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Effects of Topical Intranasal Doxycycline Treatment in the Rat Allergic Rhinitis Model

Effects of Topical Intranasal Doxycycline Treatment in the Rat Allergic Rhinitis Model Clinical and Experimental Otorhinolaryngology Vol. 7, No. 2: 106-111, June 2014 http://dx.doi.org/10.3342/ceo.2014.7.2.106 pISSN 1976-8710 eISSN 2005-0720 Original Article Effects of Topical Intranasal Doxycycline Treatment in the Rat Allergic Rhinitis Model 1 2 3 2 3 Mehmet Ozgür Avincsal·Seda Ozbal·Ahmet Omer Ikiz·Cetin Pekcetin·Enis Alpin Güneri 1 2 Department of Otorhinolaryngology-Head and Neck Surgery, Private Gazi Hospital, Izmir; Departments of Histology and Embriyology and Otorhinolaryngology-Head and Neck Surgery, Dokuz Eylul University School of Medicine, Izmir, Turkey Objectives. Allergic rhinitis (AR) is a chronic upper respiratory tract disease that inflames the mucous membranes of the nose and occurs when circulating inflammatory cells including eosinophils and basophils migrate to and accumulate in the inflammation area by passing through the interstitium and capillary walls. To pass through these barriers, the in- flammatory cells degrade extracellular matrix proteins. Matrix metalloproteinases (MMPs) released by inflammatory cells mediate the degradation of these proteins. MMPs have synthetic inhibitors and doxycycline, a tetracycline anti- biotic, inhibits MMPs. This study investigated the efficiency of intranasal doxycycline in decreasing the symptoms and inflammatory cell infiltration in an animal model of AR. Methods. AR was created in female Wistar rats by repeated intranasal challenge with ovalbumin by intraperitoneal injection. For 15 days, topical intranasal doxycycline was administered one hour before ovalbumin administration. Following intranasal administration, nasal symptoms were scored and the nasal mucosae of all rats were evaluated histopatho- logically. To investigate tissue changes, hematoxyline-eosin and Alcian blue/periodic acid Schiff stains were used. As well, cilia loss, goblet cell changes, vascular congestion, vascular proliferation, inflammatory cell infiltration, eosinophil infiltration and the degree of hypertrophy in chondrocytes were evaluated with light microscopy. Results. Typical symptoms of AR were decreased by intranasal doxycycline administration. These effects were stable after repeated intranasal ovalbumin administration. Histological evaluation of doxycycline treated rats did not reveal typi- cal inflammatory changes associated with AR. Conclusion. MMPs may have crucial functions in AR and topical intranasal doxycycline, which decreases inflammatory cell infiltration, may offer an alternative therapy for AR. Keywords. Doxycycline, Allergic rhinitis, Matrix metalloproteinase inhibitors lary walls [1,2]. To pass through these barriers, inflammatory INTRODUCTION cells degrade extracellular matrix (ECM) proteins. The degrada- Allergic rhinitis (AR) is a chronic upper respiratory tract disease tion of these proteins is mediated by matrix metalloproteinases that involves inflammation of the mucous membranes of the (MMPs) released by inflammatory cells [3,4]. MMPs are a multi- nose. AR occurs when circulating inflammatory cells including genic family of endopeptidases whose synthesis, secretion and eosinophils and basophils migrate to and accumulate in the in- activation are regulated by transcriptional regulation, latent en- flammation area by passing through the interstitium and capil- zyme activation and specific tissue inhibitors of metalloprotein- ases (TIMPs) [3]. Besides TIMPs, some synthetic inhibitors of • Received December 16, 2012 MMPs function by binding to Zn++ atoms. The tetracycline anti- Revised February 12, 2013 biotic doxycycline (Dox) inhibits MMP activity at sub-antimi- Accepted March 26, 2013 crobial doses without any side effects [5]. Corresponding author : Mehmet Ozgür Avincsal Department of Otorhinolaryngology-Head and Neck Surgery, Private Gazi This study investigated the efficiency of intranasal Dox in de- Hospital, 1421 sokak No:29 Alsancak, Izmir, Turkey creasing the symptoms of AR and mucosal histological changes Tel: +90-505-7501639, Fax: +902323481800 E-mail: ozgur_tr@hotmail.com in an animal model. Copyright © 2014 by Korean Society of Otorhinolaryngology-Head and Neck Surgery. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 106 Avincsal MO et al. Topical Doxycycline and Allergic Rhinitis 107 Table 1. Scoring of allergic rhinitis MATERIALS AND METHODS Variable 0 1 2 3 Animals No. of nasal itching None 2 4–6 >6 This study was conducted on 21 healthy female Wistar rats motion (scratches) weighing between 250 g and 300 g, which had been bred in the (time/minute) No. of sneezes None 2 4–6 >6 Center of Laboratory Animal Breeding and Experimental Stud- (time/minute) ies at Dokuz Eylul University. The rats were housed in 30×18× Amount of nasal flow None In one In both Out- 24 cm cages (four rats per cage) in a 12 hours light/12 hours nostril nostrils flowing dark schedule at 24°C±2°C and fed standard pellet diet. The animals had free access to food and water. The study was ap- proved by the Committee on Ethics in Animal Experimentation sion followed by 400 mL formaldehyde introduced through the of Dokuz Eylul University (approval number: 30/2009, approval left ventricle 24 hours