January 2006
Volume 47, Issue 1
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Cornea  |   January 2006
Botulinum Toxin B-Induced Mouse Model of Keratoconjunctivitis Sicca
Author Affiliations
  • Olan Suwan-apichon
    From the Wilmer Ophthalmological Institute, Johns Hopkins University, Baltimore, Maryland; the
    Department of Ophthalmology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; and the
  • Michael Rizen
    From the Wilmer Ophthalmological Institute, Johns Hopkins University, Baltimore, Maryland; the
  • Ram Rangsin
    Department of Military and Community Medicine, Phramongkutklao College of Medicine, Bangkok, Thailand.
  • Samantha Herretes
    From the Wilmer Ophthalmological Institute, Johns Hopkins University, Baltimore, Maryland; the
  • Johann M. G. Reyes
    From the Wilmer Ophthalmological Institute, Johns Hopkins University, Baltimore, Maryland; the
  • Kaevalin Lekhanont
    From the Wilmer Ophthalmological Institute, Johns Hopkins University, Baltimore, Maryland; the
  • Roy S. Chuck
    From the Wilmer Ophthalmological Institute, Johns Hopkins University, Baltimore, Maryland; the
Investigative Ophthalmology & Visual Science January 2006, Vol.47, 133-139. doi:10.1167/iovs.05-0380
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      Olan Suwan-apichon, Michael Rizen, Ram Rangsin, Samantha Herretes, Johann M. G. Reyes, Kaevalin Lekhanont, Roy S. Chuck; Botulinum Toxin B-Induced Mouse Model of Keratoconjunctivitis Sicca. Invest. Ophthalmol. Vis. Sci. 2006;47(1):133-139. doi: 10.1167/iovs.05-0380.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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

methods. Under direct visualization with an operating microscope, CBA/J mice received a transconjunctival injection of saline or 1.25, 5, or 20 milliunits (mU) of botulinum toxin B (BTX-B) into the lacrimal gland. The mice were either left unstressed or were subjected to an air blower for 5 h/d, 5 d/wk in fixed temperature and humidity conditions. Tear production and corneal fluorescein staining were evaluated in all groups before injection and at several time points after. Tear production was measured with phenol red–impregnated cotton threads. Corneal fluorescein staining was photographed under cobalt blue light with a digital camera fitted with a macro lens.

results. BTX-B-injected mice displayed significantly decreased tear production until the 4-week time point. Throughout all time points, the addition of environmental blower stress did not appear to alter tear production significantly. Linear regression models, used to evaluate the effects of various doses of BTX-B on tear production, showed that doses higher than 1.25 mU did not provide significantly different outcomes. After 3 days, saline-injected mice showed no corneal staining, whereas BTX-B-injected mice displayed various amounts of staining. At the early time point (day 3), there did not appear to be an additional effect of the blower on corneal fluorescein staining. However, at 1, 2, and 4 weeks, the blower stress appeared to increase the amount of corneal fluorescein staining at each BTX-B dose, although not significantly. Furthermore, at 8 to 10 weeks, in the BTX B-injected groups, corneas had persistent staining, even though tear production had already returned to normal levels. Histopathologic analyses revealed no inflammatory cell infiltration of the stroma or acini of the lacrimal glands and conjunctivae of both saline-injected and BTX-B-injected animals.

conclusions. Intralacrimal gland injection of BTX-B resulted in persistent corneal fluorescein staining within 3 days, and a significant decrease in aqueous tear production that persisted for 1 month. Intralacrimal gland injection of BTX-B suppressed lacrimation, thereby establishing a dry eye state. This animal model could be a useful tool for investigating the pathogenesis of the chronic condition KCS in humans.

