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Inhibitory effect of N-acetyl cysteine and ascorbic acid on the development of myringosclerosis: An experimental study
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Inhibitory effect of N-acetyl cysteine and ascorbic acid on the development of myringosclerosis: An experimental study Rıza Du¨ndar a, Sevinc¸ I˙nan b, Nuray Bayar Muluk c,*, Cemal Cingi d, Ali Ekber I˙lknur e, Hu¨seyin Katılmıs¸ f a
Kızıltepe State Hospital, ENT Department, Mardin, Turkey Celal Bayar University, Faculty of Medicine, Histology and Embryology Department, Manisa, Turkey Kırıkkale University, Faculty of Medicine, ENT Department, Kırıkkale, Turkey d Osmangazi University, Medical Faculty, Department of Otorhinolaryngology, Eskisehir, Turkey e Urla State Hospital, ENT Department, I˙zmir, Turkey f ˙ Izmir Katip C¸elebi University, Training and Research Hospital, ENT Clinics, I˙zmir, 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 26 October 2013 Received in revised form 14 March 2014 Accepted 29 March 2014 Available online xxx
Objectives: This study investigated the effects of ascorbic acid and N-acetyl cysteine (NAC) antioxidants on the development of myringosclerosis (MS) in an experimental model. Methods: Myringotomies were performed in the ears of 15 guinea pigs, and Spongostan1 pieces were placed on the perforated regions of the tympanic membrane. The subjects were divided randomly into three groups and treated with three different solutions on the Spongostan—group 1: (control, 0.9% saline), group 2 (ascorbic acid), and group 3 (NAC). On day 15 after treatment, specimens from the tympanic membranes were obtained and examined via light microscopy. Sclerosis and inflammation scores and the tympanic membrane thicknesses were evaluated. Immunohistochemical methods were used to evaluate the expression of VEGF, TGF-b, iNOS, and IL1-b in all groups. Results: Lower sclerosis and inflammation scores and reduced tympanic membrane thicknesses were observed in groups treated with NAC or ascorbic acid compared with the control group. Immunohistochemical studies revealed significantly less expression of VEGF, TGF-b, and iNOS in groups 2 and 3 compared with group 1. Additionally, IL1-b expression was significantly less in group 3 than in group 1. Compared with group 1, group 2 animals exhibited reduced inflammation in the lamina propria, fewer active fibroblasts, less leukocyte infiltration, and decreased thickness of the vessels; group 3 animals exhibited decreased numbers of active fibroblasts and collagen fibers in the lamina propria. Conclusions: Inflammation scores, cellular infiltration, and expression of VEGF, TGF-b, and iNOS were reduced by ascorbic acid and/or NAC treatments, thereby decreasing MS development. Decreased expression of IL1-b was observed only in animals treated with NAC. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ascorbic acid Inducible nitric oxide synthase (iNOS) Interleukin 1-beta (IL1-b) Myringosclerosis N-acetyl cysteine (NAC) Vascular endothelial growth factor (VEGF)
1. Introduction Myringosclerosis (MS) is a condition that develops after a middle ear infection or traumatic intervention. In this condition, hyalinization and calcification of the collagen layer develop in certain areas of the tympanic membrane (TM) with increased collagen fibers due to progressive fibroblast infiltration, hyaline
* Corresponding author at: Birlik Mahallesi, Zirvekent 2. Etap Sitesi, C-3 blok, No.: 62/43, 06610 C¸ankaya/Ankara, Turkey. Tel.: +90 312 4964073/532 7182441; fax: +90 312 4964073. E-mail addresses: [email protected], [email protected] (N.B. Muluk).
degeneration, and extracellular calcium deposition within the lamina propria [1–3]. MS is characterized by the development of calcified plaques on the TM [4,5]. The etiology of MS includes inflammatory disease, intratympanic membrane bleeding after tube insertion, traumatic tube insertion, excessive aspiration of middle ear fluid, and increased production of oxygen-derived free radicals within the middle ear after myringotomy [5–9]. Tos et al. [10] reported that decreased ear membrane mobility after insertion of a ventilation tube (VT) promotes hyalinization and calcification in the collagen layer, resulting in MS. While most cases of MS remain asymptomatic, the clinical condition in cases with symptoms can range from mild to severe hearing loss associated with large sclerotic plaques [11]. MS develops due to free oxygen radicals secondary to hyperoxic medium and
http://dx.doi.org/10.1016/j.ijporl.2014.03.029 0165-5876/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: R. Du¨ndar, et al., Inhibitory effect of N-acetyl cysteine and ascorbic acid on the development of myringosclerosis: An experimental study, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.029
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mechanical injury within the middle ear. Several studies have reported that agents with antioxidant characteristics inhibited the development of MS [3,11,12]. Aydog˘an et al. [1] reported that oral administration of coenzyme Q10 did not reduce MS formation in myringotomized rats. Spratley et al. [12] studied the effects of topical ascorbic acid applied on Gelfoam cups to the TMs in 12 Sprague-Dawley rats that were myringotomized bilaterally. They reported that the extent of sclerotic lesions was significantly reduced in the ascorbic acidtreated group compared to saline-treated or untreated animals, and concluded that topical ascorbic acid reduced the occurrence of MS following TM perforations. In the present experimental study, we investigated the protective effects of known anti-oxidant agents, ascorbic acid and N-acetyl cysteine (NAC), compared with saline solution, on the development of MS. MS was evaluated using light microscopy. Saline solution was used as a control because TM thicknesses were found to be similar in untreated and saline-treated rats after myringotomy [13]. 2. Materials and methods This study was conducted at Izmir Ataturk Training and Research Hospital and followed the principles of the Declaration of Helsinki [14]. Adaptation and care of the animals and the experimental study were performed at the same center. 2.1. Animal subjects Fifteen female guinea pigs (weighing 350–400 g) were used in this study. Approval for the study was obtained from the Committee for Ethical Issues of Izmir Ataturk Training and Research Hospital (date: 2008, number: 4551710). All animal procedures were performed in accordance with the approved protocol. All interventions to the animals were conducted under aseptic conditions. 