AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY XX ( 2 0 14 ) XXX –XXX
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study Vefa Kınıs, MD a,⁎, Musa Ozbay, MD a , Mehmet Akdag, MD a , Ulas Alabalık, MD b , Aylin Gul, MD a , Beyhan Yılmaz, MD a , Hakan Ozkan, MD a , Ismail Topcu, MD a a b
Department of ENT and Head and Neck Surgery, Dicle University Medical College, Diyarbakir, Turkey Department of Pathology, Dicle University Medical College, Diyarbakir, Turkey
ARTI CLE I NFO
A BS TRACT
Article history:
Purpose: Wound healing of the nasal mucosa is a highly complex process that restores the
Received 12 November 2013
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 nontreated 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. © 2014 Elsevier Inc. All rights reserved.
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 [1]. Nasal mucosa, which is lined by ⁎ Corresponding author at: Department of ENT and Head and Neck Surgery, Dicle University, Medical College 21280, Diyarbakir, Turkey. Tel.: +90 412 2488001/4535; fax: + 90 412 248 8440. E-mail address: [email protected] (V. Kınıs).
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 [2,3]. 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
http://dx.doi.org/10.1016/j.amjoto.2014.02.008 0196-0709/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
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AM ER IC AN JOURNAL OF OTOLARYNGO LOGY –H EAD AN D N E CK ME D I CI NE AN D SUR GE RY XX ( 2 0 14 ) XXX–X XX
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 [4–7]. 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 [8–11]. 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 [12]. 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 [11]. 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 [13]. Therefore, we concluded the study on day 15.
2.1.
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 [13,14]. 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
Tissue preparation
All surgical procedures were carried out under clean but nonsterile 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
Fig. 1 – Severe loss of goblet cells and ciliated cells is shown in the non-treated group (arrows). Increased inflammation, neovascularization and fibrosis are present, especially in the subepithelial layer (red asterisk). Thickness due to hypertrophy is present in the epithelial (yellow asterisk) and subepithelial (red asterisk) layers (H&E, 200 ×).
Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY XX ( 2 0 14 ) XXX –XXX
Fig. 2 – Severe loss of goblet cells and ciliated cells is shown in the saline (control) group (black arrows). Thickness due to hypertrophy and increased inflammation is present in the epithelial (yellow asterisk) and subepithelial (red asterisk) layers. Additionally, part of the cartilaginous nasal septum is shown (red arrow) (H&E, 200 ×).
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).
4.
Discussion
The model system of inducing mechanical trauma on the nasal mucosa of rats has been reported to have many advantages [7]. The small size and ease of working with rats in the laboratory, in addition to the similarities of rat nasal mucosa structure with that in humans, are among these advantages. Rapid wound healing in rat nasal mucosa is another important advantage that allowed us to evaluate this process in a relatively short time.
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Fig. 3 – Mostly preserved goblet cells and ciliated cells are shown in the CAPE group (arrows). Thickness is not present in the epithelial and subepithelial layers (asterisk) (H&E, 200×).
Wound healing is a highly organized and complex process that includes inflammation, reepithelialization, matrix deposition and tissue remodeling and is regulated by a wide variety of cytokines and growth factors through recruitment of inflammatory leukocytes, fibroblasts and epithelial cells [15–17]. Although studies have been conducted to investigate wound healing in skin [18], only a limited number of studies are available regarding the postoperative wound healing of the nasal and paranasal mucosa [7,13,14]. Caffeic acid phenyl ester (CAPE) is a major, biologically active component of propolis from honeybee hives and has been used in traditional medicine for many years. Previously, it has been demonstrated that CAPE has many preventive and protective effects against the toxic properties of several agents in various tissues such as cochlea, tympanic membrane, kidney, heart, red blood cells, brain, and spinal cord [10,19–22]. However, to our knowledge, the effects of CAPE on the wound healing of nasal mucosa have not yet been investigated. The possible beneficial effects of CAPE on wound healing were demonstrated in this study. Healing problems of the nasal mucosa, including synechia and excessive crust formation, are among the important causes of failure or recurrence of disease after the most commonly performed otorhinolaryngological surgeries such as endoscopic sinus surgery and septoplasty. Performing mechanical trauma on inflamed nasal mucosa increases the risk of synechia formation, especially after chronic rhinosinusitis surgeries. As a result, these healing problems may decrease the chances of post-surgical success. A better understanding of the wound healing process of the nasal
Table 1 – Severity of inflammation. Group Non-treated Saline CAPE
Mild N-percentage (%)
Moderate N-percentage (%)
Severe N-percentage (%)
0 0 3
5 6 4
2 1 0
P Value, Kruskal–Wallis test
0 0 42,9
71,4 85,7 57,1
28,6 14,3 0
Mean ± st. deviation 2,29 ± 0,49 2,14 ± 0,38 1,57 ± 53 0,036
Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