Dry eye is a significant public health problem with 14.4% of the U.S. population reporting symptoms. 1 There is increasing appreciation that the ocular surface and the lacrimal gland are linked via a neuroendocrine mechanism that maintains the health of the ocular surface. 2 3 4 The lacrimal gland interacts with the ocular surface through sensory and secretomotor pathways and lymphocyte trafficking throughout the mucosal immune system. A better understanding of these relationships should help unravel the biology underlying the signs and symptoms of common dry eye disease. Our current lack of understanding makes the diagnosis of dry eye difficult and poses a significant impediment to epidemiologic and interventional studies. Progress in this area should make it possible to characterize, diagnose, and treat dry eye conditions more effectively. 
Much effort and resources have been expended to develop a reliable laboratory model for advanced human dry eye ocular surface disease, or keratoconjunctivitis sicca (KCS). Although some success has been achieved with animal modeling, a reliable and chronic model of the human condition has yet to be reported. 5 6 7 8 9 The currently available models have from problems of short duration of disease, associated systemic side effects, surgical difficulty, and incomplete progression to KCS, despite marked aqueous tear deficiency. 
Previous studies have demonstrated that pharmacologic blockade of cholinergic muscarinic receptors in mouse lacrimal glands with topical atropine sulfate or systemic transdermal or subcutaneous scopolamine coupled with environmental stress can decrease tear production and cause dry eye, but the single-dose effect lasts only from a few hours to 2 days. 6 9 Botulinum toxins (BTXs) are well known and widely used blockers of acetylcholine release in neuromuscular and cholinergic nerve junctions. There is increasing evidence that botulinum toxin induces a localized clinical dry eye state free of systemic side effects when injected periorbitally. 10 11 12 The effect usually lasts 3 to 4 months. In fact, therapeutic intraglandular injection of BTX-A in humans is known to suppress lacrimation for 4 to 5 months. 13 14 15 16 17 Even though BTX-A is more widely used, BTX-B injections were recently proposed in clinical practice, with quicker onset of action and greater diffusion than BTX-A. 18 19 Commercial BTX-B (Myobloc; Elan Pharmaceuticals, South San Francisco, CA) has an acidic pH of 5.6. It is this characteristic that stabilizes the solution, avoiding the requirement for reconstitution and providing a prolonged shelf life without loss of potency. 
We proposed that similar injection of BTX-B into the mouse lacrimal gland may provide a reliable laboratory animal model of chronic KCS. We chose to use BTX-B rather than BTX-A, because BTX-B may be more diffusible when injected into the large mouse lacrimal gland. Herein, we report a novel long-term dry eye model using inbred mice and intralacrimal gland injection BTX-B. Moreover, our new mouse model provides a genetically pure substrate that can be further environmentally and genetically manipulated for future study. 
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 of an air blower; (3) injected with 1.25 mU BTX-B; (4) injected with 5 mU BTX-B; (5) injected with 20 mU BTX-B; (6) injected with 1.25 mU BTX-B and placed in the path of an air blower; (7) injected with 5 mU BTX-B and placed in the path of an air blower; and (8) injected with 20 mU BTX-B and placed in the path of an air blower. Therefore, there were eight mice in each BTX-B-treated group and the control group (four with environmental stress and four without) and 16 mice in each of the unstressed and stressed groups. 
All mice were anesthetized with ketamine and xylazine (45 and 4.5 mg/kg, respectively) and handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Under an operating microscope, all mice in each group were injected with either saline or BTX-B unilaterally through the conjunctiva into the lacrimal gland of the left eye, with a custom made 33-gauge needle (Hamilton, Reno, NV). To induce environmental stress, some mice were placed in the path of an air blower. Corneal fluorescein staining was evaluated in all experimental groups before treatment, and then 3 days and 1, 2, 4, 8, and 10 weeks after treatment. Tear production without systemic or topical anesthesia was measured at the same time points. 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% of female 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 a starting point, assuming this 1:1 BTX-A:B dose equivalence in the mouse, 18 we used the accepted transcutaneous intralacrimal human dose 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) × 20 U = 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 pH of 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 is quite large relative to the other orbital structures. The lacrimal gland is known to be wrapped around the Harderian gland, a structure of little-known function. With the operating microscope, the anterior portion of the lacrimal gland can be easily seen under the conjunctiva in the superior fornix after the globe is retracted downward (Fig. 1) . Thus, localization and injection of BTX into the gland through the transconjunctival route is feasible. Diffusion may aid the spread of the injected BTX-B throughout the 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 the path of an air blower (Lasko Products, West Chester, PA) with a flow rate of 400 ft/min for 5 h/d, 5 d/wk. 6 All animals were maintained under 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-impregnated cotton threads (Zone-Quick; Oasis, Glendora, CA). The threads were applied to the ocular surface in the lateral canthus for 15 seconds in the unanesthetized mouse. Wetting of the thread was measured in millimeters, 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 10 minutes after topical application of 1 μL of 1% sodium fluorescein (Sigma-Aldrich, St. Louis, MO) to the unanesthetized mouse eye and photographed with a digital camera (Nikon) fitted with a macro lens. 
Corneal fluorescein staining was classified with a grading system that is based on area of corneal staining. 20 The total area of punctate staining was designated as grade 0 when there was no punctate staining, 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 3 when equal to or less than one half was stained, and grade 4 when greater than one half or the entire area was stained. Corneas representative of each staining grade are shown in Figure 3
Lacrimal Gland Histology
Under the operating microscope, after death, the lacrimal gland was surgically excised and fixed immediately in 36.5% formaldehyde and embedded in paraffin. Six- to 8-μm-thick sections were stained with hematoxylin and eosin, or with the periodic acid-Schiff (PAS) reagent. 
Statistical Analyses
Means and standard deviations of tear production at baseline and each time point from each group of mice were calculated and compared between each BTX-B-treated group and the control group using the Wilcoxon signed rank test. The Mann-Whitney test was used to compare the tear production among groups of mice, with and without blower stress. Linear regression modeling was also used to evaluate the dose-dependent effect of BTX-B on tear production. Data analyses were performed on computer (SPSS ver. 11.5; SPSS, Chicago, IL). 
