelid margin
distances (AB and CD measures). Qualitative evaluation was
also performed and ptosis was considered satisfactory when
corneal coverage was equal to or greater than 50%, incomplete when a recovery between 25% and 49% was observed,
and unsatisfactory when less than 25% of the corneal surface
was covered. The researcher was masked for the sequence of
the photographs during the palpebral measures.
To evaluate the feasibility of BoNT/A to induce temporary ptosis for corneal protection, the variables followed in
this study were the onset and duration of clinical effect of
BoNT/A, time for development of maximum ptosis, the
mean percentage reduction in palpebral fissure height, lacrimal production, and IOP variations. The statistical analysis
was performed by the Holm–Sidak method using the software sigma plot and all P values less than 0.05 were considered significant.
FIG. 2. Palpebral fissure length (AB distance) and measure
of the eyelid opening (CD distance).
USE OF BOTULINUM TOXIN TO INDUCE PTOSIS IN DOGS
FIG. 3.
3
Photograph documentation showing the evolution of ptosis degree of dog 4 at day 1, 3, 7, and 28 respectively.
Results
Discussion
The average weight of the animals used in our study was
14 kilograms (range 8.7 to 25 kilos). A total of 10 eyes
underwent transcutaneous anterior chemodenervation of
levator palpebral superioris muscle with a 15 U dose of
BoNT/A injection in the left eyelid. The onset of clinical
effects was observed between 2 and 3 days after application
of the toxin. The time taken for maximum ptosis to develop
varied from 4 to 7 days (mean 5 days). The average duration
of BoNT/A effect was 21 days. The mean percentage
reduction in palpebral fissure height was 42,859% (SD –
35,714%–59,821%) (Table 1). The palpebral fissure height
had gradually returned from the seventh day and in 3 dogs
it was stabilized at pretreatment level on the 28th day. On
the last day of follow-up, 7 animals still had some degree
of ptosis, although these dogs had less than 20% of eye
coverage (Fig. 4). In one animal the onset of effects was
observed after the fourth day, with maximum coverage of
27.78% on the day 6 and return to the initial size on day 15.
In our qualitative analysis, more than 50% reduction in
palpebral fissure height (suitable ptosis) was observed in 4
out of 10 eyes and reduction with a cornea recovery between
25% and 49% (incomplete ptosis) was observed in 6 of the
10 dogs.
There was no significant difference in IOP before and after
the BoNT/A application (P = 0.974); the same was noted in
the lacrimal production evaluation (P = 0.276, Kruskal–Wallis
One-Way Analysis of Variance on Ranks). There was no
change in ocular motility and no other adverse event was
observed in association with the administration of the study
drug.
Temporary protection of the cornea may be necessary
following clinical keratopathy treatment including corneal
ulceration or exposure, epithelial debridement, grid keratotomy, and after superficial keratectomy. The tarsorrhaphy
and third eyelid flap are the techniques of surgical covering
routinely used in veterinary medicine to achieve this protective function.16–18 These procedures frequently lead to
pain and discomfort, however, they increase the healing rate
and prevent the worsening of the corneal condition.11,13,16,19
The mechanism of action of protective ptosis is still unclear.
In addition to irritation reduction caused by the decrease in
eyelid movement on the cornea and less evaporation of the
lacrimal film, there is an hypothesis that tear lysozymes,
cytokines, and growth factors released by the vessels of the
tarsal conjunctiva that are close to injury may act favorably
on the repair process.11,13
This study is based on several reports that have been
published demonstrating the feasibility of the use of botulinum toxin into the levator palpebrae superioris muscle for
the production of protective ptosis in humans.10–14 Based on
these favorable results, we believe that dogs can also be
benefited from the advantage of this application in relation to
surgical procedures. The reasons are that the technique neither requires general anesthesia and surgical skills for its
execution, nor produces inflammation of the eyelids and
discomfort, daily care is not required, and principally it allows the application of topical drugs and the evaluation of
corneal healing progression.10–14
There has been a suggestion to administer BoNT/A into the
levator palpebral superioris muscle by the transconjunctival
Table 1.
