botulinum toxin b-induced mouse model of keratoconjunctivitis sicca

Botulinum Toxin B-Induced Mouse Model ofKeratoconjunctivitis Sicca

Olan Suwan-apichon,1,2 Michael Rizen,1 Ram Rangsin,3 Samantha Herretes,1

Johann M. G. Reyes,1 Kaevalin Lekhanont,1 and Roy S. Chuck1

PURPOSE. To develop a mouse model of human chronic dry eye(keratoconjunctivitis sicca [KCS]).

METHODS. Under direct visualization with an operating micro-scope, CBA/J mice received a transconjunctival injection ofsaline or 1.25, 5, or 20 milliunits (mU) of botulinum toxin B(BTX-B) into the lacrimal gland. The mice were either leftunstressed or were subjected to an air blower for 5 h/d, 5 d/wkin fixed temperature and humidity conditions. Tear productionand corneal fluorescein staining were evaluated in all groupsbefore injection and at several time points after. Tear produc-tion was measured with phenol red–impregnated cottonthreads. Corneal fluorescein staining was photographed undercobalt blue light with a digital camera fitted with a macro lens.

RESULTS. BTX-B-injected mice displayed significantly decreasedtear production until the 4-week time point. Throughout alltime points, the addition of environmental blower stress didnot appear to alter tear production significantly. Linear regres-sion models, used to evaluate the effects of various doses ofBTX-B on tear production, showed that doses higher than 1.25mU did not provide significantly different outcomes. After 3days, saline-injected mice showed no corneal staining, whereasBTX-B-injected mice displayed various amounts of staining. Atthe early time point (day 3), there did not appear to be anadditional effect of the blower on corneal fluorescein staining.However, at 1, 2, and 4 weeks, the blower stress appeared toincrease the amount of corneal fluorescein staining at eachBTX-B dose, although not significantly. Furthermore, at 8 to 10weeks, in the BTX B-injected groups, corneas had persistentstaining, even though tear production had already returned tonormal levels. Histopathologic analyses revealed no inflamma-tory cell infiltration of the stroma or acini of the lacrimal glandsand conjunctivae of both saline-injected and BTX-B-injectedanimals.

CONCLUSIONS. Intralacrimal gland injection of BTX-B resulted inpersistent corneal fluorescein staining within 3 days, and asignificant decrease in aqueous tear production that persistedfor 1 month. Intralacrimal gland injection of BTX-B suppressedlacrimation, thereby establishing a dry eye state. This animalmodel could be a useful tool for investigating the pathogenesis

of the chronic condition KCS in humans. (Invest OphthalmolVis Sci. 2006;47:133–139) DOI:10.1167/iovs.05-0380

Dry eye is a significant public health problem with 14.4% ofthe U.S. population reporting symptoms.1 There is in-

creasing appreciation that the ocular surface and the lacrimalgland are linked via a neuroendocrine mechanism that main-tains the health of the ocular surface.2–4 The lacrimal glandinteracts with the ocular surface through sensory and secreto-motor pathways and lymphocyte trafficking throughout themucosal immune system. A better understanding of these re-lationships should help unravel the biology underlying thesigns and symptoms of common dry eye disease. Our currentlack of understanding makes the diagnosis of dry eye difficultand poses a significant impediment to epidemiologic and in-terventional studies. Progress in this area should make it pos-sible to characterize, diagnose, and treat dry eye conditionsmore effectively.

Much effort and resources have been expended to developa reliable laboratory model for advanced human dry eye ocularsurface disease, or keratoconjunctivitis sicca (KCS). Althoughsome success has been achieved with animal modeling, areliable and chronic model of the human condition has yet tobe reported.5–9 The currently available models have from prob-lems of short duration of disease, associated systemic sideeffects, surgical difficulty, and incomplete progression to KCS,despite marked aqueous tear deficiency.

