Abstract
Objective
The aim of this study was to investigate the preventive effect of mitomycin-C (MMC) on external auditory canal (EAC) fibrosis in an animal model.
Study design
This is a randomized, controlled animal study.
Subjects and methods
Sixteen guinea pigs were used for this study. After the skin of cartilaginous EAC was injured with an electrocautery, the cottonoid soaked in MMC solution with concentration of 0.4 mg/mL was topically applied for 5 minutes to the injured EAC in an MMC-treated group (n = 8). In addition, saline was applied in the control group (n = 8). At 4 weeks after injury, postsurgical changes of EAC were evaluated by histologic examination.
Results
External auditory canal fibrosis was induced by injury with electrocautery. The MMC-treated group showed less degree of fibrosis without differences in epithelialization and inflammatory cell infiltration.
Conclusion
This study suggests that MMC can be helpful in preventing EAC fibrosis after injury.
1
Introduction
Acquired external auditory canal (EAC) stenosis is an uncommon disease entity with an annual incidence of 0.6 cases per 100 000 inhabitants . External auditory canal stenosis results from various etiologies including infection, trauma, surgery, radiation, and tumor . Chronic otitis externa is the most common cause of acquired EAC stenosis . Irrespective of etiologies, a common pathophysiology of EAC stenosis is a proliferative inflammation, leading to granulation tissue formation and ultimately fibrotic membranous atresia .
The surgical principle of treatment for EAC stenosis is complete resection of the fibrous plug, widening of EAC, and reepithelialization of EAC . However, the rate of restenosis, which is the most common postoperative complication, is reported up to 27% in the published literatures .
Mitomycin-C (MMC), isolated from Streptomyces caespitosus , is an antibiotic and antineoplastic agent that inhibits fibroblast proliferation and is used to prevent stenosis or scar tissue formation in the field of otolaryngology . Recently, Battelino et al showed that the intraoperative use of MMC appeared to have been helpful in preventing scarring in treatment of EAC stenosis, whereas Banthia and Selesnick reported that topical MMC was ineffective in preventing scar formation in the postsurgical EAC. Although these studies showed the opposite results, MMC might have the potential for the prevention of EAC stenosis. However, there have been no reports of experimental animal studies on the effects of MMC on the prevention of EAC stenosis.
The aim of this study was to investigate the preventive effect of MMC against fibrosis, key stage in developing EAC stenosis, in an experimentally induced EAC injury model.
2
Materials and methods
2.1
Study design
Sixteen male Dunkin-Hartly albino guinea pigs (body weight between 350 and 400 g) were used for this study. Animals were randomly allocated to 2 groups: a control group (saline-treated group) and a study group (MMC-treated group) with 8 animals per group. All experiments were performed with the approval of the Animal Experiment Committee of the Clinical Research Institute at Chungnam National University Hospital.
2.2
Surgical technique
Anesthesia was induced with intraperitoneal injection of ketamine (40 mg/kg) and xylazine (10 mg/kg). Additional local anesthesia was performed with packing a 5% lidocaine-soaked cottonoid into EAC. Under visualization using an operating microscope (Carl Zeiss, OPMI pico, Göttingen, Germany), the skin of ventral cartilaginous EAC in 1 mm lateral to bony-cartilaginous junction was injured with a microtip of an electrocautery setting at 10 W in coagulation mode. Because the circumferential injury of EAC in our preliminary experiment resulted in the complete stenosis due to very small diameter of guinea pig EAC, partial wound was produced in this study, although most of the inflammatory EAC stenosis showed circumferential narrowing.
To minimize variations in the depth of injury, thermal injury was made only contacting a microtip to EAC skin without any pressure on the skin.
Dorsal half of EAC was left intact for histologic comparison. Intraperitoneal cefazolin sodium (100 mg/kg) was administered. After the cessation of all procedures, the cottonoid soaked in MMC solution with concentration of 0.4 mg/mL was topically applied for 5 minutes to the injured EAC in the MMC-treated group. The cottonoid soaked in saline was applied in the control group.