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Table 2 – Comparison of epithelial and subepithelial thickness positivity. Group
ETI (+) N-percentage (%)
Non-treated Saline CAPE
5 5 3
P Value, Chi-squared test
71,4 71,4 42,9 0,446
STI (+) N-percentage (%) 7 6 3
100 85,7 42,9 0,021
mucosa may decrease the failure and recurrence of disease. Careful postoperative maintenance of the nasal cavity aims to prevent such complications and modulate the healing process [23]. Other studies have been performed to demonstrate the effects of nasal and systemic corticosteroids on nasal mucosa healing after endoscopic sinus surgery [4–6]. It has been reported that nasal corticosteroids improve wound healing by decreasing mucosal swelling and the formation of adhesions and granulation tissue without disrupting epithelial differentiation. In contrast to these findings, systemic corticosteroids may increase the inflammatory response, retard epithelialization and cause a low degree of collagenization [24,25]. The steps of wound healing in rat nasal mucosa after mechanical trauma induced by brushing have been investigated in two important studies [13,14]. In one of these studies, performed by Khalmaturova et al. [14], the effects of corticosteroids on the wound healing of rat nasal mucosa were examined. In other study conducted by Khalmaturova et al. [13], the effects of untreated mechanical trauma on the wound healing of rat nasal mucosa were demonstrated. Reepithelialization started on the 2nd day and increased to a maximum level on the 14th day of investigation. The thickness of the epithelial layer returned to a normal level on day 28 [13]. Similar findings have been reported for skin wound healing [26]. In the other study done by Khalmaturova et al. [14], it was shown that subepithelial edema and inflammatory cell infiltration were significantly lower in the dexamethasone group than in the saline group on day 5. Therefore, the authors reported that dexamethasone decreased the inflammatory response against mechanical trauma [14]. They reported that wound reepithelialization started on day 5. Epithelial cell differentiation was more prominent in the saline group than in the dexamethasone group on day 14. However, there was less adhesion formation in the study group than in the control group. The authors concluded that this possible beneficial effect of dexamethasone could be important in wound management after surgery [14].
The STI and ETI values increased significantly on day 14 and were restored to near-normal levels on day 28 [13]. On day 2 of the other study performed by Khalmaturova et al. [14], the STI values were significantly different between the experimental and control groups. However, after day 5, there was no difference between them. There was a significant difference in the ETI value between the experimental and control groups on days 1 and 5. Similarly to the other study by Khalmaturova et al. [13], the thickness of the epithelial cell layer returned to a normal level by day 28 [14]. In the present study, the severity of inflammation due to fibrosis, neovascularization and inflammatory cell infiltration in the epithelial and subepithelial layers was significantly milder in the CAPE group compared to that in the non-treated and saline groups. CAPE significantly reduced the inflammation severity after mechanical trauma. The ETI was similar among the 3 groups, and no statistically significant difference was found. The STI of the CAPE group was significantly lower than those of the non-treated and saline groups, most likely due to the inflammation-reducing effects of CAPE, which resulted in mild hypertrophy in the subepithelial region. Ciliated cells have a very important role in cleaning and protecting the upper respiratory airways. The mucociliary clearance mechanism plays a vital role in the normal nasal mucosa healing process, and pseudostratified ciliated epithelial cells are the major component of this mechanism [2,3,27]. Mechanical trauma, inflammation, respiratory tract infections and toxic substances (such as cigarette smoke) may easily destroy this ciliated structure and the mucociliary clearance mechanism [1,13,14,28,29]. Goblet cells play another important role in the mucociliary clearance mechanism by producing mucin. Mucin is composed of mucous glycoproteins [30]. Goblet cells respond to various stimuli by changing the amount of mucin production. This response is also important in mucociliary clearance and protection [1]. Goblet cells and ciliated cells started to regenerate on day 14 after inducing mechanical mucosal trauma in the rats, and regeneration was not yet complete on day 28 [13]. It has been demonstrated that reciliation was complete on the 84th day in a sheep model [31]. Thus, it has been reported that a rat model is not suitable and a longer observation time is needed [13]. In one of study conducted by Khalmaturova et al. [14], the numbers of newly formed goblet cells and ciliated cells were significantly larger in the control group than in the dexamethasone group, suggesting that ciliary and goblet cell regeneration may be delayed by dexamethasone. In the present study, the loss of goblet cells and ciliated cells was significantly lower in the CAPE group than in the other 2 groups. Given this finding, we can say that CAPE has a
Table 3 – Severity of goblet cell and ciliated cell loss. Group Non-treated Saline CAPE
Mild N-percentage (%)
Moderate N-percentage (%)
Severe N-percentage (%)
0 0 5
3 4 2
4 3 0
0 0 71,4
P Value, Kruskal–Wallis test
42,9 57,1 28,6
57,1 42,9 0
Mean ± st. deviation 2,57 ± 0,53 2,43 ± 0,53 1,29 ± 0,49 0,003
ETI: Epithelial thickness index, STI: Subepithelial thickness index.
Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY XX ( 2 0 14 ) XXX –XXX
protective effect on the mucociliary transport mechanism of the nasal mucosa. In conclusion, our study and previous studies have demonstrated that it is very difficult to evaluate in detail the complex phases of wound healing of the nasal mucosa with one study. In this study, we tried to demonstrate the effects of CAPE on wound healing of the nasal mucosa after performing mechanical trauma. Although we obtained important and useful findings, there is still much to learn. For example, we do not know the effects of CAPE in cases of chronic inflammation or infection, such as in chronic rhinosinusitis. Therefore, new studies are needed to evaluate wound healing of the nasal mucosa. More successful surgical outcomes can be achieved if we can obtain more information about this complex process. It is not possible to perform human studies due to ethical and practical concerns. Therefore, the rat will continue to be a useful model for investigation of wound healing of the nasal mucosa.
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Acknowledgments The authors wish to thank Dr. Yilmaz Palanci, Assistant Professor of Public Health, Dicle University School of Medicine, for his valuable contribution to the statistical analysis.
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Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study Vefa Kınıs, MD a,⁎, Musa Ozbay, MD a , Mehmet Akdag, MD a , Ulas Alabalık, MD b , Aylin Gul, MD a , Beyhan Yılmaz, MD a , Hakan Ozkan, MD a , Ismail Topcu, MD a a b
Department of ENT and Head and Neck Surgery, Dicle University Medical College, Diyarbakir, Turkey Department of Pathology, Dicle University Medical College, Diyarbakir, Turkey
ARTI CLE I NFO
A BS TRACT
Article history:
Purpose: Wound healing of the nasal mucosa is a highly complex process that restores the
Received 12 November 2013
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 nontreated 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. © 2014 Elsevier Inc. All rights reserved.
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 [1]. Nasal mucosa, which is lined by ⁎ Corresponding author at: Department of ENT and Head and Neck Surgery, Dicle University, Medical College 21280, Diyarbakir, Turkey. Tel.: +90 412 2488001/4535; fax: + 90 412 248 8440. E-mail address: [email protected] (V. Kınıs).
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 [2,3]. 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
http://dx.doi.org/10.1016/j.amjoto.2014.02.008 0196-0709/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
2
AM ER IC AN JOURNAL OF OTOLARYNGO LOGY –H EAD AN D N E CK ME D I CI NE AN D SUR GE RY XX ( 2 0 14 ) XXX–X XX
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 [4–7]. 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 [8–11]. 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 [12]. 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 [11]. 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 [13]. Therefore, we concluded the study on day 15.
2.1.
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 [13,14]. 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
Tissue preparation
All surgical procedures were carried out under clean but nonsterile 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
Fig. 1 – Severe loss of goblet cells and ciliated cells is shown in the non-treated group (arrows). Increased inflammation, neovascularization and fibrosis are present, especially in the subepithelial layer (red asterisk). Thickness due to hypertrophy is present in the epithelial (yellow asterisk) and subepithelial (red asterisk) layers (H&E, 200 ×).
Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY XX ( 2 0 14 ) XXX –XXX
Fig. 2 – Severe loss of goblet cells and ciliated cells is shown in the saline (control) group (black arrows). Thickness due to hypertrophy and increased inflammation is present in the epithelial (yellow asterisk) and subepithelial (red asterisk) layers. Additionally, part of the cartilaginous nasal septum is shown (red arrow) (H&E, 200 ×).
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).
4.
