Abstract
Purpose
Wound healing of the nasal mucosa is a highly complex process that restores the anatomical and functional integrity of tissue that has been exposed to trauma. In this experimental study, our aim was to use histopathological examination to investigate the effects of caffeic acid phenethyl ester on the wound healing of rat nasal mucosa after mechanical trauma.
Materials and methods
The rats were randomly divided into 3 experimental groups: a non-treated group (n = 7), a control saline group (n = 7) and a caffeic acid phenethyl ester group (n = 7). The non-treated group received no treatment for 15 days. The second group was administered saline (2.5 mL/kg, intraperitoneal) once a day for 15 days. The third group received caffeic acid phenethyl ester intraperitoneally at a dose of 10 μmol/kg once a day for 15 days. At the beginning of the study, unilateral mechanical nasal trauma was induced on the right nasal mucosa of all rats in the three groups using a brushing technique. Samples were stained using hematoxylin and eosin solution and were examined by a pathologist using a light microscope.
Results
The severity of inflammation was milder in the caffeic acid phenethyl ester group compared with that in the non-treated and saline groups (P < 0.05). The subepithelial thickness index was lower in the experimental group (P < 0.05). Goblet cell and ciliated cell loss was substantially reduced in the experimental group compared with the non-treated and saline groups (P < 0.05).
Conclusions
Caffeic acid phenethyl ester decreases inflammation and the loss of goblet cells and ciliated cells. Therefore, caffeic acid phenethyl ester has potential beneficial effects on the wound healing of nasal mucosa in the rat.
1
Introduction
The nasal mucosa has many functions, such as temperature regulation by heating the air and protection of the airway by filtering foreign bodies . Nasal mucosa, which is lined by respiratory epithelium, needs a healthy mucociliary transport mechanism, cell structure and anatomy to perform these functions. A problem in the cell structure or the anatomy may also disrupt the mucociliary clearance function of nasal mucosa. The importance of epithelial cells, secretory glands and ciliated cells has been shown in many studies . Wound healing is a highly complex process that restores the anatomical and functional integrity of tissues that have been exposed to trauma due to either mechanical factors or harmful chemical substances. There are a limited number of studies that examine the mechanical trauma after the most commonly performed otorhinolaryngological surgeries such as endoscopic sinus surgery and septoplasty. These studies were generally performed to show the effects of nasal corticosteroid sprays and nasal irrigation after endoscopic sinus surgery . In this experimental study, our aim was to demonstrate the histopathological effects of caffeic acid phenethyl ester (CAPE) on the wound healing of rat nasal mucosa after mechanical trauma. It has been shown in many studies that CAPE has antioxidant, antiinflammatory, antiviral, immunomodulatory and anticarcinogenic effects . Thus, we aimed to shed light on problems with healing that can affect the success of nasal surgeries using a rat model.
2
Materials and methods
The study was performed according to the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health, Commission on Life Sciences and the National Research Council . The study protocol was approved by the Ethics Committee of our institution with document number 2013/08.
Twenty-one male adult Wistar albino rats weighing between 235 and 330 g were used in this study. The rats were kept under suitable conditions in accordance with the standard guidelines and in suitable cages that were maintained under standard environmental conditions (room temperature between 22 and 24 °C, 50% relative humidity and 12-hour light and dark cycles). The animals had free access to water and were fed with conventional laboratory diet until they were euthanized. The rats were randomly divided into 3 groups: the non-treated group (n = 7), the control saline group (n = 7) and the CAPE group (n = 7). The non-treated group received no treatment for 15 days. The second group was administered saline (2.5 mL/kg, intraperitoneal) once a day for 15 days. We injected CAPE (Sigma-Aldrich Co LLC, St. Louis, MO) intraperitoneally at a dose of 10 μmol/kg once a day for 15 days; at this concentration, CAPE completely blocks the production of reactive oxygen species in human neutrophils and the xanthine/xanthine oxidase system . Intraperitoneally injected ketamine hydrochloride (60 mg/kg; Ketalar, Pfizer, Istanbul, Turkey) and 2% xylazine hydrochloride (10 mg/kg; Rompun, Bayer, Istanbul, Turkey) were used as anesthesia. Unilateral mechanical nasal trauma was performed on the right nasal mucosa of all rats in the three groups using a brushing technique. An interdental brush was used to perform the mechanical trauma. Inflammation in rat nasal mucosa has been shown to reach a peak after 14 days . Therefore, we concluded the study on day 15.
2.1
Tissue preparation
All surgical procedures were carried out under clean but non-sterile conditions. After the induction of anesthesia, the rats were decapitated at the end of 15 days. The nose of each rat was removed by microdissection. The rats’ noses were fixed in 10% formaldehyde solution for 24 hours and decalcified in 10% ethylenediamine tetraacetic acid (EDTA) solution for 3 weeks. After the fixation and decalcification processes, the nasal septa were carefully removed using scissors. Then, the nasal septa were rinsed in tap water for 24 hours, dehydrated using a graded alcohol series, rendered transparent and blocked after infiltration with paraffin. The paraffin-embedded samples were sliced with a microtome (Microm HM 360) to a thickness of 5 μm, and the obtained cross-sections were stained with hematoxylin and eosin (H&E) before examination with a Nikon ECLIPSE 80i microscope. The stained specimens were evaluated by the same pathologist, who was blinded to the study groups. The intensity of goblet cell loss and ciliated cell loss were calculated by comparison of injured site with contralateral side. The intensity of inflammation was evaluated just subjectively. The grade of inflammation, goblet cell loss and ciliated cell loss were histologically evaluated and scored as follows: (+) mild, (++) moderate and (+++) severe. The epithelial thickness index (ETI) and subepithelial thickness index (STI) were scored as positive (+) or negative (−). These evaluations and measurements were performed according to procedures reported in two previous studies of wound healing indices . The ETI and STI values were obtained by calculating the ratio of the average height of the newly regenerated epithelium or subepithelial tissue at the wound site to the height of the average epithelium or subepithelium from the side without the wound. If this ratio was greater than 1, it was considered (+); if not, it was considered (−).
