Transcript Ami Casis - Marietta College

Ami Casis, Dr. Steven Spilatro, Marietta College
RESULTS
ABSTRACT
Seasonal allergic conjunctivitis is the most common form of eye allergy. Based on patient selfassessments of treatment effects, clinical studies have shown that topical treatments are more
effective than oral treatments in alleviating symptoms. This study investigated the effectiveness of
topical Visine and oral Benedryl in reducing inflammation as measured by polymorphonuclear cell
(PMN) density along the conjunctival epithelium. Previous studies have shown PMN density
correlates inversely to treatment efficacy, and in this study PMNs were predicted to be lower in mice
treated topically rather than orally. BALB/c mice were sensitized toward ragweed pollen, allergic
conjunctivitis was induced, and histology sections were examined via light microscopy. The onset
of the allergy was determined by a significant increase (p<0.05) in granulocyte density. Mice
treated with Visine and Benedryl did not show significance in decreasing cell migration from the
area of allergen exposure. Contrary to the clinical study, the oral treatment alleviated cell migration
better than the topical agent.
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INTRODUCTION
Figure 2. Clinical
symptoms of allergic
conjunctivitis in mice 24
hours after ragweed
application included tearing,
mucus discharge (white
asterisk), and swelling of
the eyelids (arrow).
Eye allergy, also known as ocular conjunctivitis, is a type I hypersensitivity that affects over 20% of
the general population, of which seasonal allergic conjunctivitis is the most predominant form
around the world.5 Symptoms of eye allergies include itchiness, irritation, mucus discharge, tears,
and swelling of the lining of the eye and eyelid.
Seasonal conjunctivitis develops when the immune system of susceptible individuals reacts
excessively to an usually harmless allergen. The mucous membrane of the eye, the conjunctiva, is
highly vulnerable to airborne or microbial allergens.2 One of the most common allergen, which was
used in this study, is ragweed pollen. In an allergic reaction, PMNs show significant degranulation,
increase in number, and change in distribution.4 Degranulation is the release of chemical mediators
such as histamine to rid of the allergen.
A murine model was developed to best resemble the human disease and to test therapies. 7 In order
to make sense of the clinical assessments of topical versus oral treatments, a better understanding
of the cellular activities in the eye will help relate the immunologic processes with the clinical
observations.
This experiment investigated the effects of Visine, a topical vasoconstrictor, and Benedryl, an oral
systemic antihistamine, by inducing the allergy in BALB/c mice, administering the treatments, and
evaluating the levels of immune cells in the conjunctivial region. The subcutaneous route of
sensitization is a modified technique derived from traditional murine models that injected into the
footpads. Total lymphocytes were examined histologically to determine the onset of allergic
conjunctivitis and the effects of treatments.
METHODS
Allergy Induction
Female 8-weeks-old BALB/c mice were given a subcutaneous injection of ragweed pollen in ALUM
in the right shoulder. Ten days later, conjunctivitis was induced by topical instillation of ragweed
directly on each eye. Twenty-four hours later, the mice were sacrificed under ether anesthesia.
Figure 5. Cellular observations of enlarged goblet cells. (A) Enlarged mucin-secreting goblet cells
(asterisk) was more prevalently observed in allergy mice (B) than in the non-allergy (C).
Scale bar = 50 m.
Allergic conjunctivitis was successfully induced in BALB/c mice. The mice were sacrificed 24 hours
after allergen application, and peak cellular infiltration was evaluated. 7 Enlarged goblet cells (Figure
5) are associated with heightened mucus discharge in allergic mice (Figure 2). 7 As shown in Figures
3 and 4, cellular infiltration consisted of mostly PMNs. The inverse relation of PMN density and the
onset of allergy was similar to that of previous studies. Ragweed challenged mice showed a
heightened cellular increase in overall PMN density than naive mice.7
A
C
This experimental model showed allergic conjunctivitis via subcutaneous injection of ragweed
allergen. In traditional models, allergen injection was inserted into the footpad, but the
subcutaneous route of sensitization yielded similar results in inducing the allergy. However, allergy
cell density from subcutaneous injection was less than in the footpad method. 7
B
Figure 3. Histological results of non-allergy (A) versus allergy group (B) after the topical
challenge. Allergy mice did show significant cellular migration into the conjunctiva (p < 0.031)
when compared to the non-challenged group. (C) Independent sample t-test of the PMN density
of the standard counting area was used determine significance in inducing the ragweed allergy.
PMN cell density in non-allergy mice averaged 2.77 cells/mm2, whereas the ragweed allergy mice
showed elevated numbers of 6.07 cells/mm2. Scale bar = 50m. r2 = 83%.
Histology Examination
Eyeballs were enucleated with eyelid attached, fixed, dehydrated, and embedded in LR White resin.
Tissues sections (3μm) were mounted on gelatin-coated slides, stained (H & E), and photographed.