The Percentage of Palpebral Fissure Reduction per Animal During the Study
Dog
Qualitative
analysis
Day 1
(%)
Day 2
(%)
Day 3
(%)
Day 4
(%)
Day 5
(%)
Day 6
(%)
Day 7
(%)
Day 8
(%)
Day 15
(%)
Day 21
(%)
Day 28
(%)
1 (M, 10.8 kg)
2 (M, 12 kg)
3 (F, 16.95 kg)
4 (M, 20.5 kg)
5 (F, 8.7 kg)
6 (F, 11 kg)
7 (F, 16.5 kg)
8 (M, 25 kg)
9 (F, 8.3 kg)
10 (M, 12.4 kg)
Incomplete
Incomplete
Incomplete
Suitable
Suitable
Incomplete
Suitable
Suitable
Incomplete
Incomplete
0
0
0
0
0
0
0
0
0
0
3.50
11.12
29.31
41.79
22.81
28.44
14.17
12.79
18.47
25.69
10.34
30.79
31.11
37.66
58.29
30.11
16.93
32.31
44.38
32.52
14.36
34.33
38.58
47.42
55.42
34.34
54.72
40.57
47.85
34.96
17.18
35.65
34.38
57.04
64.11
26.32
77.47
51.42
45.19
34.80
27.78
43.99
44.33
60.48
49.92
33.21
70.57
48.41
35.04
30.16
17.18
41.49
37.78
61.65
50.24
27.38
53.34
45.54
33.20
23.57
7.86
43.36
32.38
59.45
57.18
22.47
41.42
43.51
38.85
29.93
6.15
37.25
32.24
45.98
50.48
20.27
38.66
23.29
39.13
24.35
2.65
20.92
25.23
27.29
21.05
12.41
24.27
14.68
28.77
14.06
- 0.17
6.74
17.36
19.79
20.02
- 0.83
19.33
10.91
8.86
- 1.41
In bold a higher eyelid fissure reducing percentage.
M, male; F, female.
4
BITTENCOURT ET AL.
FIG. 4. Development and
recovery of the ptosis degree
caused by the botulinum
toxin application in levator
palpebral superioris muscle
in 10 dogs, which are represented by the squares.
route, although for this study we chose to rely on Naik and
colleagues 2008 methodology, and perform a transpalpebral
injection with anterior placement of the toxin using a halfinch needle. This study has shown that this form of application allows a higher initial dose thereby avoiding repeated
injections, and it maintains a longer duration of induced
ptosis that may also potentially reduce the chances of complications such as ocular perforation and superior rectus
underaction.10
Prosigne is a lyophilized BoNT/A form produced from
the purified crude toxin of the culture Clostridium botulinum
strain Hall and 1 mouse unit (U) is equivalent to the amount
of toxin found to kill 50% (LD50) of a mice group when injected intraperitoneally. This toxin was approved in 1996 for
commercial use in China and was approved for therapeutic
use in Brazil in 2003 (MS No. 1.0298.0317). Studies have
shown that Prosigne (Lanzhou Institute of Biological Products) and Botox (Allergan, Inc., Irvine, CA) have 1:1 dose
equivalence and have equal clinical effect, efficacy, tolerability, and safety.20,21
The dose determined for this study was not the same
Dysport (Ipsen Slough, UK/Galderma, Paris, France) dose
of 33.3 U per point of application used in the treatment of
blepharospasm in one dog15 or 24 U that has also been used
to induce protective ptosis in humans22 because there is no
bioequivalence between Dysport (Ipsen Slough, UK/Galderma, Paris, France) and Prosigne (Lanzhou Institute
of Biological Products). Reports state that a simple doseconversion factor is not applicable because units of different
serotype A toxins are not interchangeable.23,24
Considering the local action of BoNT/A, which fundamentally consists of a selective inhibition of evoked acetylcholine release at the skeletal neuromuscular junction that
result in a focal flaccid paralysis1,2,25 and anatomical muscular similarity between species, a 15 U dose was chosen for
this study, the same maximum BOTOX (Allergan, Inc.)
dose used by Naik et al.,10 due to the dose equivalence between BOTOX (Allergan, Inc.) and Prosigne (Lanzhou
Institute of Biological Products)20 and it being within the
studied species safe dose (L50).26
In humans, after applying to the specific muscle, botulinum toxin starts its activity between 1 and 3 days, however,
it is not uncommon that its effect begins up to 1–2 weeks
after application. Its total effect occurs between 2–4 weeks,
and the flaccid paralysis caused, which is dose dependent,
can have variable durations lasting 4–8 weeks on average
and gradually disappearing.1–4 Although we have seen a
similar onset of action, the degree, time of total effect, and
duration of ptosis was lower in dogs than that observed with
the same dose and technique of application in humans. The
hypothesis that dogs have a high natural resistance to the
toxin has to be investigated to provide a safe and efficient
protocol for the use of BoNT/A for therapeutic purposes in
this species.