after the last intranasal challenge. The date: June 5, 2009). head of each rat was removed and fixed in 10% neutral buffered formalin for 3 days, then decalcified in 5% trichloroacetic acid Allergic rhinitis model for 7 days. The nasal cavity was transversely sectioned at the After acclimatization to laboratory conditions, the first phase of level of incisive papilla of the hard palate and the tissue block the study involved the immunization of the rats. Fourteen rats was embedded in paraffin. Some of the sections were stained were sensitized with ovalbumin (OVA; 0.3 mg intraperitoneally; with hematoxylin and eosin (H&E) to examine tissue histology Grade V, Sigma-Aldrich, St. Louis, MO, USA) as antigen, which and the remaining sections were stained with Alcian blue and was administered together with aluminum hydroxide (30 mg) in periodic acid Schiff (AB/PAS) to highlight goblet cells. Digital saline (1 mL intraperitoneally) every 2 days for 14 days, be- images were obtained from bothstained sections using a model tween 11 and 12 AM. In the second phase, all 14 sensitized ani- DP71 camera (Olympus Optical, Tokyo, Japan) connected to a mals were used to develop a model of AR by repeated intrana- model BX51 light microscope (Olympus Optical) at an original sal instillation of 10 μL of 20 mg/mL OVA with a micropipette magnification of ×40. Images were processed with the 3D Re- daily for 15 days between 11 and 12 AM [6]. The animals were construct Program 1.1.0.1 (Medical College of Georgia, Georgia separated into two groups. Group A included 7 sensitized rats Health Sciences University, Augusta, GA, USA). Cilia loss, goblet (AR group). The 7 rats in group B additionally received 10 μL of cell increase, vascular congestion, vascular proliferation, inflam- 20 mg/mL Dox (D-9891; Sigma-Aldrich) dissolved in 0.9% matory cell infiltration, eosinophil infiltration, and the degree of physiological saline, which was instilled into both nostrils using hypertrophy in chondrocytes were evaluated [7-9] in each sec- a pipette one hour before the administration of OVA for 15 days tion. The change in each parameter was scored as 0, no change; 1, (Dox group). Additionally, a control group (group C) was estab- mild change; 2, moderate change; or 3, severe change. All histo- lished; 7 rats were repeatedly sensitized and challenged with sa- morphological analyses were performed by two histologists with line with the same dose and on a similar time schedule as the no prior knowledge of the treatment groups. other animals in the other groups. Statistical analyses Evaluation of symptoms The mean of each parameter was statistically analyzed using The immediate and late reactions of all animals after nasal chal- SPSS ver. 15.0 (SPSS, Chicago, IL, USA). The analyses were per- lenge were examined on day 1, 14, 17, 20, 23, 26, and 28 after formed by assigning the results into groups as none to mild the administration OVA. Observations were performed by the (scores below 1.5) and moderate to severe (scores above 1.5). same person for 10 minutes, following an adaptation period of Fisher exact test was used to test for the differences between the 10 minutes. AR was evaluated regarding the severity of typical groups. The measurement of sneezes and nasal scratches was clinical symptoms that included nasal irritation, sneezing and na- evaluated using Mann-Whitney U-test for paired groups. Groups sal secretion. Nasal symptom scores were graded on a four-point were compared within themselves using Wilcoxon signed-rank scale, each grade was assigned a numerical score (0–3) and the test, P<0.05 was considered significant in all analyses. scores were graded as summarized in Table 1. On day 14, which was the first day of sensitization, nasal scratching, sneezing and nasal flow were scored. The AR model was considered successful RESULTS if the total score exceeded 5 [6]. Experimental AR model Histological examination Following injection and initial intranasal challenge with OVA, All animals were sacrificed by 80 mg/kg intraperitoneal pento- typical AR symptoms including frequent sneezing, nasal scratch- barbital injection and 100 mL 0.9% physiological saline perfu- ing and watery rhinorrhea were observed in each animal, which 108 Clinical and Experimental Otorhinolaryngology Vol. 7, No. 2: 106-111, June 2014 then gradually disappeared over approximately 1.5 hours. Since Histological evaluation the total score in all animals was >5 at the first subjective evalu- Semiquantitative histological evaluation of the nasal cavity, na- ation of AR carried out on day 14, the AR model was consid- sal septum and lateral nasal walls revealed that there were nor- ered successful. Thus, all animals in groups A, B, and C were in- mal histological structures in the control group with regular cili- cluded in the study. Subsequent intranasal challenges with OVA ae and goblet cells in the pseudostratified ciliated cylindrical re- triggered more severe symptoms that persisted longer. However, spiratory epithelium cells. Eosinophil and inflammatory cell in- symptoms were reduced and disappeared on day 2 in animals filtration were not observed in the lamina propria, connective treated with Dox. Furthermore, subsequent intranasal challeng- tissue structures were normal, and vascular congestion and pro- es with OVA did not trigger AR symptoms. liferation