Previous studies have demonstrated that pharmacologicblockade of cholinergic muscarinic receptors in mouse lacri-mal glands with topical atropine sulfate or systemic transder-mal or subcutaneous scopolamine coupled with environmentalstress can decrease tear production and cause dry eye, but thesingle-dose effect lasts only from a few hours to 2 days.6,9

Botulinum toxins (BTXs) are well known and widely usedblockers of acetylcholine release in neuromuscular and cholin-ergic nerve junctions. There is increasing evidence that botu-linum toxin induces a localized clinical dry eye state free ofsystemic side effects when injected periorbitally.10–12 The ef-fect usually lasts 3 to 4 months. In fact, therapeutic intraglan-dular injection of BTX-A in humans is known to suppresslacrimation for 4 to 5 months.13–17 Even though BTX-A is morewidely used, BTX-B injections were recently proposed in clin-ical practice, with quicker onset of action and greater diffusionthan BTX-A.18,19 Commercial BTX-B (Myobloc; Elan Pharma-ceuticals, South San Francisco, CA) has an acidic pH of 5.6. Itis this characteristic that stabilizes the solution, avoiding therequirement for reconstitution and providing a prolonged shelflife without loss of potency.

We proposed that similar injection of BTX-B into the mouselacrimal gland may provide a reliable laboratory animal modelof chronic KCS. We chose to use BTX-B rather than BTX-A,because BTX-B may be more diffusible when injected into thelarge mouse lacrimal gland. Herein, we report a novel long-term dry eye model using inbred mice and intralacrimal glandinjection BTX-B. Moreover, our new mouse model provides agenetically pure substrate that can be further environmentallyand genetically manipulated for future study.

From the 1Wilmer Ophthalmological Institute, Johns Hopkins Uni-versity, Baltimore, Maryland; the 2Department of Ophthalmology, Fac-ulty of Medicine, Khon Kaen University, Khon Kaen, Thailand; and the3Department of Military and Community Medicine, PhramongkutklaoCollege of Medicine, Bangkok, Thailand.

Submitted for publication March 24, 2005; revised July 2 andSeptember 5, 2005; accepted November 22, 2005.

Disclosure: O. Suwan-apichon, None, M. Rizen, None; R. Rang-sin, None; S. Herretes, None; J.M.G. Reyes, None; K. Lekhanont,None; R.S. Chuck, None

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be marked “advertise-ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Roy S. Chuck, Wilmer OphthalmologicalInstitute, Johns Hopkins University, 255 Woods Building, 600 NorthWolfe Street, Baltimore, MD 21286; [email protected].

Investigative Ophthalmology & Visual Science, January 2006, Vol. 47, No. 1Copyright © Association for Research in Vision and Ophthalmology 133

MATERIALS AND METHODS

We divided female CBA/J mice (age, 6–8 weeks; Jackson Laboratories,Bar Harbor, ME) into eight groups as follows (four mice per group): (1)injected with saline; (2) injected with saline and placed in the path ofan air blower; (3) injected with 1.25 mU BTX-B; (4) injected with 5 mUBTX-B; (5) injected with 20 mU BTX-B; (6) injected with 1.25 mUBTX-B and placed in the path of an air blower; (7) injected with 5 mUBTX-B and placed in the path of an air blower; and (8) injected with 20mU BTX-B and placed in the path of an air blower. Therefore, therewere eight mice in each BTX-B-treated group and the control group(four with environmental stress and four without) and 16 mice in eachof the unstressed and stressed groups.

All mice were anesthetized with ketamine and xylazine (45 and 4.5mg/kg, respectively) and handled in accordance with the ARVO State-ment for the Use of Animals in Ophthalmic and Vision Research. Underan operating microscope, all mice in each group were injected witheither saline or BTX-B unilaterally through the conjunctiva into thelacrimal gland of the left eye, with a custom made 33-gauge needle(Hamilton, Reno, NV). To induce environmental stress, some micewere placed in the path of an air blower. Corneal fluorescein stainingwas evaluated in all experimental groups before treatment, and then 3days and 1, 2, 4, 8, and 10 weeks after treatment. Tear productionwithout systemic or topical anesthesia was measured at the same timepoints. At the final 10-week time point, the mice were euthanatized,and the lacrimal glands surgically removed for histologic examination.