2.2. Methods During the experimental procedure, the guinea pigs were sedated using 10 mg/kg xylazine and 30 mg/kg intraperitoneal injection of ketamine. Myringotomies were performed in the upper posterior quadrant of the TM (2 mm in length) in both ears of each guinea pig. Pieces of Spongostan1 were placed on the perforated region of the TM. The subjects were randomly divided into three groups with five animals in each group. Three different solutions were applied onto the Spongostan1 (three drops twice a day for 10 days) as follows: group 1 received saline (0.9% NaCl) solution (n = 5, 10 ears); group 2 received ascorbic acid (0.28 mmol/L in sterile water) [12] (n = 5, 10 ears); and group 3 received NAC (1.2 mg/mL) [15] (n = 5, 10 ears). On day 15 of the study, the TMs were examined otomicroscopically, and the findings were scored according to the development of MS: (0) no visible sclerotic lesion; (+) sclerotic lesion only on the malleus arm or adjacent to it; (++) sclerotic lesion adjacent to the malleus arm and on the upper frontal part of the pars tensa; or (+++) sclerotic lesion adjacent to the malleus and continuing through the annulus. 2.3. Histological examination procedure Rats were sacrificed using 80 mg/kg pentothal 15 days after treatments began. Immediately after death, the temporal bones were removed, and the otic bullas were excised and placed in fixative (10% formalin). The temporal bones were decalcified in 5% formic acid [16], deparaffinized, and dehydrated by immersion into xylene twice for 10 min. Following dehydration in an
ascending series of ethanol (70, 80, 96, 100%), tissue samples were cleared in xylene and embedded in paraffin. The preparations were transferred into citrate-based antigen retrieval solution to detect vascular endothelial growth gactor (VEGF), transforming growth factor beta (TGF-b), inducible nitric oxide synthase (iNOS), and interleukin 1-beta (IL1-b) antigens. All slides were microwaved twice for 5 min in a microwave oven (750 W). Using the Shandon Sequeza Tm manual staining device for standardization, the classical streptavidine avidin-biotin-peroxidase (Strept. AB-Peroxidase) method and diaminobenzidine (DAB) chromogen (20 min) were applied for immunohistochemical analysis using four antibodies. Non-immune mouse serum served as a negative control and Mayer’s hematoxylin was used as the counterstain. Cytoplasmic staining was considered evidence of positivity. A minimum of 3–4 fields for each sample were examined and scored by an observer blinded to the treatment of the animals [17]. Slides were examined using light microscopy with an Olympus BX40 microscope, and photos were taken with an Olympus DP-70 digital camera. The inflammation process was evaluated by observing the cellular density, the existence of polymorphonuclear leukocytes, and the vascular diameter. Samples were scored as having (1) normal structure, (2) moderate inflammation, or (3) severe inflammation. Immunohistochemical staining properties were evaluated semi-quantitatively by examiners who were blinded to the treatment. Antigen (+) cells were assessed by counting a total of 100 cells in 3–4 fields of high magnification (400), and the mean expressions of antigen were calculated. Scoring was performed using a scale of 0–3: (0) represented negative staining, 0% cell count; (+) mild staining, <5% cell count; (++): moderate staining, 5–50% cell count; and (+++) severe, >50% cell count [18]. 2.4. Statistical analysis The statistical package for SPSS (Version 16.0) was used for statistical analyses. Kruskal–Wallis variance analysis was used to analyze differences between groups. When statistically significant results were obtained, pairwise comparisons were performed using the Mann–Whitney U Test with Bonferrroni correction to detect the value that had caused a difference. A p-value < 0.05 was considered to indicate statistical significance. 3. Results 3.1. Otomicroscopical sclerosis scores (OSS) Table 1 and Fig. 1 presents the OSS values for groups 1–3. Significant differences were detected between OSS values (p = 0.004): OSS values in groups 2 and 3 were significantly lower than those in group 1 (pgroups 1 and 2 = 0.014, pgroups 1–3 = 0.002). No significant difference was detected between groups 2 and 3 (pgroups 2 and 3 = 0.318). 3.2. Inflammation score (IS) Table 2 and Fig. 1 presents the IS values in groups 1–3. Significant differences were detected between IS values (p = 0.003): IS values in groups 2 and 3 were significantly lower than those in group 1 (pgroups1 and 2 = 0.014, pgroups 1–3 = 0.002). No significant differences were observed between groups 2 and 3 (pgroups 2 and 3 = p = 0.313). 3.3. TM thickness Table 3 lists TM thicknesses in groups 1–3. Significant differences were detected between TM thicknesses (p = 0.000):
Please cite this article in press as: R. Du¨ndar, et al., Inhibitory effect of N-acetyl cysteine and ascorbic acid on the development of myringosclerosis: An experimental study, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.029
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Table 1 Otomicroscopical sclerosis scores for groups 1–3a. Groups
n
Group 1 (saline) Group 2 (ascorbic acid) Group 3 (N-acetyl cysteine)
10 10 10
a & ô
Numerical distribution of OSS (n) 0
+
++
+++
0 1 2
1 5 6
2 2 1
7 2 1
OSS median (min–max)
p&
pô
3.0 (1.0–3.0) 1.0 (0.0–3.0) 1.0 (0.0–3.0)
0.004
pgroups 1 and 2 = 0.014, z = 2.463 pgroups 1–3 = 0.002, z = 3.083 pgroups2 and 3 = 0.318, z = 0.998
OSS: Otomicroscopical sclerosis score. p-Value shows the results of Kruskal–Wallis variance analysis. p-Value shows the results of Mann–Whitney U test with Bonferrroni correction.
Fig. 1. Sclerosis, inflammation, and immunohistochemical staining scores for each group. Scoring for sclerosis: (0) no visible sclerotic lesion; (+) sclerotic lesion only on the malleus arm or adjacent to it; (++) sclerotic lesion adjacent to the malleus arm and on the upper frontal part of the pars tensa; (+++) sclerotic lesion adjacent to the malleus and continuing through the annulus. Scoring for inflammation: (1) normal structure; (2) moderate inflammation; (3) severe inflammation. Scoring for immunohistochemical staining to detect VEGF, TGF-b, i-NOS, IL1-b: (1) mild; (2) moderate; (3) intense.
