ARTICLE IN PRESS Effects of Smoke Generated by Electrocautery on the Larynx *Yavuz Atar, *Ziya Salturk, *Tolgar Lutfi Kumral, *Yavuz Uyar, †Caglar Cakir, ‡Gurcan Sunnetci, and *Guler Berkiten, *†Istanbul and ‡Kastamonu, Turkey Summary: Objective. The aim of this study was to investigate effects of smoke produced by electrocautery on the laryngeal mucosa. Materials and Methods. We used 16 healthy, adult female Wistar albino rats. We divided the rats into two groups. Eight rats were exposed to smoke for 60 min/d for 4 weeks, and eight rats were not exposed to smoke and served as controls. The experimental group was maintained in a plexiglass cabin during exposure to smoke. At the end of 4 weeks, rats were sacrificed under high-dose ketamine anesthesia. Each vocal fold was removed. An expert pathologist blinded to the experimental group evaluated the tissues for the following: epithelial distribution, inflammation, hyperplasia, and metaplasia. Mucosal cellular activities were assessed by immunohistochemical staining for Ki67. Results taken before and after effect were compared statistically. Results. There was a significant difference in the extent of inflammation between the experimental group and the control group. Squamous metaplasia was detected in each group, but the difference was not significant. None of the larynges in either group developed hyperplasia. Conclusions. We showed increased tissue inflammation due to irritation by the smoke. Key Words: Electrocautery–Smoke–Metaplasia–Inflammation–Larynx. INTRODUCTION In 1985, the National Institute for Occupational Safety and Health recognized smoke generated by electrocautery as a potentially hazardous chemical to health.1 This announcement by the National Institute for Occupational Safety and Health raised awareness, and consequently, several studies focusing on the chemical contents of this type of smoke were published.2–6 Exposure to smoke from electrocautery is unavoidable in procedures that require both general and local anesthesia, and thus, not only health-care professionals but also patients can easily become exposed. Smoke contents include dead and living cellular material,7,8 blood fragments,9 bacteria,10,11 viruses,12–15 toxic gases and vapors,3,16 and lung-damaging particulates.17 Hensman et al16 reported the presence of 21 toxic chemicals in electrocautery smoke produced in closed environments. Al Sahaf et al3 also detected toxic material and found that the composition of the smoke that is retained in various tissues differs among tissue type. As a result, the United States recommended local exhaust ventilation to protect health-care professionals.1 Although toxic materials have been detected in smoke generated by electrocautery and the risks are apparent, limited studies have investigated the effects of this smoke on the respiratory system.17–19 Therefore, the goal of this study was to investigate the effects of exposure to external smoke generated by electrocautery on the laryngeal mucosa. Accepted for publication May 16, 2016. From the *ENT Clinic, Okmeydani Training and Research Hospital, Istanbul, Turkey; †Pathology Clinic, Okmeydani Training and Research Hospital, Istanbul, Turkey; and the ‡ENT Clinic, Taskopru Government Hospital, Kastamonu, Turkey. Address correspondence and reprint requests to Yavuz Atar, Department of Otorhinolaryngology—Head and Neck Surgery, Okmeydani Training and Research Hospital, Darulaceze Cad. No:25, Okmeydani—Sisli, Istanbul, Turkey. E-mail: [email protected] Journal of Voice, Vol. ■■, No. ■■, pp. ■■-■■ 0892-1997 © 2016 The Voice Foundation. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvoice.2016.05.012 MATERIALS AND METHODS Approval from the Institutional Review Board was obtained from the Istanbul University Experimental Animal Research Ethics Committee. In total, we used 16 healthy, adult female Wistar albino rats (weight range: 200–250 g, age range: 7–8 months). The experimental group was maintained in a plexiglass cabin during exposure to smoke. In the cabin, there were 10 holes (3 cm in diameter) to provide fresh air and allow the smoke to exit. For entry of electrocautery smoke into the cabin, we created a cone shaped hole 12 cm in the base of the cabin. The source of smoke was a PETKOK 500s electrocauterizer (PENTAS, Ankara, Turkey) in monopolar mode. We used a power of 35 watts for cut mode and 30 watts for coagulation mode for 30 minutes on sheep liver.20 We chose a current of 3 amps and a frequency of 468 kHz. We divided the 16 rats into two groups. Eight rats were exposed to smoke for 60 min/d for 4 weeks, and eight rats were not exposed to smoke and served as controls. The animals had free access to food and water and were kept under a 12 h:12 h lightdark cycle at 25°C. At the end of 4 weeks, rats were sacrificed under high-dose ketamine anesthesia. Each vocal fold was removed intact and stored in 10% (v/v) buffered formalin solution. Specimens were cut into 5-mm-thick sections. Standard tissue-processing methods were applied, and sections were stained with hematoxylin and eosin before analysis by light microscopy. An expert pathologist blinded to the experimental group evaluated the tissues for the following: epithelial distribution, inflammation, hyperplasia, and metaplasia. Inflammation was graded based on cell density, where grade 1 represented scattered inflammatory cells, grade 3 represented inflammatory cell density that forms lymphoid follicles, and grade 2 represented a cell density between grades 1 and 3. Mucosal cellular activities were assessed by immunohistochemical staining for Ki67, which is expressed during all phases ARTICLE IN PRESS 2 Journal of Voice, Vol. ■■, No. ■■, 2016 of the cell cycle except for G0. Expression of Ki67 closely parallels [3H]-thymidine