BTX-B Preparation

One unit is defined as the amount of toxin that is lethal in 50% offemale Swiss-Webster mice after intraperitoneal injection (mouse LD50

bioassay). In mice, 1 U of BTX-A appears to equal 1 U of BTX-B. As astarting point, assuming this 1:1 BTX-A:B dose equivalence in themouse,18 we used the accepted transcutaneous intralacrimal humandose of BTX-A for the treatment of gustatory hyperlacrimation (20 U)and titrated up and down per comparative unit of mouse body weight.That is, for a standard 20-unit dose of BTX-A in an adult 80-kg human,the equivalent dose in a 20-g mouse would be [(20 g/80,000 g) � 20U � 5 mU � x]. Our initial starting doses were 4�, 1�, and 0.25� (i.e.,20, 5, and 1.25 mU, respectively).

BTX-B is produced as a ready-to-use liquid formulation set at a pHof 5.6 to stabilize the complex. It is available in vials of 2,500, 5,000,and 10,000 U, each with a concentration of 5,000 U/mL.

Localization of the Mouse Lacrimal Gland

The orbital portion of the mouse gland lies in the superior orbit and isquite large relative to the other orbital structures. The lacrimal gland isknown to be wrapped around the Harderian gland, a structure of little-known function. With the operating microscope, the anterior portion ofthe lacrimal gland can be easily seen under the conjunctiva in the superiorfornix after the globe is retracted downward (Fig. 1). Thus, localizationand injection of BTX into the gland through the transconjunctival route isfeasible. Diffusion may aid the spread of the injected BTX-B throughoutthe tear-secreting portions of the orbital lobe of the lacrimal gland.

Desiccating Environmental Air Blower Stress

To induce environmental stress, some mouse cages were placed in thepath of an air blower (Lasko Products, West Chester, PA) with a flowrate of 400 ft/min for 5 h/d, 5 d/wk.6 All animals were maintainedunder fairly constant temperature and humidity conditions (70–75°F,20–35%, respectively).

Measurement of Aqueous Tear Production

Tear production was measured with standardized phenol red-impreg-nated cotton threads (Zone-Quick; Oasis, Glendora, CA). The threadswere applied to the ocular surface in the lateral canthus for 15 secondsin the unanesthetized mouse. Wetting of the thread was measured inmillimeters, according to the scale imprinted on the cotton thread (Fig.2, scale not visible).

Corneal Fluorescein Staining

Corneal fluorescein staining was evaluated under cobalt blue light 10minutes after topical application of 1 �L of 1% sodium fluorescein(Sigma-Aldrich, St. Louis, MO) to the unanesthetized mouse eye andphotographed with a digital camera (Nikon) fitted with a macro lens.

Corneal fluorescein staining was classified with a grading systemthat is based on area of corneal staining.20 The total area of punctatestaining was designated as grade 0 when there was no punctatestaining, grade 1 when equal to or less than one eighth was stained,grade 2 when equal to or less than one fourth was stained, grade 3when equal to or less than one half was stained, and grade 4 when

FIGURE 1. With the operating microscope, the anterior portion of thelacrimal gland can be seen under the conjunctiva in the superior fornixafter the globe was retracted downward.

FIGURE 2. Phenol red–impregnated cotton threads were used to mea-sure aqueous tear production.

134 Suwan-apichon et al. IOVS, January 2006, Vol. 47, No. 1

greater than one half or the entire area was stained. Corneas represen-tative of each staining grade are shown in Figure 3.

Lacrimal Gland Histology

Under the operating microscope, after death, the lacrimal gland wassurgically excised and fixed immediately in 36.5% formaldehyde and

embedded in paraffin. Six- to 8-�m-thick sections were stained withhematoxylin and eosin, or with the periodic acid-Schiff (PAS) reagent.

Statistical Analyses

Means and standard deviations of tear production at baseline and eachtime point from each group of mice were calculated and compared

FIGURE 3. Corneal fluorescein staingrading scale from 0, no punctatestaining to 4, staining greater thanone half the area of the cornea.