Discussion
The model system of inducing mechanical trauma on the nasal mucosa of rats has been reported to have many advantages [7]. The small size and ease of working with rats in the laboratory, in addition to the similarities of rat nasal mucosa structure with that in humans, are among these advantages. Rapid wound healing in rat nasal mucosa is another important advantage that allowed us to evaluate this process in a relatively short time.
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Fig. 3 – Mostly preserved goblet cells and ciliated cells are shown in the CAPE group (arrows). Thickness is not present in the epithelial and subepithelial layers (asterisk) (H&E, 200×).
Wound healing is a highly organized and complex process that includes inflammation, reepithelialization, matrix deposition and tissue remodeling and is regulated by a wide variety of cytokines and growth factors through recruitment of inflammatory leukocytes, fibroblasts and epithelial cells [15–17]. Although studies have been conducted to investigate wound healing in skin [18], only a limited number of studies are available regarding the postoperative wound healing of the nasal and paranasal mucosa [7,13,14]. Caffeic acid phenyl ester (CAPE) is a major, biologically active component of propolis from honeybee hives and has been used in traditional medicine for many years. Previously, it has been demonstrated that CAPE has many preventive and protective effects against the toxic properties of several agents in various tissues such as cochlea, tympanic membrane, kidney, heart, red blood cells, brain, and spinal cord [10,19–22]. However, to our knowledge, the effects of CAPE on the wound healing of nasal mucosa have not yet been investigated. The possible beneficial effects of CAPE on wound healing were demonstrated in this study. Healing problems of the nasal mucosa, including synechia and excessive crust formation, are among the important causes of failure or recurrence of disease after the most commonly performed otorhinolaryngological surgeries such as endoscopic sinus surgery and septoplasty. Performing mechanical trauma on inflamed nasal mucosa increases the risk of synechia formation, especially after chronic rhinosinusitis surgeries. As a result, these healing problems may decrease the chances of post-surgical success. A better understanding of the wound healing process of the nasal
Table 1 – Severity of inflammation. Group Non-treated Saline CAPE
Mild N-percentage (%)
Moderate N-percentage (%)
Severe N-percentage (%)
0 0 3
5 6 4
2 1 0
P Value, Kruskal–Wallis test
0 0 42,9
71,4 85,7 57,1
28,6 14,3 0
Mean ± st. deviation 2,29 ± 0,49 2,14 ± 0,38 1,57 ± 53 0,036
Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
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AM ER IC AN JOURNAL OF OTOLARYNGO LOGY –H EAD AN D N E CK ME D I CI NE AN D SUR GE RY XX ( 2 0 14 ) XXX–X XX
Table 2 – Comparison of epithelial and subepithelial thickness positivity. Group
ETI (+) N-percentage (%)
Non-treated Saline CAPE
5 5 3
P Value, Chi-squared test
71,4 71,4 42,9 0,446
STI (+) N-percentage (%) 7 6 3
100 85,7 42,9 0,021
mucosa may decrease the failure and recurrence of disease. Careful postoperative maintenance of the nasal cavity aims to prevent such complications and modulate the healing process [23]. Other studies have been performed to demonstrate the effects of nasal and systemic corticosteroids on nasal mucosa healing after endoscopic sinus surgery [4–6]. It has been reported that nasal corticosteroids improve wound healing by decreasing mucosal swelling and the formation of adhesions and granulation tissue without disrupting epithelial differentiation. In contrast to these findings, systemic corticosteroids may increase the inflammatory response, retard epithelialization and cause a low degree of collagenization [24,25]. The steps of wound healing in rat nasal mucosa after mechanical trauma induced by brushing have been investigated in two important studies [13,14]. In one of these studies, performed by Khalmaturova et al. [14], the effects of corticosteroids on the wound healing of rat nasal mucosa were examined. In other study conducted by Khalmaturova et al. [13], the effects of untreated mechanical trauma on the wound healing of rat nasal mucosa were demonstrated. Reepithelialization started on the 2nd day and increased to a maximum level on the 14th day of investigation. The thickness of the epithelial layer returned to a normal level on day 28 [13]. Similar findings have been reported for skin wound healing [26]. In the other study done by Khalmaturova et al. [14], it was shown that subepithelial edema and inflammatory cell infiltration were significantly lower in the dexamethasone group than in the saline group on day 5. Therefore, the authors reported that dexamethasone decreased the inflammatory response against mechanical trauma [14]. They reported that wound reepithelialization started on day 5. Epithelial cell differentiation was more prominent in the saline group than in the dexamethasone group on day 14. However, there was less adhesion formation in the study group than in the control group. The authors concluded that this possible beneficial effect of dexamethasone could be important in wound management after surgery [14].