All animals were housed for 4 weeks in approved animal care facility without packing in EAC.
2.3
Histologic analysis
After 4 weeks of the original injury, an overdose of sodium pentobarbital was administered to kill the animal, and the cartilaginous portion of EAC was harvested. All EAC were placed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Five-micrometer-thick sections were cut from the surface of each block through the injured portion of EAC. Hematoxylin and eosin staining and Masson trichrome staining were used to assess histologic changes such as epithelialization, inflammation, and fibrosis. The degree of epithelial regeneration was interpreted as the ratio of the area of epithelial covering to the area of the trauma. The number of infiltrated inflammatory leukocytes was counted in the high-power field (×400) of the lamina propria of EAC skin. The degree of fibrosis was examined and recorded on a subjective scale of 0 (none) to +3 (severe). The specimens were evaluated by a pathologist blinded to the group assignments. Three guinea pigs were used for comparison in assessing the histologic appearances of the EAC.
2.4
Statistical analysis
SPSS 12.0 for Windows (SPSS Inc, Chicago, IL) was used for statistical analysis. The Mann-Whitney U test was used to compare histologic differences between the groups, and histologic data were expressed as mean ± SD. P values of less than .05 were considered statistically significant.
2
Materials and methods
2.1
Study design
Sixteen male Dunkin-Hartly albino guinea pigs (body weight between 350 and 400 g) were used for this study. Animals were randomly allocated to 2 groups: a control group (saline-treated group) and a study group (MMC-treated group) with 8 animals per group. All experiments were performed with the approval of the Animal Experiment Committee of the Clinical Research Institute at Chungnam National University Hospital.
2.2
Surgical technique
Anesthesia was induced with intraperitoneal injection of ketamine (40 mg/kg) and xylazine (10 mg/kg). Additional local anesthesia was performed with packing a 5% lidocaine-soaked cottonoid into EAC. Under visualization using an operating microscope (Carl Zeiss, OPMI pico, Göttingen, Germany), the skin of ventral cartilaginous EAC in 1 mm lateral to bony-cartilaginous junction was injured with a microtip of an electrocautery setting at 10 W in coagulation mode. Because the circumferential injury of EAC in our preliminary experiment resulted in the complete stenosis due to very small diameter of guinea pig EAC, partial wound was produced in this study, although most of the inflammatory EAC stenosis showed circumferential narrowing.
To minimize variations in the depth of injury, thermal injury was made only contacting a microtip to EAC skin without any pressure on the skin.
Dorsal half of EAC was left intact for histologic comparison. Intraperitoneal cefazolin sodium (100 mg/kg) was administered. After the cessation of all procedures, the cottonoid soaked in MMC solution with concentration of 0.4 mg/mL was topically applied for 5 minutes to the injured EAC in the MMC-treated group. The cottonoid soaked in saline was applied in the control group.
All animals were housed for 4 weeks in approved animal care facility without packing in EAC.
2.3
Histologic analysis
After 4 weeks of the original injury, an overdose of sodium pentobarbital was administered to kill the animal, and the cartilaginous portion of EAC was harvested. All EAC were placed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Five-micrometer-thick sections were cut from the surface of each block through the injured portion of EAC. Hematoxylin and eosin staining and Masson trichrome staining were used to assess histologic changes such as epithelialization, inflammation, and fibrosis. The degree of epithelial regeneration was interpreted as the ratio of the area of epithelial covering to the area of the trauma. The number of infiltrated inflammatory leukocytes was counted in the high-power field (×400) of the lamina propria of EAC skin. The degree of fibrosis was examined and recorded on a subjective scale of 0 (none) to +3 (severe). The specimens were evaluated by a pathologist blinded to the group assignments. Three guinea pigs were used for comparison in assessing the histologic appearances of the EAC.
2.4
Statistical analysis
SPSS 12.0 for Windows (SPSS Inc, Chicago, IL) was used for statistical analysis. The Mann-Whitney U test was used to compare histologic differences between the groups, and histologic data were expressed as mean ± SD. P values of less than .05 were considered statistically significant.
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