FIGURE 4. Mice without environ-mental blower stress. (a) Postinjec-tion day 3; (b) week 1; (c) week 2;(d) week 4. (i–iv) Saline injection and1.25-, 5, and 20-mU BTX-B intralacri-mal gland injection, respectively.

IOVS, January 2006, Vol. 47, No. 1 Mouse Model of Dry Eye 135

between each BTX-B-treated group and the control group using theWilcoxon signed rank test. The Mann-Whitney test was used to com-pare the tear production among groups of mice, with and withoutblower stress. Linear regression modeling was also used to evaluate thedose-dependent effect of BTX-B on tear production. Data analyses wereperformed on computer (SPSS ver. 11.5; SPSS, Chicago, IL).

RESULTS

There were no obvious oculomotor or lid palsies noted in thisexperimental series. After day 3, saline-injected mice showedno corneal staining, whereas BTX-B-injected mice showed var-ious amounts of corneal fluorescein staining with an apparentdose–response, as shown in Figures 4 and 5. The mean tearproduction was also reduced at this time point in all BTX-B-

injected mice. The reduction in tear production persisted for 4weeks in all treatment groups after BTX-B injection, as shownin Table 1. However, when the staining changes at each timepoint were formally graded, no statistically significant differ-ences in dose–response, with or without blower stress, weredetected at any time (Fig. 6). Increases in corneal staining scorewere sustained for at least 8 weeks, even though tear produc-tion had already returned to normal levels.

To determine further the effects of BTX-B, a nonparametrictest for related samples, we used the Wilcoxon signed ranktest, to compare the differences in tear production betweenbaseline and each time point among each BTX-B-treated groupand the control group. We found that all animals treated withBTX-B had a statistically significant reduction in tear produc-tion at all time points up to 2 weeks (P � 0.05), compared with

FIGURE 5. Mice subjected to envi-ronmental blower stress. (a) Postin-jection day 3; (b) week 1; (c) week 2;(d) week 4. (i-iv) Saline injection and1.25, 5, and 20 mU BTX-B intralacri-mal gland injection, respectively.

TABLE 1. Tear Production in Millimeters of Cotton Thread Wet at Each Time Point with Different Doses of BTX-B

BTX-B Dose(mU) Baseline Day 3 Week 1 Week 2 Week 4 Week 8 Week 10

Control** 2.63 � 1.33 2.81 � 0.75(0.391)

3.06 � 0.73(0.350)

2.78 � 0.77(0.498)

2.75 � 0.71(0.492)

3.06 � 0.79(0.035)

2.50 � 0.76(0.674)

1.25 mU 2.78 � 1.01 1.81 � 0.69(0.027)

1.38 � 0.79(0.016)

1.78 � 0.54(0.035)

1.47 � 0.49(0.035)

3.69 � 0.84(0.121)

2.46 � 0.40(0.498)

5.00 mU 2.44 � 0.68 1.84 � 0.71(0.017)

1.69 � 0.75(0.039)

1.44 � 0.48(0.011)

1.78 � 0.59(0.061)

3.56 � 0.86(0.016)

3.13 � 0.86(0.111)

20.00 mU 2.63 � 0.79 1.66 � 0.73(0.035)

1.56 � 0.42(0.017)

1.31 � 0.35(0.018)

1.72 � 0.60(0.058)

3.41 � 1.03(0.023)

3.00 � 0.79(0.414)

Data are expressed as the mean � standard deviation, with the probabilities in parentheses. Probabilities show the comparisons of each dosegroup with the baseline (control), by the Wilcoxon signed rank test. For all groups, n � 8, except at week 10 (n � 6).


the saline-injected animals. At the 4-week time point, eventhough the reduction in tear production persisted, there wereno statistically significant decreases in tear production in the 5-and 20-mU BTX-B injection groups when compared with thecontrol group at this time point (Table 1).

At 8 weeks, as the effect of the drug waned, the mean tearproduction in all treatment groups increased above baseline,gradually returning to normal 2 weeks later (Fig. 7).