was not present and mucosal glands and chondrocytes In group A, the number of sneezes was considerably higher on were normal (Fig. 3, Table 2). days 14, 17, 20, 23, 26, and 28 than on day 1 (P=0.017, Significant changes were observed for all histological parame- P=0.017, P=0.017, P=0.016, P=0.016, and P=0.017, respec- ters in group A. H&E staining demonstrated cilia loss in the re- tively). In group B (Dox-treated), a decrease in the number of spiratory epithelium cells; eosinophil and inflammatory cell in- sneezes was observed, beginning from day 3 of treatment (Fig. 1). filtration and vascular congestion and proliferation in the con- Comparison of groups A and B revealed no significant differenc- nective tissue veins were significantly higher in this group of rats es between the number of sneezes among the two groups on day than in the control group (P= 0.001, P= 0.001, P= 0.001, P= 1 and 14 (P=0.606 and P=0.790, respectively). However, com- 0.001, P=0.001, respectively). Examination of the cartilage tis- pared to group A, a decline was observed in the mean number of sues revealed significant hypertrophy in the chondrocytes sneezes in group B on day 17, 20, 23, 26, and 28. The differences (P=0.001) (Fig. 4). AB/PAS staining revealed a significant in- between the two groups were significant (P=0.001, P=0.001, crease in goblet cells (P=0.001). The results of the HE staining P=0.001, P=0.001, and P=0.001, respectively). Throughout the in group B demonstratedminimal cilia loss in the respiratory epi- study, a significant difference in number of sneezes was not ob- thelium cells, also eosinophil and inflammatory cell infiltration served in group C. and vascular congestion and proliferation in the connective tis- Statistically significant differences were not observed between sue veins were significantly reduced, compared to group A the mean number of nose scratches among the groups A and B (P= 0.005, P= 0.001, P= 0.001, P= 0.001, P= 0.001, P= 0.001, on day 1 and 14 (P= 0.728 and P= 0.181, respectively). However, respectively). Although AB/PAS staining demonstrated a decline a significant decrease in the number of nasal scratches was ob- in the goblet cell increase, a statistically significant difference served in the Dox-treated rats in group B on day 17, 20, 23, 26 was not present between groups A and B (P= 1.000). There were and 28 (P=0.002, P=0.001, P=0.001, P=0.001, and P=0.001, no significant differences between group B and the control respectively). In group C, no significant differences were ob- groupwith respect to the results of the H&E staining (all P= served in the number of nose scratches on the aforementioned days (Fig. 2). Group A Group B A B C Group C 1st 14st 17st 20st 23st 26st 28st D E F Time (day) Fig. 1. Mean number of sneezes after nasal challenge. Group A G H I Group B Group C Fig. 3. Summary of histological evaluations. Light-microscopic imag- es of H&E (A–F) and Alcian blue/periodic acid Schiff (G–I) in the control group (A, D, G). Allergic rhinitis group (B, E, H) and the group treated with doxycycline (C, F, I). Stars indicate vascular con- 1st 14st 17st 20st 23st 26st 28st gestion, white arrows indicate goblet cells, and black arrows indi- Time (day) cate eosinophilis. A–C: H&E, 40; D–F: H&E, 100; G–I, Alcian blue/ × × Fig. 2. Mean number of nose scratches after nasal challenge. periodic acid Schiff,×40. Rubbing (number/10 min) Sneezing (number/10 min) Avincsal MO et al. Topical Doxycycline and Allergic Rhinitis 109 Table 2. Semiquantitative histological evaluation of the nasal cavity, nasal septum and lateral nasal walls in groups A–C Goblet cell Vascular Vascular Inflammatory Chondrocyte Eosinophil Group Cilia loss increase congestion proliferation cell infiltration hypertrophy infiltration A1 2.00 2.60 2.60 3.00 2.40 3.00 2.60 A2 2.00 2.40 2.60 3.00 2.00 2.60 2.40 A3 2.00 2.00 2.60 2.80 2.20 2.80 2.40 A4 2.00 2.60 2.60 2.80 2.40 2.80 2.40 A5 2.00 2.80 2.60 3.00 2.40 2.60 2.60 A6 2.00 2.80 2.20 2.60 2.00 2.60 2.00 A7 2.00 2.60 2.60 3.00 2.80 3.00 2.80 B1 1.00 1.40 1.00 1.00 1.00 0.00 1.00 B2 1.60 1.60 1.40 1.20 1.20 0.40 1.20 B3 1.20 1.60 1.00 1.00 1.00 0.00 1.20 B4 1.40 1.60 1.00 1.00 1.00 0.20 1.00 B5 1.40 1.80 1.00 1.00 1.20 0.40 1.20 B6 1.00 1.60 1.20 1.20 1.00 0.20 1.00 B7 1.40 1.80 1.20 1.20 1.00 0.20 1.20 C1 0.00 0.00 0.00 0.00 0.00 0.00 0.20 C2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C3 0.00 0.00 0.00 0.00 0.00 0.00 0.40 C4 0.00 0.00 0.00 0.00 0.00 0.00 0.20 C5 0.00 0.00 0.00 0.00 0.00 0.00 0.20 C6 0.00 0.00 0.00 0.00 0.00 0.00 0.40 C7 0.00 0.00 0.00 0.00 0.00 0.00 0.20 tain drugs. In humans, the main symptoms of AR are sneezing, itchy nose and rhinorrhea; therefore, there is a need for animal models that exhibit similar allergic symptoms. This study clearly revealed that repeated topical intranasal OVA application causes typical AR symptoms such as sneezing and nose scratching in rats and provides a good model for experimental AR [6,10]. Immunohistological studies aimed at investigating the nasal tissues of AR patients have indicated intense accumulation of eo- sinophils and basophil/mast cells in the lamina propria and epi- thelium [11]. The recruitment and migration of these inflamma- tory cells to sites of inflammatory reactions involves traversing the capillary walls and the interstitium [1-3]. To traverse these barriers, inflammatory