Results
There were no obvious oculomotor or lid palsies noted in this experimental series. After day 3, saline-injected mice showed no corneal staining, whereas BTX-B-injected mice showed various amounts of corneal fluorescein staining with an apparent dose–response, as shown in Figures 4 and 5 . The mean tear production was also reduced at this time point in all BTX-B-injected mice. The reduction in tear production persisted for 4 weeks in all treatment groups after BTX-B injection, as shown in Table 1 . However, when the staining changes at each time point were formally graded, no statistically significant differences in dose–response, with or without blower stress, were detected at any time (Fig. 6) . Increases in corneal staining score were sustained for at least 8 weeks, even though tear production had already returned to normal levels. 
To determine further the effects of BTX-B, a nonparametric test for related samples, we used the Wilcoxon signed rank test, to compare the differences in tear production between baseline and each time point among each BTX-B-treated group and the control group. We found that all animals treated with BTX-B had a statistically significant reduction in tear production at all time points up to 2 weeks (P < 0.05), compared with the saline-injected animals. At the 4-week time point, even though the reduction in tear production persisted, there were no statistically significant decreases in tear production in the 5- and 20-mU BTX-B injection groups when compared with the control group at this time point (Table 1)
At 8 weeks, as the effect of the drug waned, the mean tear production in all treatment groups increased above baseline, gradually returning to normal 2 weeks later (Fig. 7)
Using linear regression models to evaluate the effects of various doses of BTX-B on tear production, we found that the effects of doses higher than 1.25 mU did not provide outcomes significantly different from that of the 1.25-mU dose (data not shown). 
Throughout all time points, the addition of environmental blower stress under these fairly fixed temperature and humidity conditions did not appear to alter tear production or the corneal staining score significantly, as shown in Tables 2 and 3
In this model, despite no significant differences in formal grading, corneal staining secondary to lacrimal insufficiency and, separately, environmental stress could be differentiated by location and density. As exemplified in Figure 8 , at 2 weeks the 1.25-mU BTX-B dose group without blower stress had discrete corneal staining confined to an area in and above the superior portion of the pupillary zone. When environmental blower stress was added, however, there was obvious extension of the surface damage to include the entire pupil, with significant extension nasally in the exposure zone. There was obvious overlap within the interpalpebral space. 
Histopathologic analysis of the lacrimal glands and conjunctiva was similar in all groups. There was no lymphocytic or inflammatory cell infiltration of the stroma or acini of the lacrimal gland, or of the conjunctiva. Thus, no difference between saline- and BTX-B-injected animals, with or without the induction of environmental stress was observed (Fig. 9)
Discussion
Botulinum toxin acts to block the release of acetylcholine in neuromuscular and cholinergic nerve junctions, including sweat and lacrimal glands, by inhibition of fusion of neurotransmitter vesicles with presynaptic membranes. Lacrimal acinar cells also use a vesicle fusion mechanism similar to that in nerve endings. There are currently two approved forms of BTX available for clinical use in the United States. Serotype A (BTX-A) acts specifically on SNA (synaptosomal-associated protein of 25 kDa) and has been found to cleave proteins (e.g., the v-Snare protein, VAMP2) that are necessary for the docking of secretory 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 the potency difference between BTX-A and BTX-B in mice is not as great as in humans. 24 Moreover, it is known that BTX-B produces a greater area of diffusion and a more rapid onset of action than does BTX-A. 18 19  
In our study, lacrimal gland injection of BTX-B resulted in ocular surface changes (corneal fluorescein staining) and significant inhibition of tear production in as early as 3 days. This inhibitory effect persisted for at least 2 to 4 weeks. At the 8-week time point, an overproduction of tears was noted. It is possible that the refractory, inactive state of the paralyzed gland caused accumulation of glandular secretions. As the effect of the drug began to wear off, these inspissated secretions were released, thus leading to increased tear production. However, after this short rebound phenomenon, values gradually returned to normal 2 weeks later (10-week time point). 
In this study, we found no significant differences in tear production between 1.25 mU of BTX-B injection and higher doses, although the corneal fluorescein staining seemed to be dose dependent. It may be that our study sample failed to demonstrate significant differences between these groups with regard to tear production and corneal fluorescein staining, due to small sample size. It may also be that a dose of 1.25 mU is already at or near saturation in this experimental system. A final possibility is that although no gross oculomotor or lid palsies were observed, subclinical BTX-B dose-dependent effects may have been present, resulting in the observed differential ocular surface staining. 
When environmental blower stress was added, there was no significant additional effect on tear production and formal gross grading of corneal fluorescein staining. However, with respect to corneal fluorescein staining, on careful inspection, there was an apparent extension of the staining to involve more of the interpalpebral exposure zone. Thus, environmental stress caused by the blower led to further drying of the exposed areas of the ocular surface. 
The patterns seen in this study appeared to mimic closely those observed clinically in humans with KCS. Ultimately, we hope to develop a grading system based on location and density of corneal staining that will help establish the degree of KCS and identify the different staining patterns from different mechanisms, such as aqueous deficiency versus environmental induction. 
Somewhat surprisingly, histopathologic examination of our experimental BTX-B-injected mouse lacrimal glands and conjunctivae revealed no inflammation in this model of nonautoimmune dry eye disease. These findings suggest that the procedure, in itself, does not cause significant mechanical trauma and that this model is different from other models in which lacrimal gland inflammation is observed. 5  
Thus, in our study, we found that intraglandular injection of BTX-B induces a localized clinical dry eye state, free of other ocular or systemic side effects. This animal model, which may mimic human non-Sjögren’s disease, could be a useful tool for investigating the pathogenesis of the chronic human condition KCS. 
We believe that the similarities between this mouse model of dry eye and human chronic KCS will be valuable in the near future. Besides contributing to our understanding of KCS, this model may also allow efficient high-throughput preclinical screening of dry eye therapeutics, as well as corneal penetration of other ocular drugs in various states of surface disease. 
 