Using linear regression models to evaluate the effects ofvarious doses of BTX-B on tear production, we found that theeffects of doses higher than 1.25 mU did not provide outcomessignificantly different from that of the 1.25-mU dose (data notshown).

Throughout all time points, the addition of environmentalblower stress under these fairly fixed temperature and humid-ity conditions did not appear to alter tear production or thecorneal staining score significantly, as shown in Tables 2 and 3.

In this model, despite no significant differences in formalgrading, corneal staining secondary to lacrimal insufficiencyand, separately, environmental stress could be differentiated bylocation and density. As exemplified in Figure 8, at 2 weeks the1.25-mU BTX-B dose group without blower stress had discretecorneal staining confined to an area in and above the superiorportion of the pupillary zone. When environmental blowerstress was added, however, there was obvious extension of thesurface damage to include the entire pupil, with significant

extension nasally in the exposure zone. There was obviousoverlap within the interpalpebral space.

Histopathologic analysis of the lacrimal glands and conjunc-tiva was similar in all groups. There was no lymphocytic orinflammatory cell infiltration of the stroma or acini of thelacrimal gland, or of the conjunctiva. Thus, no difference be-tween saline- and BTX-B-injected animals, with or without theinduction of environmental stress was observed (Fig. 9).

DISCUSSION

Botulinum toxin acts to block the release of acetylcholine inneuromuscular and cholinergic nerve junctions, includingsweat and lacrimal glands, by inhibition of fusion of neurotrans-mitter vesicles with presynaptic membranes. Lacrimal acinarcells also use a vesicle fusion mechanism similar to that innerve endings. There are currently two approved forms of BTXavailable for clinical use in the United States. Serotype A(BTX-A) acts specifically on SNA (synaptosomal-associated pro-tein of 25 kDa) and has been found to cleave proteins (e.g., thev-Snare protein, VAMP2) that are necessary for the docking ofsecretory vesicles to the cell membrane in lacrimal acini.21,22

Serotype B (BTX-B) cleaves synaptobrevin, which is a vesicle-associated membrane protein.23 It is also thought that thepotency difference between BTX-A and BTX-B in mice is not asgreat as in humans.24 Moreover, it is known that BTX-B pro-duces a greater area of diffusion and a more rapid onset ofaction than does BTX-A.18,19

In our study, lacrimal gland injection of BTX-B resulted inocular surface changes (corneal fluorescein staining) and sig-nificant inhibition of tear production in as early as 3 days. Thisinhibitory effect persisted for at least 2 to 4 weeks. At the8-week time point, an overproduction of tears was noted. It ispossible that the refractory, inactive state of the paralyzedgland caused accumulation of glandular secretions. As theeffect of the drug began to wear off, these inspissated secre-tions were released, thus leading to increased tear production.However, after this short rebound phenomenon, values grad-ually returned to normal 2 weeks later (10-week time point).

In this study, we found no significant differences in tearproduction between 1.25 mU of BTX-B injection and higherdoses, although the corneal fluorescein staining seemed to bedose dependent. It may be that our study sample failed todemonstrate significant differences between these groups withregard to tear production and corneal fluorescein staining, dueto small sample size. It may also be that a dose of 1.25 mU isalready at or near saturation in this experimental system. A final

FIGURE 7. Mean tear production in all mice.

FIGURE 6. Corneal fluorescein staining scores in the (top) nonblowerand (bottom) blower groups after intralacrimal gland injection overtime.


possibility is that although no gross oculomotor or lid palsieswere observed, subclinical BTX-B dose-dependent effects mayhave been present, resulting in the observed differential ocularsurface staining.

When environmental blower stress was added, there was nosignificant additional effect on tear production and formalgross grading of corneal fluorescein staining. However, withrespect to corneal fluorescein staining, on careful inspection,there was an apparent extension of the staining to involvemore of the interpalpebral exposure zone. Thus, environmen-tal stress caused by the blower led to further drying of theexposed areas of the ocular surface.

