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Please check this box or indicate your approval if you have no corrections to make to the PDF file Thank you for your assistance. G Model PEDOT 7365 1–5 International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx Contents lists available at ScienceDirect International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl 1 2 3 4 5 6 7 8 9 10 Histopathological evaluation of Ankaferd blood stopper use in the rabbit septoplasty model Orhan a,*, Remzi Dogan b, Erkan Soylu c, Fadlullah Aksoy d, Bayram Veyseller d, Orhan Ozturan d, Mukaddes Esrefoglu e, Mehmet Serif Aydın e Q1 Israfil a Department of Otorhinolaryngology, Kahramanmaras Sutcu Imam University Medical Faculty, Kahramanmaras, Turkey Bayrampasa State Hospital, Department of Otorhinolaryngology, Bayrampasa, Istanbul, Turkey Department of Otorhinolaryngology, Istanbul Medipol University Medical Faculty, Istanbul, Turkey d Department of Otorhinolaryngology, Bezmialem Vakif University, Fatih, Istanbul, Turkey e Department of Histology and Embryology, Bezmialem Vakif University Medical Faculty, Fatih, Istanbul, Turkey b c A R T I C L E I N F O A B S T R A C T Article history: Received 9 September 2014 Received in revised form 15 October 2014 Accepted 14 November 2014 Available online xxx Objective: The purpose of this study was to assess the use of Ankaferd blood stopper (ABS), an organic hemostatic agent of plant origin, in septoplasty operations, and to determine its effect on nasal septal tissues in the rabbit model. Method: The study was performed on 30 New Zealand adult male rabbits each weighing 2500–3500 g (average: 3000 g). The rabbits were randomly divided into 4 groups, namely, the control group (without septoplasty), the septoplasty + tampon group, the septoplasty + ABS group, and the septoplasty + tampon + ABS group. All animals were sacrificed after two weeks, and the nasal septums were total removed using the lateral rhinotomy technique. The specimens were obtained from similar sites of cartilaginous nasal septum. The sections were stained with hematoxylin–eosin and Mason trichrome stains and studied under the light microscope by the same pathologist who evaluated the mucosal ulcerations, the severity of inflammatory cell infiltration, the mucosal thickness, and the cartilage thickness. The data obtained were statistically analyzed using the Kruskal–Wallis variance analysis and the Mann–Whitney U test. Results: No mucosal ulceration or inflammatory cell infiltration was detected in any of the rabbit groups. There was a statistically significant difference between the groups in terms of mucosal thickness and cartilage thickness (p < 0.05). The values of mucosal thickness in the groups mentioned above were 147.7  17.6 mm, 205.7  36.7 mm, 139.6  14.8 mm, and 190.2  17.5 mm, respectively. The values of cartilage thickness were 398.2  28.9 mm, 546.2  35.3 mm, 363.7  24.7 mm, and 447.8  28.2 mm, respectively. There was no significant difference between the control group and the septoplasty + ABS group in terms of mucosal thickness and cartilage thickness (p > 0.05). However, there was a significant increase in nasal mucosal and cartilage thickness in tampon-using groups when compared with the other groups (p < 0.05). Conclusion: Although nasal tampons provide the contact of mucoperichondrium with the cartilage, they are generally accepted as a discomfort for patients. Ankaferd blood stopper can be used instead of nasal tampons to increase patient comfort. ß 2014 Elsevier Ireland Ltd. All rights reserved. Keywords: Rabbit Ankaferd blood stopper Septoplasty 11 1. Introduction * Corresponding author at: Kahramanmaraş Sütçü Imam University Medical Faculty, Department of Otorhinolaryngology, Kahramanmaraş, Turkey. Tel.: +90 344 280 34 34; fax: +90 344 280 40 52. E-mail address: israfil.orhan@mynet.com (I. Orhan). 12 The septoplasty operation is one of the techniques frequently Q213 used by otorhinolaryngologists. Intranasal tampons and stents are 14 widely used in cases undergoing septoplasty to prevent postoper- 15 ative hemorrhage, to provide stabilization of the mobilized 16 surrounding tissues, and to prevent adhesions [1,2]. Although 17 nasal tampons provide the contact of mucoperichondrium with the 18 cartilage, they are generally accepted as a discomfort for patients. 