cells adhere and degrade ECM proteins [4-6]. The degradation of ECM proteins by inflammatory cells is accomplished in part by the secretion of MMPs, such as MMP-2 and MMP-9, which specifically shred denatured collagen, native Fig. 4. Light-microscopic images of H&E staining in the allergic rhini- tis group. Stars indicate vascular congestion and arrows indicate type IV and V collagens and elastin [12,13]. In murine models, chondrocyte hypertrophy (×40). inflammatory cells, MMP-9, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 protein 1.000). The goblet cell count revealed by AB/PAS staining in and mRNA levels are increased after challenge with toluene di- group B was found to be close to that in the control group, al- isocyanate. The administration of a MMP inhibitor reportedly re- though it was significantly higher (P= 0.001). duced the amount of inflammatory cells and the levels of ICAM- 1 and VCAM-1 mRNA and protein, supporting the notion that MMP inhibitors inhibit inflammatory cell migration by causing a decrease in adhesion molecules [3,4]. DISCUSSION Introduction of platelet activating factor and interleukin can The search for the treatment of AR has led to various attempts to prompt eosinophil transmigration from the artificial basal mem- establish animal models and investigations of the effects of cer- brane and the introduction of Batimastat, a synthetic MMP in- 110 Clinical and Experimental Otorhinolaryngology Vol. 7, No. 2: 106-111, June 2014 hibitor, can inhibit this transmigration in vitro [14]. The present CONFLICT OF INTEREST in vivo observations of significant decreases of eosinophils and infiltration of other inflammatory cells in the nasal mucosa of No potential conflict of interest relevant to this article was re- Dox-treated rats, support the in vitro results and strengthen the ported. validity of the animal model. Structural abnormalities seen in AR, which are collectively termed tissue remodeling, are caused by MMPs secreted from REFERENCES epithelial cells and fibroblasts in addition to infiltrating inflam- 1. Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, Sorsa T. Tet- matory cells [15]. MMPs are also reportedly responsible for mi- racyclines inhibit connective tissue breakdown by multiple non-an- crovascular permeability leading to edema, cell migration and timicrobial mechanisms. Adv Dent Res. 1998 Nov;12(2):12-26. ECM remodelling at the site of inflammation [2]. Therefore it is 2. Asano K, Kanai KI, Suzaki H. Suppressive activity of fexofenadine reasonable to speculate that manipulation of MMP production hydrochloride on metalloproteinase production from nasal fibro- from inflammatory cells, epithelial cells and fibroblasts by anti- blasts in vitro. Clin Exp Allergy. 2004 Dec;34(12):1890-8. 3. De S, Fenton JE, Jones AS. Matrix metalloproteinases and their in- allergic agents may be an important strategy for treating symp- hibitors innon-neoplastic otorhinolaryngological disease. J Laryngol toms of allergic diseases including AR. In this study, cilia loss in Otol. 2005 Jun;119(6):436-42. the respiratory epithelium cells, marked vascular congestion and 4. Lee KS, Jin SM, Kim HJ, Lee YC. Matrix metalloproteinase inhibitor proliferation in the veins located in the connective tissue, signifi- regulatesinflammatory cell migration by reducing ICAM-1 and VCAM-1 expression in a murine model of toluene diisocyanate-in- cant hypertrophy in the chondrocytes and a significant increase duced asthma. J Allergy Clin Immunol. 2003 Jun;111(6):1278-84. in goblet cells were demonstrated in AR rats. However, in the 5. Baltacıoglu E, Akalın A. Tetracyclines and their non-antimicrobial Dox-treated group, ciliary loss, vascular congestion and prolifer- properties: a new approach to their use in periodontal treatment. ation, chondrocyte hypertrophy and increase in goblet cells Hacettepe Dişhekimliği Fakültesi Dergisi. 2006;30(1):97-107. 6. Wen WD, Yuan F, Wang JL, Hou YP. Botulinum toxin therapy in the were reduced significantly. ovalbumin-sensitized rat. Neuroimmunomodulation. 2007;14(2):78- Fluticasone propionate, a H1-receptor-antagonist, can reduce the release of MMP-2 and MMP-9, and the expression of MMP- 7. Salib RJ, Howarth PH. Remodelling of the upper airways in allergic mRNA in nasal fibroblasts, demonstrating that the action of cor- rhinitis: is it a feature of the disease? Clin Exp Allergy. 2003 Dec; 33(12):1629-33. ticosteroids in the treatment of AR is mediated by MMPs [16]. 8. Bousquet J, Jacot W, Vignola AM, Bachert C, Van Cauwenberge P. To investigate the effects of fexofenadine hydrochloride on Allergic rhinitis: a disease remodeling the upper airways? J Allergy MMPs, nasal fibroblasts were stimulated in vitro by tumor ne- Clin Immunol. 2004 Jan;113(1):43-9. crosis factor-alpha. The compound reduced the release of MMP- 9. Ercan I, Cakir BO, Basak T, Ozbal EA, Sahin A, Balci G, et al. Effects 2 and MMP-9 and the expression of MMP-mRNA in a similar oftopical application of methotrexate on nasal mucosa in rats: a pre- clinicalassessment study. Otolaryngol Head Neck Surg. 2006 May; fashion [3]. Dox inhibits MMP activity at sub-antimicrobial dos- 134(5):751-5. es [5,17]. This study is the first to administer topical intranasal 10. Shimizu S, Hattori R, Majima Y, Shimizu T. Th2 cytokine inhibitor Dox for a therapeutic purpose in an AR model. Treatment with suplatasttosilate inhibits antigen-induced mucus hypersecretion in 20 mg/mL Dox caused a significant decrease in AR symptoms the nasal epithelium of sensitized rats. Ann Otol Rhinol Laryngol. 