The patterns seen in this study appeared to mimic closelythose observed clinically in humans with KCS. Ultimately, we

hope to develop a grading system based on location and den-sity of corneal staining that will help establish the degree ofKCS and identify the different staining patterns from differentmechanisms, such as aqueous deficiency versus environmentalinduction.

Somewhat surprisingly, histopathologic examination of ourexperimental BTX-B-injected mouse lacrimal glands and con-junctivae revealed no inflammation in this model of nonauto-immune dry eye disease. These findings suggest that the pro-cedure, in itself, does not cause significant mechanical traumaand that this model is different from other models in whichlacrimal gland inflammation is observed.5

Thus, in our study, we found that intraglandular injection ofBTX-B induces a localized clinical dry eye state, free of other

TABLE 2. Effects of Environmental Blower Stress on Tear Production from Baseline of Tear Production by Follow-up Times

Blower Baseline Day 3 Week 1 Week 2 Week 4 Week 8 Week 10

No 2.73 � 1.11 1.94 � 0.61 2.13 � 0.79 1.91 � 0.64 1.91 � 0.55 3.56 � 0.99 2.92 � 0.79Yes 2.50 � 0.75 2.13 � 1.01 1.72 � 1.06 1.75 � 0.93 1.95 � 0.94 3.30 � 0.76 2.63 � 0.69P 0.619 0.718 0.263 0.276 0.760 0.452 0.519

Data are expressed as the mean � SD. Mann-Whitney test. n � 16 in each group, except at week 10 (n � 12).

TABLE 3. Effects of Environmental Blower Stress on Corneal Staining Score from Baseline by Follow-up Times

Blower Baseline Day 3 Week 1 Week 2 Week 4 Week 8 Week 10

No 0.00 � 0.00 1.13 � 1.08 1.18 � 0.83 1.63 � 0.89 2.00 � 1.37 2.06 � 1.34 1.42 � 1.16Yes 0.00 � 0.00 1.13 � 0.89 1.44 � 0.89 2.31 � 1.01 2.13 � 1.20 2.56 � 0.96 1.42 � 0.90P 1.000 0.926 0.564 0.094 0.867 0.361 0.932

Scores ranged from 1 to 4. Data are expressed as the mean score � SD. Mann-Whitney test. n � 16, except at week 10 (n � 12).

FIGURE 8. Two-week time point inthe saline injected (left) and the1.25-mU BTX-B-injected (right)groups, without (a) or with (b) envi-ronmental blower stress.


ocular or systemic side effects. This animal model, which maymimic human non-Sjogren’s disease, could be a useful tool forinvestigating the pathogenesis of the chronic human conditionKCS.

We believe that the similarities between this mouse modelof dry eye and human chronic KCS will be valuable in the nearfuture. Besides contributing to our understanding of KCS, thismodel may also allow efficient high-throughput preclinicalscreening of dry eye therapeutics, as well as corneal penetra-tion of other ocular drugs in various states of surface disease.

References

1. Lemp MA. Report of the National Eye Institute/Industry workshopon clinical trials in dry eyes. CLAO J. 1995;21:221–232.

2. Meneray MA, Bennett DJ, Nguyen DH, Beuerman RW. Effect ofsensory denervation on the structure and physiologic responsive-ness of rabbit lacrimal gland. Cornea. 1998;7:99–107.

3. Schimmelpfennig B, Beuerman R. A technique for controlled sen-sory denervation of the rabbit cornea. Graefes Arch Clin ExpOphthalmol. 1982;218:287–293.

4. Song XJ, Li DQ, Farley W, et al. Neurturin-deficient mice developdry eye and keratoconjunctivitis sicca. Invest Ophthalmol Vis Sci.2003;44:4223–4229.

5. Barabino S, Dana MR. Animal models of dry eye: a critical assess-ment of opportunities and limitations. Invest Ophthalmol Vis Sci.2004;45:1641–1646.

6. Dursun D, Wang M, Monroy D, et al. A mouse model of kerato-conjunctivitis sicca. Invest Ophthalmol Vis Sci. 2002;43:632–638.

7. Fujihara T, Nagano T, Nakamura M, Shirasawa E. Establishment ofa rabbit short-term dry eye model. J Ocul Pharmacol. 1995;11:503–508.