The difference in response between animals is probably
due to some variation of the injection site. The dogs that
showed the least degree of ptosis probably did not received
the drug directly into the muscle, since it is known that an
increased paralytic effect is obtained when the application is
performed directly into the motor endplate.25,26 Further
studies with an electromyography guided application are
suggested. The dose and dilution are factors that also exert
influence in response to the toxin, since they are directly
related to the degree and duration of paralysis.27 Some authors have described the repeated application to achieve a
greater and longer lasting coating cornea,11–13 which is also
necessary to be evaluated in future studies in dogs with the
objective of obtaining a greater degree of coverage and a
more prolonged effect.
Transient diplopia and superior rectus underaction are
described as major side effects in humans submitted to this
treatment, which occurs by toxin diffusion for the extraocular muscles.10,28 The diplopia cannot be evaluated in dogs
and hypofunction was not observed in the studied animals.
Another side effect reported was a case of acute glaucoma
after application of BoNT/A for blepharospasm treatment in
a patient predisposed to glaucoma; the IOP increase in this
patient was due to mydriasis caused by the diffusion of the
toxin.29 Similarly, the diffusion of the toxin could also
influence the tear production and for this reason BoNT/A is
also used in the treatment of crocodile tears to decrease
lacrimal production.30 Studies demonstrated BoNT/A action
on glandular activity, reducing salivary31 and nasal secretion
in dogs.32 During this study no significant difference in IOP
and tear production was observed, as were any other side
effects that could contraindicate its use.
USE OF BOTULINUM TOXIN TO INDUCE PTOSIS IN DOGS
The form of computerized image analysis used in this
study was an accessible, noninvasive, and precise procedure
and could be used as a valuable clinical or research tool due
to its simple and objective measurements.33,34
Application of BoNT/A for the production of ptosis in
dogs was effective, safe, well tolerated, and easy to perform.
Care after the application was not required and restrictions
on movement were not performed because activity is important to contribute to an effective temporary ptosis.13 We
suggest further studies with different doses and forms of
applications to obtain a higher degree of coverage, and
clinical studies to assess whether this coverage brings real
benefits to the healing of injuries.
Conclusion
Based on our findings, the application of BoNT/A into the
levator palpebral superioris muscle in dogs was effective and
safe to promote protective ptosis with a temporary covering
of the cornea.
Acknowledgments
The authors thank Prof. Fernando Antônio Bretas Viana
for the technical assistance and professional revision and the
relevant facilities at the Faculdade de Medicina Veterinária
de Itajubá (FEPI) to carry out this study.
Author Disclosure Statement
This research was supported by the resources of the
Masters scholarships from CAPES. No competing financial
interests exist.
References
1. Jankovic, J. Botulinum toxin in clinical practice. J. Neurol.
Neurosurg. Psychiatry. 75:951–957, 2004.
2. Dutton, J.J., and Fowler, A.M. Botulinum toxin in ophthalmology. Surv. Ophthalmol. 52:13–31, 2007.
3. Sposito, M.M.M. Botulinic toxin type A: action mechanism.
Acta Fisiatr. 16:25–37, 2009.
4. Brin, M.F. Botulinum toxin: chemistry, pharmacology,
toxicity and immunology. Muscle Nerve. Supplement 6:S146–
S168, 1997.
5. Erbguth, F.J., and Naumann, M. Historical aspects of botulinum toxin: Justinus Kerner (1786–1862) and the ‘‘sausage
poison’’. Neurology. 53:1850–1853, 1999.
6. Erbguth, F.J. Historical notes on botulism, Clostridium botulinum, botulinun toxin, and the idea of the therapeutic use
of the toxin. Mov. Disord. 19:S2–S6, 2004.
7. Scott, A.B. Botulinun toxin injection of eye muscles to correct
strabismus. Trans. Am. Ophthalmol. Soc. 79:734–770, 1981.
8. Naumann, M. et al. Safety and efficacy of botulinum toxin
type A following log term use. Eur. J. Neurol. 13:35–40, 2006.
9. Minguini, N., et al. Surgery with intraoperative botulinum
toxin-A injection for the treatment of large-angle horizontal
strabismus: a pilot study. Clinics. 67:279–282, 2012.
10. Naik, M.N., et al. Anterior chemodenervation of levator
palpebrae superioris with botulinum toxin type-A (Botoxs)
to induce temporary ptosis for corneal protection. Eye. 22:
1132–1136, 2008.
11. Adams, G.G.W., Kirkness, C.M., and Lee, J.P. Botulinum
toxin A induced protective ptosis. Eye. 603–608, 1987.
5
12. Vleming, E.N., et al. Persistent corneal defect treated with
botulinum toxin-induced ptosis. Arch. Soc. Esp. Oftalmol.
82:547–550, 2007.
13. Fraco, M.F.E., and Fraco, M.D. An evaluation of the safety
and efficacy of botulinum toxin type A (BOTOX) when used
to produce a protective ptosis. Clin. Experiment. Ophthalmol.