Table 2 Inflammation scores for groups 1–3a. Groupsô
n
Group 1 (Saline) Group 2 (Ascorbic acid) Group 3 (N-Acetyl Cysteine) a & ô
Numerical distribution of ISs (n)
10 10 10
+ (normal)
++ (moderate)
+++ (severe)
1 5 6
3 4 3
6 1 1
ISs median (min–max)
p&
pô
3.0 (1.0–3.0) 1.5 (1.0–3.0) 1.0 (1.0–2.0)
0.003
pgroups pgroups pgroups
1 and 2 1–3
= 0.014, z = 2.445 = 0.002, z = 3.135 = 0.313, z = 1.009
2 and 3
IS: inflammation score. p-Value shows the results of Kruskal–Wallis variance. p-Value shows the results of Mann–Whitney U test with Bonferrroni correction.
Table 3 Thickness of the tympanic membrane for groups 1–3. Groups
Group 1 (Saline) Group 2 (Ascorbic acid) Group 3 (N-Acetyl Cysteine) pô & ô
The thicknesses of the tympanic membranes of guinea pigs (mm) 1
2
0.090 0.068 0.056 pgroups
0.080 0.088 0.056 0.058 0.066 0.049 and 2 = 0.001, z =
1
3
4
5
6
0.092 0.070 0.060 0.046 0.065 0.050 0.050 0.045 0.052 3.258; pgroups 1–3 = 0.000,
7
8
Mean std. dev 9
Median (min-max)
p&
0.084 (0.060–0.093) 0.057 (0.043–0.080) 0.051 (0.045–0.073)
0.000
10
0.078 0.089 0.093 0.080 0.082 0.010 0.068 0.080 0.050 0.043 0.058 0.011 0.073 0.049 0.046 0.056 0.054 0.008 z = 3.557; pgroups 2 and 3 = p = 0.383, z = 0.873
p-Value shows the results of Kruskal–Wallis variance analysis. p-Value shows the results of Mann–Whitney U test with Bonferrroni correction.
TM thicknesses in group 2 and 3 were significantly thinner than those in group 1 (pgroups 1 and 2 = 0.001; pgroups 1–3 = 0.000). No significant difference was detected between groups 2 and 3 (pgroups 2 and 3 = 0.383).
3.4. Immunoreactivity scores for VEGF, TGF-b, i-NOS and IL1-b Table 4 and Fig. 1 present immunoreactivity scores for VEGF, TGF-b, i-NOS, and IL1-b.
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Table 4 Immunoreactivity scores for VEGF, TGF-b, i-NOS. and IL1-b for each group. Immunohistochemistry
p&
Immunoreactivity scores in the groups Group 1 (saline) (n=10) Numerical distribution +
++
Median
+++
Group 2 (ascorbic acid) (n=10)
Group 3 (N-acetyl cysteine) (n = 10)
Numerical distribution
Numerical distribution
+
++
Median
+++
+
++
Median
+++
VEGFô
1 2 7 3.0 pgroups 1 and 2 = 0.002, z = 3.086
8 1 1 1.0 pgroups 1–3 = 0.003, z = 2924
7 2 1 1.0 pgroups 2 and 3 = p = 0.654, z = 0,449
0.003
TGF-bô
– 4 6 3.0 pgroups 1 and 2 = 0.010, z = 2.571
3 6 1 2.0 pgroups 1–3 = 0.005, z = 2821
5 4 1 1.5 pgroups 2 and 3 = 0.450, z = 0.755
0.014
i-NOSô
– 2 8 3.0 pgroups 1 and 2 = 0.002, z = 3.091
2 7 1 2.0 pgroups 1–3 = 0.001, z = 3.423
7 2 1 1.0 pgroups 2 and 3 = p = 0.060, z = 1880
0.001
IL1-bô
2 7 1 2,0 pgroups 1 and 2 = 0.060, z = 1880
7 2 1 1,0 pgroups 1–3 = 0.008, z = 2644
8 2 – 1.0 pgroups 2 and 3 = 0.549, z = 0.600
0.015
& ô
p-Value shows the results of Kruskal–Wallis variance analysis. p-Value shows the results of Mann–Whitney U test with Bonferrroni correction.
3.5. VEGF scores (Table 4 and Fig.
1)
Significant differences in VEGF scores were observed between the groups (p = 0.003): VEGF scores in groups 2 and 3 were significantly lower than those in group 1 (pgroups 1 and 2 = 0.002; pgroups 1–3 = 0.003). No significant difference was detected between groups 2 and 3 (pgroup 2–3 = p = 0.654).
3.10. Immunohistochemical findings Immunohistochemical examination of the TM specimens obtained from the different groups revealed various patterns (Figs. 2–4). Table 4 lists the immunostaining intensities using primary antibodies against VEGF, TGF-b, iNOS, and IL1-b. These factors are known to play significant roles in angiogenesis and inflammation.
3.6. TGF-b scores (Table 4 and Fig. 1) 4. Discussion Significant differences in TGF-b scores were detected between the groups (p = 0.014): TGF-b scores in groups 2 and 3 were significantly lower than those in group 1 (pgroups 1 and 2 = 0.010, pgroups 1–3 = 0.005). No significant difference was detected between groups 2 and 3 (pgroups 2 and 3 = 0.549). 3.7. i-NOS scores (Table 4 and Fig. 1) Significant differences were detected in i-NOS scores between the groups (p = 0.001): i-NOS scores in groups 2 and 3 were significantly lower than those in group 1 (pgroups 1 and 2 = 0.002, pgroups 1–3 = 0.001). No significant difference was observed between groups 2 and 3 (pgroup 2 and 3 = 0.060). 3.8. IL1-b scores (Table 4 and Fig. 1) Significant differences were observed in IL1-b scores between the groups (p = 0.015): IL1-b scores in group 3 were significantly lower than those in Group 1 (pgroups 1–3 = 0.008). No significant differences were observed between groups 1 and 2 (pgroups 1 and 2 = 0.060) or groups 2 and 3 (pgroups 2 and 3 = p = 0.549). 3.9. Light microscopy findings Slices obtained from group 1 (saline group) revealed thickening of the TM, inflammation of the lamina propria, and increased active fibroblasts and collagen fibers. The collagen fibers were separated in some areas. Mononuclear and polymorphonuclear cellular infiltration and vasodilation were observed in the subepithelial layer (Fig. 2). The median IS was 3.0 (Table 2). Slices obtained from group 2 (ascorbic acid group) revealed decreased inflammation in the lamina propria, fewer active fibroblasts, and reduced leukocyte infiltration and thickness of the vessels (Fig. 3). The median IS was 1.5 (Table 2). Slices obtained from group 3 (NAC group) revealed fewer active fibroblasts and collagen fibers in the lamina propria (Fig. 4). The median IS was 1.0 (Table 2).