19 http://dx.doi.org/10.1016/j.ijporl.2014.11.015 0165-5876/ß 2014 Elsevier Ireland Ltd. All rights reserved. Please cite this article in press as: I. Orhan, et al., Histopathological evaluation of Ankaferd blood stopper use in the rabbit septoplasty model, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.015 G Model PEDOT 7365 1–5 I. Orhan et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx 2 (average, 3000 g). The rabbits were randomly divided into four groups: Control (no septoplasty). Septoplasty + tampon. Septoplasty + ABS. Septoplasty + tampon + ABS. 45 46 47 48 The rabbits in all groups were anesthesized with xylazine hydrochloride 10 mg/kg (Rompun, Bayer Drugs, Istanbul, Turkey) and 50 mg/kg ketamine hydrochloride 59 mg/kg (Ketalar, Eczacı- Q3 başı Drugs, İstanbul, Turkey). Prior to surgical interventions, prophylactic cefazolin sodium 20–40 mg/kg (Cefozin, Bilim İlaç, Istanbul, Turkey) was administered to each rabbit. The open rhinoplasty technique was used to reach the nasal septum in the rabbits (Fig. 1). After a columellar incision and lifting of the skin, by approaching from the nasal dorsum, the upper lateral cartilages were separated from the septum with sharp dissection. Subsequently, with the help of an elevator, the mucoperichondrium was raised with blunt dissection, paying attention not to harm the nasal septal cartilage. The rabbits in Group 2 underwent bilateral mucoperichondrial flap elevation. Following elevation, miniature Doyle nasal splints were placed in both nasal cavities. The operation was brought to an end by suturing the incisions. The rabbits in Group 3 underwent bilateral mucoperichondrial flap elevation. Following elevation, ABS 1 ml/puff  2 was administered into the space between the septal cartilage and the mucoperichondrium. The operation was completed by suturing the incisions. The rabbits in Group 4 underwent bilateral mucoperichondrial flap elevation. Following the elevation, ABS 1 ml/puff  2 was administered into the space between the septal cartilage and the mucoperichondrium. Miniature Doyle nasal splints were placed in both nasal cavities. The operation was brought to an end by suturing the incisions. Forty-eight hours after septoplasty, the nasal tampons of the rabbits in Group 2 and Group 4 were pulled out. After two weeks, all rabbits were sacrificed under ketamine hydrochloride (Ketalar, Eczacıbaşı İlaç, İstanbul, Turkey) anesthesia according the Guidelines of the National Research Council Laboratory Animals Resource Institute, and their nasal septums were totally removed. The specimens were obtained from similar sites of the septa. After having passed the specimens through the steps of fixation in 10% formalin for 24 h , alcohol, xylene, liquid paraffin, and paraffin blocking, 5-mm sections were prepared from the paraffin blocks. After being stained with hematoxylin –eosin and 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 Group Group Group Group Fig. 1. The open rhinoplasty technique was used to reach the nasal septum in the rabbits. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 In order to increase patient comfort and avoid nasal tampons, some techniques such as trans-septal suturation or use of fibrin glue have been tested [3–6], but there is no generally accepted technique at the present time. Ankaferd blood stopper (ABS) is a topical hemostatic agent that consists of hemostatic herbal extracts and contains no inorganic or synthetic additives. It is used in postoperative and posttraumatic hemorrhages. Ankaferd blood stopper accelerates the formation of an encapsulated protein web by interacting with all blood proteins and fibrinogen particles [7,8]. Although ABS has been reported to be an effective topical agent used against mucosal bleedings such as in gastrointestinal hemorrhages, urological surgery, adenoidectomy and tonsillectomy, and anterior epistaxis [9–13], there has been no study on its use in septoplasty. The purpose of the present study was to assess the use of ABS, an organic hemostatic agent of herbal origin, in septoplasty operations and to determine its effect on nasal septal tissues in the rabbit model. 