Data Analysis
Cells (total PMNs) were counted in blinded standard areas adjoining the conjuctival fornix by
volunteers. An independent sample t-test was used to compare cell density of non-allergy vs. allergy
groups. ANOVA was used to compare allergy control, Visine-treated, and Benedryl-treated groups.
p < 0.05 was considered significant.
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DISCUSSION
symptoms. 3
Most treatments for ocular conjunctivitis target the allergic reaction after the onset of
Antihistamines, a popular treatment, are administered topically or orally and sometimes both. 3
Recently, topical agents have become more widely used.3 A clinical study by Alexander et. al.1
reported that topical antihistamine was more efficient in alleviating allergic symptoms than the oral. 1
B
The subcutaneous murine model was used to evaluate efficacy of two treatments. Testing two
antihistamine agents, Alexander et al.1 reported that patients preferred the topical agent over the
oral due to faster-acting relief of allergy symptoms. In this study, the treatment types were an
antihistamine and a vasoconstrictor. The oral antihistamine resulted in lesser PMN density than the
topical vasoconstrictor. Benadryl-treated mice appeared to show fewer cells than Visine-treated
mice, thereby possibly alleviating cellular infiltration better than topical Visine.
The result of this study did not concur with the Alexander et al. study. This may be a result of the
treatment types rather than the routes of treatment administration. Generally, antihistamines bind to
histamine receptors and reduce symptoms quickly.1 The antihistamine appears to be better at
decreasing PMN density than the vasoconstrictor. Still, both treatments appeared to reduce cell
density, though not statistically significant. A bigger sample size may decrease variability and effect
size measurement.
REFERENCES
A
B
1. Alexander M., L. Rosen, W. Yang. 1999. Comparison of topical nedocromil sodium and oral terfenadine for the treatment of seasonal
allergic conjunctivitis. Clinical Therapeutics. 21(11): 1900-1907.
2. Bisca M. 1997. Current therapy of allergic conjunctivitis. Current Therapeutic Research. 58(11): 828-841.
3. Bielory, L. 2002. Ocular allergy guideline: a practical treatment algorithm. Therapy in Practice. 62(11): 1611-1634.
4. Bonini S. A., Lambiase, S. Bonini. Genetic and Environmental Factors in Ocular Allergy. Mark Abelson. Allergic Diseases of the Eye.
W.B. Saunders Company. Philadelphia. 260pp.
5. Dogru M., A. Ozmen, H. Erturk, O. Sanli, A. Karatas. 2002. Changes in tear function and the ocular surface after topical olopatadine
treatment for allergic conjunctivitis: an open-label study. Clinical therapeutics. 24(8): 1309-1321.
C
Figure 1. Gross anatomy of mouse eyeball
viewed sagittally. The tissue sample was fixed in
Histochoice Tissue Fixative MB, washed with
phosphate buffer solution, dehydrated in ethanol,
and embedded in LR White Resin. The tissue
section (3μm) was stained with Hemotoxylin and
Eosin. The scale = 200μm. (A) Lens (B) Retina
(C) Eyelid (D) Cornea (E) Iris (F) Sclera (G)
Conjunctival Fornix
6. Groneberg, D.A., L. Bielory, A. Fischer, S. Bonini, U. Wahn. 2003. Animal models of allergic and inflammatory conjunctivitis. Allergy. 58:
1101-1113.
7. Magone M.T., C. Chan, L.V. Rizzo, A.T. Kozhich, S.M. Whitcup. 1998. A novel murine model of allergic conjunctivitis. Clinical
Immunology and Immunopathology. 87(1): 75-84.
8. Magone M.T., Whitcup S.M., Fukushima A., Chan C., Silver P.B., Rizzo L.V. 2000. The role of IL-12 in the induction of late-phase
cellular infiltration in a murine model of allergic conjunctivitis. Journal of allergy Clinical Immunology. 105(2): 299-307.
Figure 4. PMN density and histological sections of non-treated, Visine-treated, and Benadryltreated mice in conjunctival fornix. (A) Both treatment groups showed a decrease in PMN density
compared to the allergy control, but there was no overall significant difference between the control
and treatment groups (P = 0.093). (B) Benedryl-treated mice showed less PMN density (P = 0.08)
than (C) mice treated with topical Visine ( p = .63). Scale bar = 50 m.
ACKNOWLEDGEMENTS: I would like to thank my principal research advisor, Dr. Spilatro, and capstone
instructor, Dr. Hogan of the Marietta College Biology Department. I would like thank Dr. McShaffrey for his
assistance in imaging. I would like to thank Dr. Reisner, O.D., for the capstone idea and Dr. Jones, M.D., for
the surgical advice. Thank you Andrea Marion, Stacey Burgess, Jessica Craig, Toni Stephenson, and Craig
Meredith for counting cells. I would like to acknowledge the Marietta College Investigative Studies Program for
their support of my undergraduate research.