Inflammation in the collagen layer of the TM is the most pronounced factor in the pathogenesis of MS [19]. Perforation of the TM (e.g., due to myringotomy and VT insertion) causes increased oxygen concentration within the middle ear cavity [20]. Mechanistically, increased amounts of free oxygen radicals accelerate the inflammatory process, leading to the onset of the sclerotic process [21,22]. Free oxygen radicals affect the unsaturated fatty acids within the membrane lipids and start the peroxidation process, thus accelerating the inflammatory process [23]. Antioxidant agents can neutralize free oxygen radicals and block this pathway. NAC, which is the N-acetylated derivative of Lcysteine, can enter the cells readily due to its molecular structure and plays a role in the formation of a significant antioxidant, glutathione (GSH). Thus, NAC supports tissue defense against oxidant stress [24–26]. Ascorbic acid is a water-soluble antioxidant that exerts a protective effect via oxidoreduction, thereby significantly reducing the effects of free oxygen radicals [27]. We investigated the protective effects of ascorbic acid and NAC compared to saline solution on the healing process after myringotomy in guinea pigs. The OSS and inflammation scores in the ascorbic acid and NAC groups were significantly lower than those in the saline group. Compared with the saline group, fewer active fibroblasts and reduced leukocyte infiltration were observed in the ascorbic acid group, and decreased numbers of active fibroblasts and collagen fibers in the lamina propria were observed in the NAC group. Inflammation scores were lower in the ascorbic acid and NAC groups compared with the control group. Therefore, we conclude that inflammation scores, cellular infiltration by mononuclear and polymorphonuclear cells, and fibroblasts and collagen fibers were reduced by ascorbic acid and/or NAC treatments, which resulted in decreased MS development. TM thicknesses were significantly thinner in the ascorbic acid and NAC groups compared with the saline group. Additionally, the immunohistochemical results revealed less expression of VEGF, TGF-b, and iNOS in groups 2 and 3 compared with group 1. IL1-b
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Fig. 2. Light microscopic evaluation of tympanic membrane sections from group 1 (saline) (hematoxylin & eosin (HE) stain, 200). MT, membrane thickness; V, vascularization; J, junctional degeneration; i, inflammatory cells. Immunohistochemical examination results for IL1-b, i-NOS, VEGF, and TGF-b are shown (200).
scores were lower in group 3 than in group 1. IL1-b plays a significant role in angiogenesis and inflammation. Whereas ascorbic acid application decreased IL1-b expression, NAC did not affect IL1-b levels compared with the control group. In a study of rats with MS formation, the application of topical NAC decreased malondialdehyde (MDA) and nitric oxide (NO) levels [23]. Ozcan et al. [28] investigated the effect of topical administration of NAC on MS in experimentally myringotomized rat TMs, using otomicroscopic evaluation and assessing the lipid
peroxidation and NO (nitrite/nitrate) levels. They concluded that topical treatment with NAC reduced MDA and NO levels and suggested that topical NAC application may be useful for preventing MS. Similarly, in the present study, we observed fewer active fibroblasts and collagen fibers in the lamina propria of NACtreated guinea pigs, which resulted in decreased MS. Mattson et al. [4] conducted an experimental study and reported that topical application of copper zinc superoxide dismutase + catalase and deferoxamine reduces or inhibits the
Fig. 3. Light microscopic evaluation of tympanic membrane sections from group 2 (ascorbic acid) (HE, 200). V, vascularization; f, ibroblast proliferation; i, inflammatory cells; k, collagen fibers; e, epithelium. Immunohistochemical examination results for IL1-b, i-NOS, VEGF, and TGF-b are shown (200).
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Fig. 4. Light microscopic evaluation of tympanic membrane sections from Group 3 (N-acetyl cysteine) (HE, 200). MT, membrane thickness; f, fibroblast proliferation; i, inflammatory cells. Immunohistochemical examination results for IL1-b, i-NOS, VEGF, and TGF-b are shown (200).
development of sclerotic lesion, whereas ears treated with copper sulfate plus iron chloride appeared unaffected. Their findings support the hypothesis that the formation of oxygen free radicals contributes significantly to the development of MS. In the present study, we investigated the protective effects of NAC or ascorbic acid and found that these treatments reduced MS. Go¨ru¨r et al. [29] reported significantly reduced development of MS in rats that were administered selenium intraperitoneally compared with rats that received saline. Akbas¸ et al. [11] found that intraperitoneal administration of L-carnitine caused sclerosis in 35% of rats compared with 80% in the control group [11]. In another study, topically applied NAC was found to be effective in the prevention of sclerotic lesions in myringotomized rat tympanic membranes [15]. Spratley et al. [12] reported that topical ascorbic acid reduces the occurrence of MS following TM perforations in rats. In their study, light microscopy revealed that the connective tissue layer of the untouched side of the pars tensa was distinctly thicker in the ascorbic acid group than in the saline or untreated groups. In the present study, we found that ascorbic acid reduced inflammation in the lamina propria, decreased the number of active fibroblasts, and reduced leukocyte infiltration and thickness of the vessels. In our study, immunohistochemical studies of TMs revealed that the expression of VEGF, TGF-b, and iNOS was decreased significantly in groups 2 and 3. Therefore, ascorbic acid and/or NAC treatments resulted in lower inflammation scores and cellular infiltration as well as reduced expression of VEGF, TGF-b, and iNOS, causing decreased MS development. Expression of IL1-b was only affected and decreased by NAC. Similar to our results, Zhu et al. [30] reported that long-term NAC treatment resulted in downregulation of VEGF expression and reduction of reactive oxygen species content in vascular tissues. Galicia-Moreno et al. [31] reported that NAC was effective in preventing TGF-b and IL-6 expression and further augmented IL10 expression, which demonstrated the beneficial effects of NAC due to its antioxidant and immunomodulatory properties [31]. Exogenous oxidants X/XO and H2O2 upregulated the secretion of