2009 Jan;118(1):67-72. including nasal scratching and sneezing, and that the repeated 11. Bentley AM, Jacobson MR, Cumberworth V, Barkans JR, Moqbel R, administration of OVA does not cause an increase in these Schwartz LB, et al. Immunohistology of the nasal mucosa in season- symptoms. The histological examinations carried out after Dox alallergic rhinitis: increases in activated eosinophils and epithelial treatment revealed a significant decrease in the typical inflam- mast cells. J Allergy Clin Immunol. 1992 Apr;89(4):877-83. 12. Herouy Y, Mellios P, Bandemir E, Dichmann S, Nockowski P, Schopf matory changes seen in AR. Our results support the view that E, et al. Inflammation in stasis dermatitis upregulates MMP-1, MMP- MMPs may have crucial functions in AR and topical intranasal 2 and MMP-13 expression. J Dermatol Sci. 2001 Apr; 25(3):198- Dox, which decreases inflammatory cell infiltration, may be a treatment option in AR. 13. Ohno I, Ohtani H, Nitta Y, Suzuki J, Hoshi H, Honma M, et al. Eosin- The main limitations of this preliminary study that we did not ophils as a source of matrix metalloproteinase-9 in asthmatic airway inflammation. Am J Respir Cell Mol Biol. 1997 Mar;16(3):212-9. study OVA-specific IgE to confirm the establishment of AR 14. Okada S, Kita H, George TJ, Gleich GJ, Leiferman KM. Migration of model and did not evaluate cytokines related with AR. Addition- eosinophils through basement membrane components in vitro: role ally, evaluating the MMP expression using immunohistochemi- of matrix metalloproteinase-9. Am J Respir Cell Mol Biol. 1997 cal staining of MMPs would be helpful to explain the role of Oct;17(4):519-28. 15. Nakaya M, Dohi M, Okunishi K, Nakagome K, Tanaka R, Imamura Dox. A multicentric, double-blinded and randomized controlled M, et al. Prolonged allergen challenge in murine nasal allergic rhini- clinical trials investigating the evidence acquired from our ex- tis: nasal airway remodeling and adaptation of nasal airway respon- perimental animal model may help provide a new option in the siveness. Laryngoscope. 2007 May;117(5):881-5. treatment of AR. 16. Namba M, Asano K, Kanai K, Kyo Y, Watanabe S, Hisamitsu T, et al. Suppression of matrix metalloproteinase production from nasal fi- Avincsal MO et al. Topical Doxycycline and Allergic Rhinitis 111 broblasts by fluticasone propionate in vitro. Acta Otolaryngol. 2004 various drugs through the rabbit’s respiratory mucosa in vitro. Arch Oct;124(8):964-9. Otorhinolaryngol. 1983;238(1):87-96. 17. Sakakura Y, Majima Y, Mitsui H, Inagaki M, Miyoshi Y. 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Copyright © 2014 by Korean Society of Otorhinolaryngology-Head and Neck Surgery.
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Abstract

Clinical and Experimental Otorhinolaryngology Vol. 7, No. 2: 106-111, June 2014 http://dx.doi.org/10.3342/ceo.2014.7.2.106 pISSN 1976-8710 eISSN 2005-0720 Original Article Effects of Topical Intranasal Doxycycline Treatment in the Rat Allergic Rhinitis Model 1 2 3 2 3 Mehmet Ozgür Avincsal·Seda Ozbal·Ahmet Omer Ikiz·Cetin Pekcetin·Enis Alpin Güneri 1 2 Department of Otorhinolaryngology-Head and Neck Surgery, Private Gazi Hospital, Izmir; Departments of Histology and Embriyology and Otorhinolaryngology-Head and Neck Surgery, Dokuz Eylul University School of Medicine, Izmir, Turkey Objectives. Allergic rhinitis (AR) is a chronic upper respiratory tract disease that inflames the mucous membranes of the nose and occurs when circulating inflammatory cells including eosinophils and basophils migrate to and accumulate in the inflammation area by passing through the interstitium and capillary walls. To pass through these barriers, the in- flammatory cells degrade extracellular matrix proteins. Matrix metalloproteinases (MMPs) released by inflammatory cells mediate the degradation of these proteins. MMPs have synthetic inhibitors and doxycycline, a tetracycline anti- biotic, inhibits MMPs. This study investigated the efficiency of intranasal doxycycline in decreasing the symptoms and inflammatory cell infiltration in an animal model of AR. Methods. AR was created in female Wistar rats by repeated intranasal challenge with ovalbumin by intraperitoneal injection. For 15 days, topical intranasal doxycycline was administered one hour before ovalbumin administration. Following intranasal administration, nasal symptoms were scored and the nasal mucosae of all rats were evaluated histopatho- logically. To investigate tissue changes, hematoxyline-eosin and Alcian blue/periodic acid Schiff stains were used. As well, cilia loss, goblet cell changes, vascular congestion, vascular proliferation, inflammatory cell infiltration, eosinophil infiltration and the degree of hypertrophy in chondrocytes were evaluated with light microscopy. Results. Typical symptoms of AR were decreased by intranasal doxycycline administration. These effects were stable after repeated intranasal ovalbumin administration. Histological evaluation of doxycycline treated rats did not reveal typi- cal inflammatory changes associated with AR. Conclusion. MMPs may have crucial functions in AR and topical intranasal