incorporation, which is a standard method of measuring cell proliferation. Immunostaining of normal tissue for the Ki67 antigen reveals nuclear activity in cells within the germinal centers of cortical follicles, cortical thymocytes, and neck cells of the gastrointestinal mucosa. Resting cells, such as hepatocytes, renal cells, and Paneth cells of the gastrointestinal mucosa, are not stained. For immunohistochemical evaluation, formalin-fixed paraffinembedded tissue blocks were cut into 5-μm-thick sections and mounted on poly L-lysine coated slides, followed by staining with an anti-Ki67 antibody (1:100 dilution, clone: BGX-Ki67; BioGenex, San Ramon, CA, USA). All procedures were performed using the standard Leica BOND-MAX autostainer protocol (Leica Biosystems, Bannockburn, IL), with positive and negative controls included. Any identifiable nuclear staining, regardless of intensity, was recorded as positive. Distribution of Ki67 in the basal and parabasal areas of the laryngeal mucosa was evaluated. Statistical analysis was performed using SPSS Statistics 17 (SPSS Inc., Chicago, Illinois, USA). Pearson chi-squared test was used for data analysis. Because of the epithelial distributions and cellular activities of the mucosa being identical in both groups, we did not analyze these data. A P value <0.05 was considered statistically significant. RESULTS No macroscopic differences were evident between the laryngeal tissue from the study group and the laryngeal tissue from the control group. Microscopically, three types of epithelial cells were detected in the control group. The ventral part of the glottis was composed of striated ciliary epithelia adjacent to loose connective tissue. The free margins of the vocal folds and adjacent areas were covered by striated non-ciliated columnar epithelium. The arytenoid region and adjacent areas were covered with striated squamous epithelium, which was thinner on the arytenoid cartilage. The laryngeal mucosa of the study group exhibited histopathologic features similar to the control group, showing typical epithelial linings and basal membranes. There was a significant difference in the extent of inflammation between the experimental group and the control group (P = 0.026) (Table 1). Squamous metaplasia was detected in one of the eight rats from the study group, as well as in one rat from the control group (Table 2); the difference was not significant (P = 0.707). None of the larynges in either group developed hyperplasia. Ki67 expression levels were measured to evaluate cellular activity of the mucosal specimens. In the experimental group, five rats had laryngeal tissue that showed basal proliferation and three TABLE 1. Results of Inflammation Grades Inflammation 1+ 2+ 3+ Total Control 1 (12.5%) 5 (62.5%) 2 (25%) 8 (100%) Smoke-exposed 2 (25%) 0 (0%) 6 (75%) 8 (100%) Total 3 (18.7%) 5 (31.3%) 8 (50%) 16 (100%) Pearson chi-square: P = 0.026. TABLE 2. Results of Squamous Metaplasia Metaplasia Control Smoke-exposed Total Negative Positive Total 7 (87.5%) 7 (87.5%) 14 (87.5%) 1 (12.5%) 1 (12.5%) 2 (12.5%) 8 (100%) 8 (100%) 16 (100%) Fisher exact test: P = 0.767. TABLE 3. Results of Ki67 Expression Levels Ki67 Control Smoke-exposed Total Basal Parabasal Total 8 (100%) 5 (62.5%) 13 (81.3) 0 (0%) 3 (37.5%) 3 (18.7%) 8 (100%) 8 (100%) 16 (100%) Fisher exact test: P = 0.100. rats showed parabasal proliferation. In the control group, all laryngeal tissue had basal proliferation patterns (Table 3); no intragroup differences were observed (P = 0.100). DISCUSSION This is the first study investigating the effects of electrocautery smoke in the rat larynx. Our study revealed that externally generated electrocautery smoke caused inflammation in the larynx. However, no difference in metaplasia and Ki67 staining was observed between the two groups. Previous studies have focused mainly on the hazardous content and the concentration of these chemicals within this type of smoke. The most prominent hazardous chemicals within this smoke are as follows: aliphatic hydrocarbons, amines, polycyclic hydrocarbons, aromatic hydrocarbons (eg, benzene), toluene, and phenol.3,4,21–27 Gianella et al21 reported no differences between monopolar and bipolar diathermy with regard to the concentration and content within smoke. Al Sahaf et al3 found that the concentration of toluene, ethylbenzene, and xylene in smoke generated from electrocautery was similar to the concentrations reported for cigarette smoke.28 In contrast, Fitzgerald et al2 stated that both electrocautery smoke and ultrasonic scalpel smoke did have carcinogenic compounds, but that the concentration was less than that observed in cigarette smoke. However, they suggested that cumulative exposure may be a health concern. Wu et al4 found that the patient’s age, surgery type, coagulation energy, and duration of the surgery were indicators of the concentration of toluene. They suggested that these factors should be considered when studying the health risks. There are a limited number of studies that have investigated the effect of electrocautery smoke on humans. Navarro-Meza et al19 performed a study with physicians and concluded that neurosurgeons had the highest rate of exposure to smoke, but that all physicians had respiratory symptoms such as sensation of a lump in the throat, sore throat, and nasal congestion caused by electrocautery smoke. ARTICLE IN PRESS Yavuz Atar et al Effects of Smoke Generated by Electrocautery The main limitations associated with this study were that we evaluated only one type of tissue and that the smoke was delivered only for a short duration. Changing these parameters may alter the effect on various tissue types and thus should be investigated in future studies. 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