Figure 1.
 
With the operating microscope, the anterior portion of the lacrimal gland can be seen under the conjunctiva in the superior fornix after the globe was retracted downward.
Figure 1.
 
With the operating microscope, the anterior portion of the lacrimal gland can be seen under the conjunctiva in the superior fornix after the globe was retracted downward.
Figure 2.
 
Phenol red–impregnated cotton threads were used to measure aqueous tear production.
Figure 2.
 
Phenol red–impregnated cotton threads were used to measure aqueous tear production.
Figure 3.
 
Corneal fluorescein stain grading scale from 0, no punctate staining to 4, staining greater than one half the area of the cornea.
Figure 3.
 
Corneal fluorescein stain grading scale from 0, no punctate staining to 4, staining greater than one half the area of the cornea.
Figure 4.
 
Mice without environmental blower stress. (a) Postinjection day 3; (b) week 1; (c) week 2; (d) week 4. (i–iv) Saline injection and 1.25-, 5, and 20-mU BTX-B intralacrimal gland injection, respectively.
Figure 4.
 
Mice without environmental blower stress. (a) Postinjection day 3; (b) week 1; (c) week 2; (d) week 4. (i–iv) Saline injection and 1.25-, 5, and 20-mU BTX-B intralacrimal gland injection, respectively.
Figure 5.
 
Mice subjected to environmental blower stress. (a) Postinjection day 3; (b) week 1; (c) week 2; (d) week 4. (i-iv) Saline injection and 1.25, 5, and 20 mU BTX-B intralacrimal gland injection, respectively.
Figure 5.
 
Mice subjected to environmental blower stress. (a) Postinjection day 3; (b) week 1; (c) week 2; (d) week 4. (i-iv) Saline injection and 1.25, 5, and 20 mU BTX-B intralacrimal gland injection, respectively.
Table 1.
 
Tear Production in Millimeters of Cotton Thread Wet at Each Time Point with Different Doses of BTX-B
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)
Figure 6.
 
Corneal fluorescein staining scores in the (top) nonblower and (bottom) blower groups after intralacrimal gland injection over time.
Figure 6.
 
Corneal fluorescein staining scores in the (top) nonblower and (bottom) blower groups after intralacrimal gland injection over time.
Figure 7.
 
Mean tear production in all mice.
Figure 7.
 
Mean tear production in all mice.
Table 2.
 