the pro-inflammatory cytokines IL-1b, IL-6 and TNF-a [32]. Antioxidants may reduce the levels of pro-inflammatory cytokines, which might prevent the development of MS by their antioxidant and anti-inflammatory features. Yang et al. [33] reported that N-acetyl-L-cysteine (L-NAC) reduced the number of missing and dying outer hair cells and reduced styrene-induced hearing loss. Lorito et al. [34] demonstrated that some antioxidant molecules such as L-NAC can prevent oxidative stress in the inner ear. They reported that L-NAC can partially protect the cochlea from continuous noise, and the protection effect is strongly dose-dependent: lower dosages do not fully protect the cochlea and higher dosages can damage the rat systemically (e.g., pulmonary toxicity). No ototoxic effects were reported. In vitro experiments by Feghali et al. [35] revealed that LNAC protected both auditory neurons and hair cells from the toxic effects of cisplatin. Because it protects both of these inner ear structures, L-NAC exhibits potential for protecting hearing from cisplatin-induced damage. L-NAC also has low systemic and mucosal toxicity, and its low molecular weight may allow it to readily pass through the round window membrane. These characteristics of L-NAC make it potentially suitable for transtympanic application for the prevention of the ototoxicity of cisplatin in vivo [36]. Celebi et al. [37] investigated the effects of intratympanic injection of vitamin C (also called ascorbic acid) on cisplatininduced ototoxicity in rats. Intratympanic vitamin C infusion provided a protective effect against cisplatin-induced ototoxicity primarily at 2 kHz and at other frequencies (2.8, 4, 6, and 8 kHz), and did not produce a toxic effect in the cochlea. Together, these findings [33–37] suggest that NAC and ascorbic acid produce no ototoxic effects in the cochlea; on the contrary, they can protect the inner ear from potential ototoxic events. Further studies are required before NAC and/or ascorbic acid should be applied after VT insertions. Our study provides no evidence of toxic effects in animal studies. However, preclinical safety studies involving humans are still required for these agents. If found safe, placebo-controlled studies involving humans should
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be conducted to investigate the effects of NAC and/or ascorbic acid to prevent MS development after VT insertions. 5. Conclusions Previous studies have reported the preventive effects of NAC on MS, but our study is the first to compare the preventative effects of NAC and ascorbic acid on MS in rats. Our findings suggest that administration of these drugs can prevent the development of MS, which can be an important complication after VT insertions. Further detailed studies should be performed to investigate the efficacy and safety of topical antioxidants to the inner ear and hearing levels in humans. If determined safe and effective, physicians could consider using NAC and/or ascorbic acid after VT insertions to prevent MS. Conflict of interest The authors declare that there is no conflict of interest. No pharmaceutical companies funded the study or contributed to the study design, outcome evaluation, or writing of this article. Acknowledgement With the exception of data collection, the preparation of this paper, including design and planning, was supported by the Continuous Education and Scientific Research Association.
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Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Inhibitory effect of N-acetyl cysteine and ascorbic acid on the development of myringosclerosis: An experimental study Rıza Du¨ndar a, Sevinc¸ I˙nan b, Nuray Bayar Muluk c,*, Cemal Cingi d, Ali Ekber I˙lknur e, Hu¨seyin Katılmıs¸ f a
Kızıltepe State Hospital, ENT Department, Mardin, Turkey Celal Bayar University, Faculty of Medicine, Histology and Embryology Department, Manisa, Turkey Kırıkkale University, Faculty of Medicine, ENT Department, Kırıkkale, Turkey d Osmangazi University, Medical Faculty, Department of Otorhinolaryngology, Eskisehir, Turkey e Urla State Hospital, ENT Department, I˙zmir, Turkey f ˙ Izmir Katip C¸elebi University, Training and Research Hospital, ENT Clinics, I˙zmir, 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 26 October 2013 Received in revised form 14 March 2014 Accepted 29 March 2014 Available online xxx
Objectives: This study investigated the effects of ascorbic acid and N-acetyl cysteine (NAC) antioxidants on the development of myringosclerosis (MS) in an experimental model. Methods: Myringotomies were performed in the ears of 15 guinea pigs, and Spongostan1 pieces were placed on the perforated regions of the tympanic membrane. The subjects were divided randomly into three groups and treated with three different solutions on the Spongostan—group 1: (control, 0.9% saline), group 2 (ascorbic acid), and group 3 (NAC). On day 15 after treatment, specimens from the tympanic membranes were obtained and examined via light microscopy. Sclerosis and inflammation scores and the tympanic membrane thicknesses were evaluated. Immunohistochemical methods were used to evaluate the expression of VEGF, TGF-b, iNOS, and IL1-b in all groups. Results: Lower sclerosis and inflammation scores and reduced tympanic membrane thicknesses were observed in groups treated with NAC or ascorbic acid compared with the control group. Immunohistochemical studies revealed significantly less expression of VEGF, TGF-b, and iNOS in groups 2 and 3 compared with group 1. Additionally, IL1-b expression was significantly less in group 3 than in group 1. Compared with group 1, group 2 animals exhibited reduced inflammation in the lamina propria, fewer active fibroblasts, less leukocyte infiltration, and decreased thickness of the vessels; group 3 animals exhibited decreased numbers of active fibroblasts and collagen fibers in the lamina propria. Conclusions: Inflammation scores, cellular infiltration, and expression of VEGF, TGF-b, and iNOS were reduced by ascorbic acid and/or NAC treatments, thereby decreasing MS development. Decreased expression of IL1-b was observed only in animals treated with NAC. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ascorbic acid Inducible nitric oxide synthase (iNOS) Interleukin 1-beta (IL1-b) Myringosclerosis N-acetyl cysteine (NAC) Vascular endothelial growth factor (VEGF)
1. Introduction Myringosclerosis (MS) is a condition that develops after a middle ear infection or traumatic intervention. In this condition, hyalinization and calcification of the collagen layer develop in certain areas of the tympanic membrane (TM) with increased collagen fibers due to progressive fibroblast infiltration, hyaline
* Corresponding author at: Birlik Mahallesi, Zirvekent 2. Etap Sitesi, C-3 blok, No.: 62/43, 06610 C¸ankaya/Ankara, Turkey. Tel.: +90 312 4964073/532 7182441; fax: +90 312 4964073. E-mail addresses: [email protected], [email protected] (N.B. Muluk).