38 2. Materials and method 39 40 41 The study was started after having obtained the approval of the Local Ethics Committee (IRB: 2013/230). The study was performed on 30 New Zealand adult male rabbits each weighing 2500–3500 g 42 43 1 2 3 4 (n = 6): (n = 8): (n = 8): (n = 8): Fig. 2. Mucosal and cartilage thickness of Group 3 and the Group 1 (c: cartilage, m: mucosa, e: epithelium). Please cite this article in press as: I. Orhan, et al., Histopathological evaluation of Ankaferd blood stopper use in the rabbit septoplasty model, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.015 G Model PEDOT 7365 1–5 I. Orhan et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx 3 Fig. 3. Mucosal and cartilage thickness of Group 3 and the Group 2. (c: cartilage, m: mucosa, e: epithelium). 86 87 88 89 Masson-trichrome stains, the sections, were studied under the light microscope (Olympus, BX51, Japan) by the same pathologists and evaluated histopathologically in terms of the parameters mentioned below: 92 91 93 94 95 96 97 98 1. 2. 3. 4. 99 100 101 102 103 The thicknesses of the mucosa and the cartilage were measured with a micrometer under the 100 objective of the microscope. The other parameters were evaluated using the semiquantitative scoring with points as follows: No change: 0; mild change: 1; moderate change: 2; and marked change: 3. 104 2.1. Statistical analysis 105 106 107 The SPSS (Data 10.0) package program was used for the statistical analysis. The Kruskal–Wallis variance analysis was used for comparison of the parameters in all groups, and the Mann–Whitney Mucosal ulceration. Severity of inflammatory cell infiltration. Mucosal thickness. Cartilage thickness. U test was performed for the comparison of two populations. A p value of <0.05 was accepted as statistically significant. 108 109 3. Results 110 3.1. Mucosal ulceration and inflammatory cell infiltration 111 No mucosal ulceration or inflammatory cell infiltration was detected in any of the rabbits in all groups. 112 113 3.2. Mucosal thickness 114 The mucosal thickness in Group 1, 2, 3, and 4 measured 147.7  17.6 mm, 205.7  36.7 mm, 139.6  14.8 mm, and 190.2  17.5 mm, respectively. There was no significant difference in mucosal thickness between Group 3 and the control group (Group 1) (p > 0.05) (Fig. 2). However, there was a significant difference in the mucosal thickness between Group 3 and Group 2 (p < 0.05) (Fig. 3). Similarly, there was a significant difference in the mucosal thickness between Group 3 and Group 4 (p < 0.05) (Fig. 4). There was 115 116 117 118 119 120 121 122 Fig. 4. Mucosal and cartilage thickness of Group 3 and the Group 4. (c: cartilage, m: mucosa, e: epithelium). Please cite this article in press as: I. Orhan, et al., Histopathological evaluation of Ankaferd blood stopper use in the rabbit septoplasty model, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.015 G Model PEDOT 7365 1–5 I. Orhan et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx 4 Fig. 5. Mucosal and cartilage thickness of Group 2 and the Group 4. (c: cartilage, m: mucosa, e: epithelium). 123 124 no significant difference in the mucosal thickness between Group 2 and Group 4 (p > 0.05) (Fig. 5). 125 3.3. Cartilage thickness 126 127 128 129 130 131 132 133 134 135 136 137 The cartilage thickness in Group 1, 2, 3, and 4 measured 398.2  28.9 mm, 546.2  35.3 mm, 363.7  24.7 mm, and 447.8  28.2 mm, respectively. There was no significant difference in the cartilage thickness between Group 3 and the control group (Group 1) (p > 0.05) (Fig. 2). However, there was a significant difference in the cartilage thickness between Group 3 and Group 2 (p < 0.05) (Fig. 3). Similarly, there was a significant difference in the cartilage thickness between Group 3 and Group 4 (p < 0.05) (Fig. 4). There was no significant difference in the cartilage thickness between Group 2 and Group 4 (p > 0.05) (Fig. 5). The comparison of mucosal and cartilage thickness between the groups has been summarized in Figs. 6 and 7. 