8. Jabs DA, Enger C, Prendergast RA. Murine models of Sjogren’ssyndrome. Invest Ophthalmol Vis Sci. 1991;32:371–380.

9. Strong B, Farley W, Stern ME, Pflugfelder SC. Topical cyclosporineinhibits conjunctival epithelial apoptosis in experimental murinekeratoconjunctivitis sicca. Cornea. 2005;24:80–85.

10. Dutton JJ, Buckley EG. Long-term results and complications ofbotulinum A toxin in the treatment of blepharospasm. Ophthal-mology. 1988;95:1529–1534.

11. Horwath-Winter J, Bergloeff J, Floegel I, Haller-Schober EM,Schmut O. Botulinum toxin A treatment in patients suffering fromblepharospasm and dry eye. Br J Ophthalmol. 2003;87:54–56.

12. Matarasso SL. Decreased tear expression with an abnormal Schirm-er’s test following botulinum toxin type A for the treatment oflateral canthal rhytides. Dermatol Surg. 2002;28:149–152.

13. Boroojerdi B, Ferbert A, Schwarz M, Herath H, Noth J. Botulinumtoxin treatment of synkinesia and hyperlacrimation after facialpalsy. J Neurol Neurosurg Psychiatry. 1998;65:111–114.

14. Hofmann RJ. Treatment of Frey’s syndrome (gustatory sweating)and crocodile tears’ (gustatory epiphora) with purified botulinumtoxin. Ophthalmic Plast Reconstr Surg. 2000;16:289–291.

15. Keegan DJ, Geerling G, Lee JP, Blake G, Collin JR, Plant GT.Botulinum toxin treatment for hyperlacrimation secondary to ab-errant regenerated seventh nerve palsy or salivary gland transplan-tation. Br J Ophthalmol. 2002;86:43–46.

16. Montoya FJ, Riddell CE, Caesar R, Hague S. Treatment of gustatoryhyperlacrimation (crocodile tears) with injection of botulinumtoxin into the lacrimal gland. Eye. 2002;16:705–709.

17. Riemann R, Pfennigsdorf S, Riemann E, Naumann M. Successfultreatment of crocodile tears by injection of botulinum toxin intothe lacrimal gland: a case report. Ophthalmology. 1999;106:2322–2324.

18. Matarasso SL. Comparison of botulinum toxin types A and B: abilateral and double-blind randomized evaluation in the treatmentof canthal rhytides. Dermatol Surg. 2003;29:7–13.

19. Flynn TC, Clark RE. Botulinum toxin B (Myobloc) versus botuli-num toxin A (Botox) frontalis study: rate of onset and radius ofdiffusion. Dermatol Surg. 2003;29:519–522.

20. Nakamura S, Shibuya M, Nakashima H, Imagawa T, Uehara M,Tsubota K. D-beta-hydroxybutyrate protects against corneal epi-thelial disorders in a rat dry eye model with jogging board. InvestOphthalmol Vis Sci. 2005;46:2379–23787.

21. Mense S. Neurobiological basis for the use of botulinum toxin inpain therapy. J Neuro. 2004;251:11–17.

22. Wang Y, Jerdeva G, Yarber FA, et al. Cytoplasmic dynein partici-pates in apically targeted stimulated secretory traffic in primaryrabbit lacrimal acinar epithelial cells. J Cell Sci. 2003;116:2051–2065.

23. Eleopra R, Tugnoli V, Quatrale R, Rossetto O, Montecucco C.Different types of botulinum toxin in humans. Mov Disord. 2004;19:S53–S59.

24. Aoki KR. Botulinum neurotoxin serotypes A and B preparationshave different safety margins in preclinical models of muscleweakening efficacy and systemic safety. Toxicon. 2002;40:923–928.

FIGURE 9. Histopathologic examina-tion of the lacrimal gland after 10weeks in (a) sham- and (b) 1.25-mUBTX-B- injected mouse groups.


botulinum toxin b-induced mouse model of keratoconjunctivitis sicca

Documents