2.2
Statistical analysis
The statistical analyses were performed using the SPSS 15.0 software package for Windows (SPSS Inc., Chicago, IL). The variations in the histological categories between the control, saline and CAPE groups were compared using the Chi-squared test. We used the Kruskal–Wallis test to compare continuous variables among the 3 groups. Statistical significance was accepted for P values below 0.05 (P < 0.05).
2
Materials and methods
The study was performed according to the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health, Commission on Life Sciences and the National Research Council . The study protocol was approved by the Ethics Committee of our institution with document number 2013/08.
Twenty-one male adult Wistar albino rats weighing between 235 and 330 g were used in this study. The rats were kept under suitable conditions in accordance with the standard guidelines and in suitable cages that were maintained under standard environmental conditions (room temperature between 22 and 24 °C, 50% relative humidity and 12-hour light and dark cycles). The animals had free access to water and were fed with conventional laboratory diet until they were euthanized. The rats were randomly divided into 3 groups: the non-treated group (n = 7), the control saline group (n = 7) and the CAPE group (n = 7). The non-treated group received no treatment for 15 days. The second group was administered saline (2.5 mL/kg, intraperitoneal) once a day for 15 days. We injected CAPE (Sigma-Aldrich Co LLC, St. Louis, MO) intraperitoneally at a dose of 10 μmol/kg once a day for 15 days; at this concentration, CAPE completely blocks the production of reactive oxygen species in human neutrophils and the xanthine/xanthine oxidase system . Intraperitoneally injected ketamine hydrochloride (60 mg/kg; Ketalar, Pfizer, Istanbul, Turkey) and 2% xylazine hydrochloride (10 mg/kg; Rompun, Bayer, Istanbul, Turkey) were used as anesthesia. Unilateral mechanical nasal trauma was performed on the right nasal mucosa of all rats in the three groups using a brushing technique. An interdental brush was used to perform the mechanical trauma. Inflammation in rat nasal mucosa has been shown to reach a peak after 14 days . Therefore, we concluded the study on day 15.
2.1
Tissue preparation
All surgical procedures were carried out under clean but non-sterile conditions. After the induction of anesthesia, the rats were decapitated at the end of 15 days. The nose of each rat was removed by microdissection. The rats’ noses were fixed in 10% formaldehyde solution for 24 hours and decalcified in 10% ethylenediamine tetraacetic acid (EDTA) solution for 3 weeks. After the fixation and decalcification processes, the nasal septa were carefully removed using scissors. Then, the nasal septa were rinsed in tap water for 24 hours, dehydrated using a graded alcohol series, rendered transparent and blocked after infiltration with paraffin. The paraffin-embedded samples were sliced with a microtome (Microm HM 360) to a thickness of 5 μm, and the obtained cross-sections were stained with hematoxylin and eosin (H&E) before examination with a Nikon ECLIPSE 80i microscope. The stained specimens were evaluated by the same pathologist, who was blinded to the study groups. The intensity of goblet cell loss and ciliated cell loss were calculated by comparison of injured site with contralateral side. The intensity of inflammation was evaluated just subjectively. The grade of inflammation, goblet cell loss and ciliated cell loss were histologically evaluated and scored as follows: (+) mild, (++) moderate and (+++) severe. The epithelial thickness index (ETI) and subepithelial thickness index (STI) were scored as positive (+) or negative (−). These evaluations and measurements were performed according to procedures reported in two previous studies of wound healing indices . The ETI and STI values were obtained by calculating the ratio of the average height of the newly regenerated epithelium or subepithelial tissue at the wound site to the height of the average epithelium or subepithelium from the side without the wound. If this ratio was greater than 1, it was considered (+); if not, it was considered (−).
2.2
Statistical analysis
The statistical analyses were performed using the SPSS 15.0 software package for Windows (SPSS Inc., Chicago, IL). The variations in the histological categories between the control, saline and CAPE groups were compared using the Chi-squared test. We used the Kruskal–Wallis test to compare continuous variables among the 3 groups. Statistical significance was accepted for P values below 0.05 (P < 0.05).
3
Results
Based on the light microscopy results, H&E staining showed moderate to severe inflammation in the non-treated and saline groups, but the degree of inflammation in the CAPE group was mild to moderate ( Figs. 1, 2, 3 ; Table 1 ). The difference between the groups was statistically significant (P < 0.05). The ETI values were similar in the non-treated and saline groups. The ETI was lower in the CAPE group compared with the other two groups ( Figs. 1, 2, 3 ). However, this difference was not significant (P > 0.05, Table 2 ). The STI was higher in the non-treated and saline groups than in the CAPE group ( Figs. 1, 2, 3 ). This result was statistically significant (P < 0.05, Table 2 ). The severity of goblet cell and ciliated cell loss was similarly moderate to severe in the non-treated and saline groups ( Figs. 1, 2 ; Table 3 ). The goblet cells and ciliated cells were mostly preserved in the CAPE group ( Fig. 3 ). This difference was statistically significant (P < 0.05, Table 1 ).