doxycycline, which decreases inflammatory cell infiltration, may offer an alternative therapy for AR. Keywords. Doxycycline, Allergic rhinitis, Matrix metalloproteinase inhibitors lary walls [1,2]. To pass through these barriers, inflammatory INTRODUCTION cells degrade extracellular matrix (ECM) proteins. The degrada- Allergic rhinitis (AR) is a chronic upper respiratory tract disease tion of these proteins is mediated by matrix metalloproteinases that involves inflammation of the mucous membranes of the (MMPs) released by inflammatory cells [3,4]. MMPs are a multi- nose. AR occurs when circulating inflammatory cells including genic family of endopeptidases whose synthesis, secretion and eosinophils and basophils migrate to and accumulate in the in- activation are regulated by transcriptional regulation, latent en- flammation area by passing through the interstitium and capil- zyme activation and specific tissue inhibitors of metalloprotein- ases (TIMPs) [3]. Besides TIMPs, some synthetic inhibitors of • Received December 16, 2012 MMPs function by binding to Zn++ atoms. The tetracycline anti- Revised February 12, 2013 biotic doxycycline (Dox) inhibits MMP activity at sub-antimi- Accepted March 26, 2013 crobial doses without any side effects [5]. Corresponding author : Mehmet Ozgür Avincsal Department of Otorhinolaryngology-Head and Neck Surgery, Private Gazi This study investigated the efficiency of intranasal Dox in de- Hospital, 1421 sokak No:29 Alsancak, Izmir, Turkey creasing the symptoms of AR and mucosal histological changes Tel: +90-505-7501639, Fax: +902323481800 E-mail: ozgur_tr@hotmail.com in an animal model. Copyright © 2014 by Korean Society of Otorhinolaryngology-Head and Neck Surgery. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 106 Avincsal MO et al. Topical Doxycycline and Allergic Rhinitis 107 Table 1. Scoring of allergic rhinitis MATERIALS AND METHODS Variable 0 1 2 3 Animals No. of nasal itching None 2 4–6 >6 This study was conducted on 21 healthy female Wistar rats motion (scratches) weighing between 250 g and 300 g, which had been bred in the (time/minute) No. of sneezes None 2 4–6 >6 Center of Laboratory Animal Breeding and Experimental Stud- (time/minute) ies at Dokuz Eylul University. The rats were housed in 30×18× Amount of nasal flow None In one In both Out- 24 cm cages (four rats per cage) in a 12 hours light/12 hours nostril nostrils flowing dark schedule at 24°C±2°C and fed standard pellet diet. The animals had free access to food and water. The study was ap- proved by the Committee on Ethics in Animal Experimentation sion followed by 400 mL formaldehyde introduced through the of Dokuz Eylul University (approval number: 30/2009, approval left ventricle 24 hours after the last intranasal challenge. The date: June 5, 2009). head of each rat was removed and fixed in 10% neutral buffered formalin for 3 days, then decalcified in 5% trichloroacetic acid Allergic rhinitis model for 7 days. The nasal cavity was transversely sectioned at the After acclimatization to laboratory conditions, the first phase of level of incisive papilla of the hard palate and the tissue block the study involved the immunization of the rats. Fourteen rats was embedded in paraffin. Some of the sections were stained were sensitized with ovalbumin (OVA; 0.3 mg intraperitoneally; with hematoxylin and eosin (H&E) to examine tissue histology Grade V, Sigma-Aldrich, St. Louis, MO, USA) as antigen, which and the remaining sections were stained with Alcian blue and was administered together with aluminum hydroxide (30 mg) in periodic acid Schiff (AB/PAS) to highlight goblet cells. Digital saline (1 mL intraperitoneally) every 2 days for 14 days, be- images were obtained from bothstained sections using a model tween 11 and 12 AM. In the second phase, all 14 sensitized ani- DP71 camera (Olympus Optical, Tokyo, Japan) connected to a mals were used to develop a model of AR by repeated intrana- model BX51 light microscope (Olympus Optical) at an original sal instillation of 10 μL of 20 mg/mL OVA with a micropipette magnification of ×40. Images were processed with the 3D Re- daily for 15 days between 11 and 12 AM [6]. The animals were construct Program 1.1.0.1 (Medical College of Georgia, Georgia separated into two groups. Group A included 7 sensitized rats Health Sciences University, Augusta, GA, USA). Cilia loss, goblet (AR group). The 7 rats in group B additionally received 10 μL of cell increase, vascular congestion, vascular proliferation, inflam- 20 mg/mL Dox (D-9891; Sigma-Aldrich) dissolved in 0.9% matory cell infiltration, eosinophil infiltration, and the degree of physiological saline, which was instilled into both nostrils using hypertrophy in chondrocytes were evaluated [7-9] in each sec- a pipette one hour before the administration of OVA for 15 days tion. The change in each parameter was scored as 0, no change; 1, (Dox group). Additionally, a control group (group C) was estab- mild change; 2, moderate change; or 3, severe change. All histo- lished; 7 rats were repeatedly sensitized and challenged with sa- morphological analyses were performed by two histologists with line with the same dose and on a similar time schedule as the no prior knowledge of the treatment groups. other animals in the other groups. Statistical analyses Evaluation of symptoms The mean of each parameter