degeneration, and extracellular calcium deposition within the lamina propria [1–3]. MS is characterized by the development of calcified plaques on the TM [4,5]. The etiology of MS includes inflammatory disease, intratympanic membrane bleeding after tube insertion, traumatic tube insertion, excessive aspiration of middle ear fluid, and increased production of oxygen-derived free radicals within the middle ear after myringotomy [5–9]. Tos et al. [10] reported that decreased ear membrane mobility after insertion of a ventilation tube (VT) promotes hyalinization and calcification in the collagen layer, resulting in MS. While most cases of MS remain asymptomatic, the clinical condition in cases with symptoms can range from mild to severe hearing loss associated with large sclerotic plaques [11]. MS develops due to free oxygen radicals secondary to hyperoxic medium and
http://dx.doi.org/10.1016/j.ijporl.2014.03.029 0165-5876/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: R. Du¨ndar, et al., Inhibitory effect of N-acetyl cysteine and ascorbic acid on the development of myringosclerosis: An experimental study, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.029
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mechanical injury within the middle ear. Several studies have reported that agents with antioxidant characteristics inhibited the development of MS [3,11,12]. Aydog˘an et al. [1] reported that oral administration of coenzyme Q10 did not reduce MS formation in myringotomized rats. Spratley et al. [12] studied the effects of topical ascorbic acid applied on Gelfoam cups to the TMs in 12 Sprague-Dawley rats that were myringotomized bilaterally. They reported that the extent of sclerotic lesions was significantly reduced in the ascorbic acidtreated group compared to saline-treated or untreated animals, and concluded that topical ascorbic acid reduced the occurrence of MS following TM perforations. In the present experimental study, we investigated the protective effects of known anti-oxidant agents, ascorbic acid and N-acetyl cysteine (NAC), compared with saline solution, on the development of MS. MS was evaluated using light microscopy. Saline solution was used as a control because TM thicknesses were found to be similar in untreated and saline-treated rats after myringotomy [13]. 2. Materials and methods This study was conducted at Izmir Ataturk Training and Research Hospital and followed the principles of the Declaration of Helsinki [14]. Adaptation and care of the animals and the experimental study were performed at the same center. 2.1. Animal subjects Fifteen female guinea pigs (weighing 350–400 g) were used in this study. Approval for the study was obtained from the Committee for Ethical Issues of Izmir Ataturk Training and Research Hospital (date: 2008, number: 4551710). All animal procedures were performed in accordance with the approved protocol. All interventions to the animals were conducted under aseptic conditions. 2.2. Methods During the experimental procedure, the guinea pigs were sedated using 10 mg/kg xylazine and 30 mg/kg intraperitoneal injection of ketamine. Myringotomies were performed in the upper posterior quadrant of the TM (2 mm in length) in both ears of each guinea pig. Pieces of Spongostan1 were placed on the perforated region of the TM. The subjects were randomly divided into three groups with five animals in each group. Three different solutions were applied onto the Spongostan1 (three drops twice a day for 10 days) as follows: group 1 received saline (0.9% NaCl) solution (n = 5, 10 ears); group 2 received ascorbic acid (0.28 mmol/L in sterile water) [12] (n = 5, 10 ears); and group 3 received NAC (1.2 mg/mL) [15] (n = 5, 10 ears). On day 15 of the study, the TMs were examined otomicroscopically, and the findings were scored according to the development of MS: (0) no visible sclerotic lesion; (+) sclerotic lesion only on the malleus arm or adjacent to it; (++) sclerotic lesion adjacent to the malleus arm and on the upper frontal part of the pars tensa; or (+++) sclerotic lesion adjacent to the malleus and continuing through the annulus. 2.3. Histological examination procedure Rats were sacrificed using 80 mg/kg pentothal 15 days after treatments began. Immediately after death, the temporal bones were removed, and the otic bullas were excised and placed in fixative (10% formalin). The temporal bones were decalcified in 5% formic acid [16], deparaffinized, and dehydrated by immersion into xylene twice for 10 min. Following dehydration in an
ascending series of ethanol (70, 80, 96, 100%), tissue samples were cleared in xylene and embedded in paraffin. The preparations were transferred into citrate-based antigen retrieval solution to detect vascular endothelial growth gactor (VEGF), transforming growth factor beta (TGF-b), inducible nitric oxide synthase (iNOS), and interleukin 1-beta (IL1-b) antigens. All slides were microwaved twice for 5 min in a microwave oven (750 W). Using the Shandon Sequeza Tm manual staining device for standardization, the classical streptavidine avidin-biotin-peroxidase (Strept. AB-Peroxidase) method and diaminobenzidine (DAB) chromogen (20 min) were applied for immunohistochemical analysis using four antibodies. Non-immune mouse serum served as a negative control and Mayer’s hematoxylin was used as the counterstain. Cytoplasmic staining was considered evidence of positivity. A minimum of 3–4 fields for each sample were examined and scored by an observer blinded to the treatment of the animals [17]. Slides were examined using light microscopy with an Olympus BX40 microscope, and photos were taken with an Olympus DP-70 digital camera. The inflammation process was evaluated by observing the cellular density, the existence of polymorphonuclear leukocytes, and the vascular diameter. Samples were scored as having (1) normal structure, (2) moderate inflammation, or (3) severe inflammation. Immunohistochemical staining properties were evaluated semi-quantitatively by examiners who were blinded to the treatment. Antigen (+) cells were assessed by counting a total of 100 cells in 3–4 fields of high magnification (400), and the mean expressions of antigen were calculated. Scoring was performed using a scale of 0–3: (0) represented negative staining, 0% cell count; (+) mild staining, <5% cell count; (++): moderate staining, 5–50% cell count; and (+++) severe, >50% cell count [18]. 2.4. Statistical analysis The statistical package for SPSS (Version 16.0) was used for statistical analyses. Kruskal–Wallis variance analysis was used to analyze differences between groups. When statistically significant results were obtained, pairwise comparisons were performed using the Mann–Whitney U Test with Bonferrroni correction to detect the value that had caused a difference. A p-value < 0.05 was considered to indicate statistical significance. 3. Results 3.1. Otomicroscopical sclerosis scores (OSS) Table 1 and Fig. 1 presents the OSS values for groups 1–3. Significant differences were detected between OSS values (p = 0.004): OSS values in groups 2 and 3 were significantly lower than those in group 1 (pgroups 1 and 2 = 0.014, pgroups 1–3 = 0.002). No significant difference was detected between groups 2 and 3 (pgroups 2 and 3 = 0.318). 3.2. Inflammation score (IS) Table 2 and Fig. 1 presents the IS values in groups 1–3. Significant differences were detected between IS values (p = 0.003): IS values in groups 2 and 3 were significantly lower than those in group 1 (pgroups1 and 2 = 0.014, pgroups 1–3 = 0.002). No significant differences were observed between groups 2 and 3 (pgroups 2 and 3 = p = 0.313). 3.3. TM thickness Table 3 lists TM thicknesses in groups 1–3. Significant differences were detected between TM thicknesses (p = 0.000):
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Table 1 Otomicroscopical sclerosis scores for groups 1–3a. Groups
n
Group 1 (saline) Group 2 (ascorbic acid) Group 3 (N-acetyl cysteine)
10 10 10
a & ô
Numerical distribution of OSS (n) 0
+
++
+++
0 1 2
1 5 6
2 2 1
7 2 1
OSS median (min–max)
p&
pô
3.0 (1.0–3.0) 1.0 (0.0–3.0) 1.0 (0.0–3.0)
0.004
pgroups 1 and 2 = 0.014, z = 2.463 pgroups 1–3 = 0.002, z = 3.083 pgroups2 and 3 = 0.318, z = 0.998
OSS: Otomicroscopical sclerosis score. p-Value shows the results of Kruskal–Wallis variance analysis. p-Value shows the results of Mann–Whitney U test with Bonferrroni correction.