Effects of Environmental Blower Stress on Tear Production from Baseline of Tear Production by Follow-up Times
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 ± 0.79
Yes 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 ± 0.69
P 0.619 0.718 0.263 0.276 0.760 0.452 0.519
Table 3.
 
Effects of Environmental Blower Stress on Corneal Staining Score from Baseline by Follow-up Times
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 ± 1.16
Yes 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 ± 0.90
P 1.000 0.926 0.564 0.094 0.867 0.361 0.932
Figure 8.
 
Two-week time point in the saline injected (left) and the 1.25-mU BTX-B-injected (right) groups, without (a) or with (b) environmental blower stress.
Figure 8.
 
Two-week time point in the saline injected (left) and the 1.25-mU BTX-B-injected (right) groups, without (a) or with (b) environmental blower stress.
Figure 9.
 
Histopathologic examination of the lacrimal gland after 10 weeks in (a) sham- and (b) 1.25-mU BTX-B- injected mouse groups.
Figure 9.
 
Histopathologic examination of the lacrimal gland after 10 weeks in (a) sham- and (b) 1.25-mU BTX-B- injected mouse groups.
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Figure 1.
 
With the operating microscope, the anterior portion of the lacrimal gland can be seen under the conjunctiva in the superior fornix after the globe was retracted downward.
Figure 1.
 
With the operating microscope, the anterior portion of the lacrimal gland can be seen under the conjunctiva in the superior fornix after the globe was retracted downward.
Figure 2.
 
Phenol red–impregnated cotton threads were used to measure aqueous tear production.
Figure 2.
 
Phenol red–impregnated cotton threads were used to measure aqueous tear production.
Figure 3.
 
Corneal fluorescein stain grading scale from 0, no punctate staining to 4, staining greater than one half the area of the cornea.
Figure 3.
 
Corneal fluorescein stain grading scale from 0, no punctate staining to 4, staining greater than one half the area of the cornea.
Figure 4.
 
Mice without environmental blower stress. (a) Postinjection day 3; (b) week 1; (c) week 2; (d) week 4. (i–iv) Saline injection and 1.25-, 5, and 20-mU BTX-B intralacrimal gland injection, respectively.
Figure 4.
 
Mice without environmental blower stress. (a) Postinjection day 3; (b) week 1; (c) week 2; (d) week 4. (i–iv) Saline injection and 1.25-, 5, and 20-mU BTX-B intralacrimal gland injection, respectively.
Figure 5.
 
Mice subjected to environmental blower stress. (a) Postinjection day 3; (b) week 1; (c) week 2; (d) week 4. (i-iv) Saline injection and 1.25, 5, and 20 mU BTX-B intralacrimal gland injection, respectively.
Figure 5.
 
Mice subjected to environmental blower stress. (a) Postinjection day 3; (b) week 1; (c) week 2; (d) week 4. (i-iv) Saline injection and 1.25, 5, and 20 mU BTX-B intralacrimal gland injection, respectively.
Figure 6.
 
Corneal fluorescein staining scores in the (top) nonblower and (bottom) blower groups after intralacrimal gland injection over time.
Figure 6.
 
Corneal fluorescein staining scores in the (top) nonblower and (bottom) blower groups after intralacrimal gland injection over time.
Figure 7.
 
Mean tear production in all mice.
Figure 7.
 
Mean tear production in all mice.
Figure 8.
 
Two-week time point in the saline injected (left) and the 1.25-mU BTX-B-injected (right) groups, without (a) or with (b) environmental blower stress.
Figure 8.
 
Two-week time point in the saline injected (left) and the 1.25-mU BTX-B-injected (right) groups, without (a) or with (b) environmental blower stress.
Figure 9.
 
Histopathologic examination of the lacrimal gland after 10 weeks in (a) sham- and (b) 1.25-mU BTX-B- injected mouse groups.
Figure 9.
 
Histopathologic examination of the lacrimal gland after 10 weeks in (a) sham- and (b) 1.25-mU BTX-B- injected mouse groups.
Table 1.
 
Tear Production in Millimeters of Cotton Thread Wet at Each Time Point with Different Doses of BTX-B
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)
Table 2.
 
Effects of Environmental Blower Stress on Tear Production from Baseline of Tear Production by Follow-up Times
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 ± 0.79
Yes 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 ± 0.69
P 0.619 0.718 0.263 0.276 0.760 0.452 0.519
Table 3.
 
Effects of Environmental Blower Stress on Corneal Staining Score from Baseline by Follow-up Times
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 ± 1.16
Yes 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 ± 0.90
P 1.000 0.926 0.564 0.094 0.867 0.361 0.932
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