was statistically analyzed using The immediate and late reactions of all animals after nasal chal- SPSS ver. 15.0 (SPSS, Chicago, IL, USA). The analyses were per- lenge were examined on day 1, 14, 17, 20, 23, 26, and 28 after formed by assigning the results into groups as none to mild the administration OVA. Observations were performed by the (scores below 1.5) and moderate to severe (scores above 1.5). same person for 10 minutes, following an adaptation period of Fisher exact test was used to test for the differences between the 10 minutes. AR was evaluated regarding the severity of typical groups. The measurement of sneezes and nasal scratches was clinical symptoms that included nasal irritation, sneezing and na- evaluated using Mann-Whitney U-test for paired groups. Groups sal secretion. Nasal symptom scores were graded on a four-point were compared within themselves using Wilcoxon signed-rank scale, each grade was assigned a numerical score (0–3) and the test, P<0.05 was considered significant in all analyses. scores were graded as summarized in Table 1. On day 14, which was the first day of sensitization, nasal scratching, sneezing and nasal flow were scored. The AR model was considered successful RESULTS if the total score exceeded 5 [6]. Experimental AR model Histological examination Following injection and initial intranasal challenge with OVA, All animals were sacrificed by 80 mg/kg intraperitoneal pento- typical AR symptoms including frequent sneezing, nasal scratch- barbital injection and 100 mL 0.9% physiological saline perfu- ing and watery rhinorrhea were observed in each animal, which 108 Clinical and Experimental Otorhinolaryngology Vol. 7, No. 2: 106-111, June 2014 then gradually disappeared over approximately 1.5 hours. Since Histological evaluation the total score in all animals was >5 at the first subjective evalu- Semiquantitative histological evaluation of the nasal cavity, na- ation of AR carried out on day 14, the AR model was consid- sal septum and lateral nasal walls revealed that there were nor- ered successful. Thus, all animals in groups A, B, and C were in- mal histological structures in the control group with regular cili- cluded in the study. Subsequent intranasal challenges with OVA ae and goblet cells in the pseudostratified ciliated cylindrical re- triggered more severe symptoms that persisted longer. However, spiratory epithelium cells. Eosinophil and inflammatory cell in- symptoms were reduced and disappeared on day 2 in animals filtration were not observed in the lamina propria, connective treated with Dox. Furthermore, subsequent intranasal challeng- tissue structures were normal, and vascular congestion and pro- es with OVA did not trigger AR symptoms. liferation was not present and mucosal glands and chondrocytes In group A, the number of sneezes was considerably higher on were normal (Fig. 3, Table 2). days 14, 17, 20, 23, 26, and 28 than on day 1 (P=0.017, Significant changes were observed for all histological parame- P=0.017, P=0.017, P=0.016, P=0.016, and P=0.017, respec- ters in group A. H&E staining demonstrated cilia loss in the re- tively). In group B (Dox-treated), a decrease in the number of spiratory epithelium cells; eosinophil and inflammatory cell in- sneezes was observed, beginning from day 3 of treatment (Fig. 1). filtration and vascular congestion and proliferation in the con- Comparison of groups A and B revealed no significant differenc- nective tissue veins were significantly higher in this group of rats es between the number of sneezes among the two groups on day than in the control group (P= 0.001, P= 0.001, P= 0.001, P= 1 and 14 (P=0.606 and P=0.790, respectively). However, com- 0.001, P=0.001, respectively). Examination of the cartilage tis- pared to group A, a decline was observed in the mean number of sues revealed significant hypertrophy in the chondrocytes sneezes in group B on day 17, 20, 23, 26, and 28. The differences (P=0.001) (Fig. 4). AB/PAS staining revealed a significant in- between the two groups were significant (P=0.001, P=0.001, crease in goblet cells (P=0.001). The results of the HE staining P=0.001, P=0.001, and P=0.001, respectively). Throughout the in group B demonstratedminimal cilia loss in the respiratory epi- study, a significant difference in number of sneezes was not ob- thelium cells, also eosinophil and inflammatory cell infiltration served in group C. and vascular congestion and proliferation in the connective tis- Statistically significant differences were not observed between sue veins were significantly reduced, compared to group A the mean number of nose scratches among the groups A and B (P= 0.005, P= 0.001, P= 0.001, P= 0.001, P= 0.001, P= 0.001, on day 1 and 14 (P= 0.728 and P= 0.181, respectively). However, respectively). Although AB/PAS staining demonstrated a decline a significant decrease in the number of nasal scratches was ob- in the goblet cell increase, a statistically significant difference served in the Dox-treated rats in group B on day 17, 20, 23, 26 was not present between groups A and B (P= 1.000). There were and 28 (P=0.002, P=0.001, P=0.001, P=0.001, and P=0.001, no significant differences between group B and the control respectively). In group C, no significant differences were ob- groupwith respect to the results of the H&E staining (all P= served in the number of