29:394–399, 2001.
14. Prell, J., et al. Botulinum toxin for temporary corneal protection after surgery for vestibular schwannoma. J. Neurosurg. 114:426–431, 2011.
15. Lindenberg, A.M., Wohlfarth, K.M., and Switzer, E.N. The
use of botulinum toxin A for treatment of possible
essential blepharospasm in a dog. Aust. Vet. J. 81:612–614,
2003.
16. Bentley, E. Spontaneous chronic corneal epithelial defects
in dogs: a review. J. Am. Anim. Hosp. Assoc. 41:158–165,
2005.
17. Slatter, D. Princı́pios da cirurgia oftálmica. In: Fundamentos
de Oftalmologia Veterinária. São Paulo: Roca; 2005; p. 135–
157.
18. Stades, F.C., and Gelatt, K.N. Dieseases and surgery of
the canine eyelid. In: Gelatt, K.N., ed. Veterinary Ophthalmology. Ames, Iowa: Blackwell Publishing; 2007; Vol. 2,
p. 612–614.
19. Morgan, R.V., and Abrams, K.L. A comparation of six different therapies for persitent corneal erosion in dogs and
cats. Vet. Comp. Ophthalmol. 4:38–43, 1994.
20. Quagliato, E.M.A.B., Carelli, E.F., and Viana, M.A.
Prospective, randomized, double-blind study, comparing
botulinum toxins type A botox and prosigne for blepharospasm and hemifacial spasm treatment. Clin. Neuropharmacol. 33:27–31, 2010.
21. Tang, X., and Wan, X. Comparison of botox with a Chinese
type A botulinum toxin. Chin. Med. J. 113:794–798, 2000.
22. Reddy, U.P., and Woodward, J.A. Abobotulinum toxin A
(Dysport) and botulinum toxin type A (Botox) for purposeful induction of eyelid ptosis. Ophthal. Plast. Reconstr. Surg.
6:489–491, 2010.
23. Marchetti, A., et al. Retrospective evaluation of the dose of
Dysport and BOTOX in the management of cervical dystonia and blepharospasm: the real dose study. Mov.t Disord.
20:937–944, 2005.
24. Frevert, J. Content of botulinum neurotoxin in Botox/
Visabel, Dysport/Azzalure, and Xeomin/Bocoture.
Drugs. 10:67–73, 2010.
25. Shaari, C.M., and Sanders, I. Quantifying how location and
dose of botulinum toxin injection affect muscle paralysis.
Muscle Nerve. 16:964–969, 1993.
26. Childers, M.K., et al. Evaluatingmotor end-plate-targeted
injections of botulinum toxin type A in a canine model.
Muscle Nerve. 21:653–655, 1998.
27. Boyle, M.H., et al. High versus low concentration botulinum
toxin A for beningn essential blefarospasm: does dilution
make a difference? Ophthal. Plast. Reconstr. Surg. 25:81–84,
2009.
28. Heyworth, P.L.O., and Lee, J.P. Persisting hypotropias following protective ptosis induced by botulinum neurotoxin.
Eye. 8:511–515, 1994.
29. Corridan, P., et al. Acute angle-closure glaucoma following
botulinum toxin injection for blepharospasm. Br. J. Ophthalmol. 74:309–310, 1990.
30. Riemann, R., et al. Successful treatment of crocodile tears by
injection of botulinum toxin into the lacrimal gland. Ophthalmology. 106:2322–2324, 1999.
6
31. Shaari, C.M., et al. Botulinum toxin decreases salivation
from canine submandibular glands. Otolaryngol. Head Neck
Surg. 118:452–457, 1998.
32. Shaari, C.M., et al. Rhinorrhea is decreased in dogs after
nasal application of botulinum toxin. Otolaryngol. Head Neck
Surg. 112:566–571, 1995.
33. Cruz, A.A.V., et al. Digital image processing measurement
of the upper eyelid contour in graves disease and congenital
blepharoptosis. Ophthalmology. 105:913–918, 1998.
34. Cruz, A.A.V., and Baccega, A. Computerized bidimensional
analysis of the palpebral fissure. Arq. Bras. Oftalmol. 64:13–
19, 2001.
BITTENCOURT ET AL.
Received: August 1, 2012
Accepted: September 25, 2012
Address correspondence to:
Maura Krähembühl Wanderley Bittencourt, DVM
Department of Ophthalmology
School of Medicine
University of Campinas (UNICAMP)
CXP-6111
Campinas, São Paulo
CEP: 13083-970
Brazil
E-mail: [email protected]