Fig. 1. Sclerosis, inflammation, and immunohistochemical staining scores for each group. Scoring for sclerosis: (0) no visible sclerotic lesion; (+) sclerotic lesion only on the malleus arm or adjacent to it; (++) sclerotic lesion adjacent to the malleus arm and on the upper frontal part of the pars tensa; (+++) sclerotic lesion adjacent to the malleus and continuing through the annulus. Scoring for inflammation: (1) normal structure; (2) moderate inflammation; (3) severe inflammation. Scoring for immunohistochemical staining to detect VEGF, TGF-b, i-NOS, IL1-b: (1) mild; (2) moderate; (3) intense.
Table 2 Inflammation scores for groups 1–3a. Groupsô
n
Group 1 (Saline) Group 2 (Ascorbic acid) Group 3 (N-Acetyl Cysteine) a & ô
Numerical distribution of ISs (n)
10 10 10
+ (normal)
++ (moderate)
+++ (severe)
1 5 6
3 4 3
6 1 1
ISs median (min–max)
p&
pô
3.0 (1.0–3.0) 1.5 (1.0–3.0) 1.0 (1.0–2.0)
0.003
pgroups pgroups pgroups
1 and 2 1–3
= 0.014, z = 2.445 = 0.002, z = 3.135 = 0.313, z = 1.009
2 and 3
IS: inflammation score. p-Value shows the results of Kruskal–Wallis variance. p-Value shows the results of Mann–Whitney U test with Bonferrroni correction.
Table 3 Thickness of the tympanic membrane for groups 1–3. Groups
Group 1 (Saline) Group 2 (Ascorbic acid) Group 3 (N-Acetyl Cysteine) pô & ô
The thicknesses of the tympanic membranes of guinea pigs (mm) 1
2
0.090 0.068 0.056 pgroups
0.080 0.088 0.056 0.058 0.066 0.049 and 2 = 0.001, z =
1
3
4
5
6
0.092 0.070 0.060 0.046 0.065 0.050 0.050 0.045 0.052 3.258; pgroups 1–3 = 0.000,
7
8
Mean std. dev 9
Median (min-max)
p&
0.084 (0.060–0.093) 0.057 (0.043–0.080) 0.051 (0.045–0.073)
0.000
10
0.078 0.089 0.093 0.080 0.082 0.010 0.068 0.080 0.050 0.043 0.058 0.011 0.073 0.049 0.046 0.056 0.054 0.008 z = 3.557; pgroups 2 and 3 = p = 0.383, z = 0.873
p-Value shows the results of Kruskal–Wallis variance analysis. p-Value shows the results of Mann–Whitney U test with Bonferrroni correction.
TM thicknesses in group 2 and 3 were significantly thinner than those in group 1 (pgroups 1 and 2 = 0.001; pgroups 1–3 = 0.000). No significant difference was detected between groups 2 and 3 (pgroups 2 and 3 = 0.383).
3.4. Immunoreactivity scores for VEGF, TGF-b, i-NOS and IL1-b Table 4 and Fig. 1 present immunoreactivity scores for VEGF, TGF-b, i-NOS, and IL1-b.
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Table 4 Immunoreactivity scores for VEGF, TGF-b, i-NOS. and IL1-b for each group. Immunohistochemistry
p&
Immunoreactivity scores in the groups Group 1 (saline) (n=10) Numerical distribution +
++
Median
+++
Group 2 (ascorbic acid) (n=10)
Group 3 (N-acetyl cysteine) (n = 10)
Numerical distribution
Numerical distribution
+
++
Median
+++
+
++
Median
+++
VEGFô
1 2 7 3.0 pgroups 1 and 2 = 0.002, z = 3.086
8 1 1 1.0 pgroups 1–3 = 0.003, z = 2924
7 2 1 1.0 pgroups 2 and 3 = p = 0.654, z = 0,449
0.003
TGF-bô
– 4 6 3.0 pgroups 1 and 2 = 0.010, z = 2.571
3 6 1 2.0 pgroups 1–3 = 0.005, z = 2821
5 4 1 1.5 pgroups 2 and 3 = 0.450, z = 0.755
0.014
i-NOSô
– 2 8 3.0 pgroups 1 and 2 = 0.002, z = 3.091
2 7 1 2.0 pgroups 1–3 = 0.001, z = 3.423
7 2 1 1.0 pgroups 2 and 3 = p = 0.060, z = 1880
0.001
IL1-bô
2 7 1 2,0 pgroups 1 and 2 = 0.060, z = 1880
7 2 1 1,0 pgroups 1–3 = 0.008, z = 2644
8 2 – 1.0 pgroups 2 and 3 = 0.549, z = 0.600
0.015
& ô
p-Value shows the results of Kruskal–Wallis variance analysis. p-Value shows the results of Mann–Whitney U test with Bonferrroni correction.