nose scratches on the aforementioned days (Fig. 2). Group A Group B A B C Group C 1st 14st 17st 20st 23st 26st 28st D E F Time (day) Fig. 1. Mean number of sneezes after nasal challenge. Group A G H I Group B Group C Fig. 3. Summary of histological evaluations. Light-microscopic imag- es of H&E (A–F) and Alcian blue/periodic acid Schiff (G–I) in the control group (A, D, G). Allergic rhinitis group (B, E, H) and the group treated with doxycycline (C, F, I). Stars indicate vascular con- 1st 14st 17st 20st 23st 26st 28st gestion, white arrows indicate goblet cells, and black arrows indi- Time (day) cate eosinophilis. A–C: H&E, 40; D–F: H&E, 100; G–I, Alcian blue/ × × Fig. 2. Mean number of nose scratches after nasal challenge. periodic acid Schiff,×40. Rubbing (number/10 min) Sneezing (number/10 min) Avincsal MO et al. Topical Doxycycline and Allergic Rhinitis 109 Table 2. Semiquantitative histological evaluation of the nasal cavity, nasal septum and lateral nasal walls in groups A–C Goblet cell Vascular Vascular Inflammatory Chondrocyte Eosinophil Group Cilia loss increase congestion proliferation cell infiltration hypertrophy infiltration A1 2.00 2.60 2.60 3.00 2.40 3.00 2.60 A2 2.00 2.40 2.60 3.00 2.00 2.60 2.40 A3 2.00 2.00 2.60 2.80 2.20 2.80 2.40 A4 2.00 2.60 2.60 2.80 2.40 2.80 2.40 A5 2.00 2.80 2.60 3.00 2.40 2.60 2.60 A6 2.00 2.80 2.20 2.60 2.00 2.60 2.00 A7 2.00 2.60 2.60 3.00 2.80 3.00 2.80 B1 1.00 1.40 1.00 1.00 1.00 0.00 1.00 B2 1.60 1.60 1.40 1.20 1.20 0.40 1.20 B3 1.20 1.60 1.00 1.00 1.00 0.00 1.20 B4 1.40 1.60 1.00 1.00 1.00 0.20 1.00 B5 1.40 1.80 1.00 1.00 1.20 0.40 1.20 B6 1.00 1.60 1.20 1.20 1.00 0.20 1.00 B7 1.40 1.80 1.20 1.20 1.00 0.20 1.20 C1 0.00 0.00 0.00 0.00 0.00 0.00 0.20 C2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C3 0.00 0.00 0.00 0.00 0.00 0.00 0.40 C4 0.00 0.00 0.00 0.00 0.00 0.00 0.20 C5 0.00 0.00 0.00 0.00 0.00 0.00 0.20 C6 0.00 0.00 0.00 0.00 0.00 0.00 0.40 C7 0.00 0.00 0.00 0.00 0.00 0.00 0.20 tain drugs. In humans, the main symptoms of AR are sneezing, itchy nose and rhinorrhea; therefore, there is a need for animal models that exhibit similar allergic symptoms. This study clearly revealed that repeated topical intranasal OVA application causes typical AR symptoms such as sneezing and nose scratching in rats and provides a good model for experimental AR [6,10]. Immunohistological studies aimed at investigating the nasal tissues of AR patients have indicated intense accumulation of eo- sinophils and basophil/mast cells in the lamina propria and epi- thelium [11]. The recruitment and migration of these inflamma- tory cells to sites of inflammatory reactions involves traversing the capillary walls and the interstitium [1-3]. To traverse these barriers, inflammatory cells adhere and degrade ECM proteins [4-6]. The degradation of ECM proteins by inflammatory cells is accomplished in part by the secretion of MMPs, such as MMP-2 and MMP-9, which specifically shred denatured collagen, native Fig. 4. Light-microscopic images of H&E staining in the allergic rhini- tis group. Stars indicate vascular congestion and arrows indicate type IV and V collagens and elastin [12,13]. In murine models, chondrocyte hypertrophy (×40). inflammatory cells, MMP-9, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 protein 1.000). The goblet cell count revealed by AB/PAS staining in and mRNA levels are increased after challenge with toluene di- group B was found to be close to that in the control group, al- isocyanate. The administration of a MMP inhibitor reportedly re- though it was significantly higher (P= 0.001). duced the amount of inflammatory cells and the levels of ICAM- 1 and VCAM-1 mRNA and protein, supporting the notion that MMP inhibitors inhibit inflammatory cell migration by causing a decrease in adhesion molecules [3,4]. DISCUSSION Introduction of platelet activating factor and interleukin can The search for the treatment of AR has led to various attempts to prompt eosinophil transmigration from the artificial basal mem- establish animal models and investigations of the effects of cer- brane and the introduction of Batimastat, a synthetic MMP in- 110 Clinical and Experimental Otorhinolaryngology Vol. 7, No. 2: 106-111, June 2014 hibitor, can inhibit this transmigration in vitro [14]. The present CONFLICT OF INTEREST in vivo observations of significant decreases of eosinophils and infiltration of other inflammatory cells in the nasal mucosa of No potential conflict of interest relevant to this article was re- Dox-treated rats, support the in vitro results and strengthen the ported. validity of the animal model. Structural abnormalities seen in AR, which are collectively termed tissue remodeling, are caused by MMPs secreted from REFERENCES epithelial cells and fibroblasts in addition to infiltrating inflam- 1. Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, Sorsa T. Tet- matory cells [15]. MMPs are also reportedly responsible for mi- racyclines inhibit connective tissue breakdown by multiple non-an- crovascular permeability leading to edema, cell migration and timicrobial mechanisms. Adv Dent Res. 1998 Nov;12(2):12-26. ECM remodelling at the site of inflammation [2]. Therefore it is 2. Asano K, Kanai KI, Suzaki H. 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Published: May 21, 2014

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