The STI and ETI values increased significantly on day 14 and were restored to near-normal levels on day 28 [13]. On day 2 of the other study performed by Khalmaturova et al. [14], the STI values were significantly different between the experimental and control groups. However, after day 5, there was no difference between them. There was a significant difference in the ETI value between the experimental and control groups on days 1 and 5. Similarly to the other study by Khalmaturova et al. [13], the thickness of the epithelial cell layer returned to a normal level by day 28 [14]. In the present study, the severity of inflammation due to fibrosis, neovascularization and inflammatory cell infiltration in the epithelial and subepithelial layers was significantly milder in the CAPE group compared to that in the non-treated and saline groups. CAPE significantly reduced the inflammation severity after mechanical trauma. The ETI was similar among the 3 groups, and no statistically significant difference was found. The STI of the CAPE group was significantly lower than those of the non-treated and saline groups, most likely due to the inflammation-reducing effects of CAPE, which resulted in mild hypertrophy in the subepithelial region. Ciliated cells have a very important role in cleaning and protecting the upper respiratory airways. The mucociliary clearance mechanism plays a vital role in the normal nasal mucosa healing process, and pseudostratified ciliated epithelial cells are the major component of this mechanism [2,3,27]. Mechanical trauma, inflammation, respiratory tract infections and toxic substances (such as cigarette smoke) may easily destroy this ciliated structure and the mucociliary clearance mechanism [1,13,14,28,29]. Goblet cells play another important role in the mucociliary clearance mechanism by producing mucin. Mucin is composed of mucous glycoproteins [30]. Goblet cells respond to various stimuli by changing the amount of mucin production. This response is also important in mucociliary clearance and protection [1]. Goblet cells and ciliated cells started to regenerate on day 14 after inducing mechanical mucosal trauma in the rats, and regeneration was not yet complete on day 28 [13]. It has been demonstrated that reciliation was complete on the 84th day in a sheep model [31]. Thus, it has been reported that a rat model is not suitable and a longer observation time is needed [13]. In one of study conducted by Khalmaturova et al. [14], the numbers of newly formed goblet cells and ciliated cells were significantly larger in the control group than in the dexamethasone group, suggesting that ciliary and goblet cell regeneration may be delayed by dexamethasone. In the present study, the loss of goblet cells and ciliated cells was significantly lower in the CAPE group than in the other 2 groups. Given this finding, we can say that CAPE has a
Table 3 – Severity of goblet cell and ciliated cell loss. Group Non-treated Saline CAPE
Mild N-percentage (%)
Moderate N-percentage (%)
Severe N-percentage (%)
0 0 5
3 4 2
4 3 0
0 0 71,4
P Value, Kruskal–Wallis test
42,9 57,1 28,6
57,1 42,9 0
Mean ± st. deviation 2,57 ± 0,53 2,43 ± 0,53 1,29 ± 0,49 0,003
ETI: Epithelial thickness index, STI: Subepithelial thickness index.
Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY XX ( 2 0 14 ) XXX –XXX
protective effect on the mucociliary transport mechanism of the nasal mucosa. In conclusion, our study and previous studies have demonstrated that it is very difficult to evaluate in detail the complex phases of wound healing of the nasal mucosa with one study. In this study, we tried to demonstrate the effects of CAPE on wound healing of the nasal mucosa after performing mechanical trauma. Although we obtained important and useful findings, there is still much to learn. For example, we do not know the effects of CAPE in cases of chronic inflammation or infection, such as in chronic rhinosinusitis. Therefore, new studies are needed to evaluate wound healing of the nasal mucosa. More successful surgical outcomes can be achieved if we can obtain more information about this complex process. It is not possible to perform human studies due to ethical and practical concerns. Therefore, the rat will continue to be a useful model for investigation of wound healing of the nasal mucosa.
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Acknowledgments The authors wish to thank Dr. Yilmaz Palanci, Assistant Professor of Public Health, Dicle University School of Medicine, for his valuable contribution to the statistical analysis.
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Please cite this article as: Kınıs V, et al, Effects of caffeic acid phenethyl ester on wound healing of nasal mucosa in the rat: an experimental study, Am J Otolaryngol–Head and Neck Med and Surg (2014), http://dx.doi.org/10.1016/j.amjoto.2014.02.008