138 4. Discussion 139 140 141 142 Nasal tampons are used to stop bleeding, to provide cartilagebone stabilization, and to prevent septal abscess and hematoma formation in all types of intranasal surgery. For these purposes, various nasal tampon materials have been described [14]. Although Q4 Fig. 6. The comparison of mucosal thickness between the groups. methods such as use of nasal tampon, use of septal splint, and transseptal sutures can prevent complications; they also have some disadvantages [4,5]. Nasal tampons have the advantages of being easily accessible, being inexpensive, and not prolonging surgery. On the other hand, they may cause some problems that would decrease the quality of life, such as synechia, septal perforation, dysfunction of the Eustachian tube, tampon allergy, toxic shock syndrome, mouth dryness, sore throat, infection, and aspiration [4–6]. Nasal tampons have been reported to make the patients more prone to obstructive sleep apnea and hypoxia. These complications are more marked in the elderly who have systemic disorders such as obstructive sleep apnea, chronic obstructive pulmonary disease, and ischemic heart disease [15]. Furthermore, the pain and bleeding that occur during pulling out the tampons can be a discomfort, both for the patient and the doctor. Because of these disadvantages, nasal tampons are increasingly being replaced by the septal suture method. In their study comparing the nasal tampon with the suture technique in the rabbit model, Genç et al. [16] found no significant difference between the tampon and the suture groups and the control group in terms of mucosal thickness, but found a significant increase in perichondrial thickness in both test groups compared to the control group. The authors associated this situation with the high reactivity of the rabbit tissue induced by perichondrial elevation. They also concluded that the cartilage is thinner in the transseptal suture Fig. 7. The comparison of cartilage thickness between the groups. Please cite this article in press as: I. Orhan, et al., Histopathological evaluation of Ankaferd blood stopper use in the rabbit septoplasty model, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.11.015 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 G Model PEDOT 7365 1–5 I. Orhan et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 group than in the nasal tampon group [16]. In their series of 226 septoplasty cases with septal suturing, but without nasal tampon, Lemmens and Lemkens [17] observed no complications such as bleeding, septal hematoma, septal perforation, and synechia. In patients with septal suture, pull-out is no problem and post-operative comfort is better, but the duration of surgery is slightly longer. Septal splints are widely used for stabilization of septal mucoperichondrial flaps. However, in their prospective randomized study, Malki et al. [18] have reported that patients with installed splints have more pain than patients without the splint. The use of fibrin glue seems practical, but it has the disadvantage of a high cost. As far as we know, our study is the first study which histopathologically evaluates the use of ABS in the rabbit septoplasty model. The goal of this study was to minimize bleeding and hence prevent hematoma by applying the hemostatic agent ABS between the septal cartilage and the mucoperichondrium. The results we have obtained demonstrate that the goal is reached. We found that the use of ABS causes no inflammation or ulceration in the nasal septum. Moreover, the mucosal and cartilage thicknesses in the ABS group were similar to those in the control group. These findings have led us to suggest that the use of ABS may be a safe method that can be an alternative to tampon use. In today’s world, almost everyone wants to return to work after surgery and spend his daily life in comfort. For this reason, surgical interventions are performed in such a way as to minimize the postoperative discomfort. In our study, the use of ABS was assessed to prevent intra-operative bleeding in septoplasty operations and to avoid post-operative discomfort caused by nasal tampons. Although there have been many studies on the use of ABS in rhinology, our study is the first study on the successful use of ABS in septoplasty. 201 5. Conclusion 202 203 204 205 206 207 208 209 Our pilot study is the first in the literature which histopathologically evaluates the use of ABS in the rabbit septoplasty model. The results of our study suggest that the use of ABS does not cause inflammation or ulceration in the nasal septum. Furthermore, the thicknesses of the nasal mucosa and cartilage in the ABS group were similar to those in the control group. These findings have led us to consider the use of ABS as a possible safe method that can be an alternative to tampon use. Studies on this subject are limited in 5 number. 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