3.5. VEGF scores (Table 4 and Fig.
1)
Significant differences in VEGF scores were observed between the groups (p = 0.003): VEGF scores in groups 2 and 3 were significantly lower than those in group 1 (pgroups 1 and 2 = 0.002; pgroups 1–3 = 0.003). No significant difference was detected between groups 2 and 3 (pgroup 2–3 = p = 0.654).
3.10. Immunohistochemical findings Immunohistochemical examination of the TM specimens obtained from the different groups revealed various patterns (Figs. 2–4). Table 4 lists the immunostaining intensities using primary antibodies against VEGF, TGF-b, iNOS, and IL1-b. These factors are known to play significant roles in angiogenesis and inflammation.
3.6. TGF-b scores (Table 4 and Fig. 1) 4. Discussion Significant differences in TGF-b scores were detected between the groups (p = 0.014): TGF-b scores in groups 2 and 3 were significantly lower than those in group 1 (pgroups 1 and 2 = 0.010, pgroups 1–3 = 0.005). No significant difference was detected between groups 2 and 3 (pgroups 2 and 3 = 0.549). 3.7. i-NOS scores (Table 4 and Fig. 1) Significant differences were detected in i-NOS scores between the groups (p = 0.001): i-NOS scores in groups 2 and 3 were significantly lower than those in group 1 (pgroups 1 and 2 = 0.002, pgroups 1–3 = 0.001). No significant difference was observed between groups 2 and 3 (pgroup 2 and 3 = 0.060). 3.8. IL1-b scores (Table 4 and Fig. 1) Significant differences were observed in IL1-b scores between the groups (p = 0.015): IL1-b scores in group 3 were significantly lower than those in Group 1 (pgroups 1–3 = 0.008). No significant differences were observed between groups 1 and 2 (pgroups 1 and 2 = 0.060) or groups 2 and 3 (pgroups 2 and 3 = p = 0.549). 3.9. Light microscopy findings Slices obtained from group 1 (saline group) revealed thickening of the TM, inflammation of the lamina propria, and increased active fibroblasts and collagen fibers. The collagen fibers were separated in some areas. Mononuclear and polymorphonuclear cellular infiltration and vasodilation were observed in the subepithelial layer (Fig. 2). The median IS was 3.0 (Table 2). Slices obtained from group 2 (ascorbic acid group) revealed decreased inflammation in the lamina propria, fewer active fibroblasts, and reduced leukocyte infiltration and thickness of the vessels (Fig. 3). The median IS was 1.5 (Table 2). Slices obtained from group 3 (NAC group) revealed fewer active fibroblasts and collagen fibers in the lamina propria (Fig. 4). The median IS was 1.0 (Table 2).
Inflammation in the collagen layer of the TM is the most pronounced factor in the pathogenesis of MS [19]. Perforation of the TM (e.g., due to myringotomy and VT insertion) causes increased oxygen concentration within the middle ear cavity [20]. Mechanistically, increased amounts of free oxygen radicals accelerate the inflammatory process, leading to the onset of the sclerotic process [21,22]. Free oxygen radicals affect the unsaturated fatty acids within the membrane lipids and start the peroxidation process, thus accelerating the inflammatory process [23]. Antioxidant agents can neutralize free oxygen radicals and block this pathway. NAC, which is the N-acetylated derivative of Lcysteine, can enter the cells readily due to its molecular structure and plays a role in the formation of a significant antioxidant, glutathione (GSH). Thus, NAC supports tissue defense against oxidant stress [24–26]. Ascorbic acid is a water-soluble antioxidant that exerts a protective effect via oxidoreduction, thereby significantly reducing the effects of free oxygen radicals [27]. We investigated the protective effects of ascorbic acid and NAC compared to saline solution on the healing process after myringotomy in guinea pigs. The OSS and inflammation scores in the ascorbic acid and NAC groups were significantly lower than those in the saline group. Compared with the saline group, fewer active fibroblasts and reduced leukocyte infiltration were observed in the ascorbic acid group, and decreased numbers of active fibroblasts and collagen fibers in the lamina propria were observed in the NAC group. Inflammation scores were lower in the ascorbic acid and NAC groups compared with the control group. Therefore, we conclude that inflammation scores, cellular infiltration by mononuclear and polymorphonuclear cells, and fibroblasts and collagen fibers were reduced by ascorbic acid and/or NAC treatments, which resulted in decreased MS development. TM thicknesses were significantly thinner in the ascorbic acid and NAC groups compared with the saline group. Additionally, the immunohistochemical results revealed less expression of VEGF, TGF-b, and iNOS in groups 2 and 3 compared with group 1. IL1-b
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Fig. 2. Light microscopic evaluation of tympanic membrane sections from group 1 (saline) (hematoxylin & eosin (HE) stain, 200). MT, membrane thickness; V, vascularization; J, junctional degeneration; i, inflammatory cells. Immunohistochemical examination results for IL1-b, i-NOS, VEGF, and TGF-b are shown (200).
scores were lower in group 3 than in group 1. IL1-b plays a significant role in angiogenesis and inflammation. Whereas ascorbic acid application decreased IL1-b expression, NAC did not affect IL1-b levels compared with the control group. In a study of rats with MS formation, the application of topical NAC decreased malondialdehyde (MDA) and nitric oxide (NO) levels [23]. Ozcan et al. [28] investigated the effect of topical administration of NAC on MS in experimentally myringotomized rat TMs, using otomicroscopic evaluation and assessing the lipid
peroxidation and NO (nitrite/nitrate) levels. They concluded that topical treatment with NAC reduced MDA and NO levels and suggested that topical NAC application may be useful for preventing MS. Similarly, in the present study, we observed fewer active fibroblasts and collagen fibers in the lamina propria of NACtreated guinea pigs, which resulted in decreased MS. Mattson et al. [4] conducted an experimental study and reported that topical application of copper zinc superoxide dismutase + catalase and deferoxamine reduces or inhibits the
Fig. 3. Light microscopic evaluation of tympanic membrane sections from group 2 (ascorbic acid) (HE, 200). V, vascularization; f, ibroblast proliferation; i, inflammatory cells; k, collagen fibers; e, epithelium. Immunohistochemical examination results for IL1-b, i-NOS, VEGF, and TGF-b are shown (200).
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Fig. 4. Light microscopic evaluation of tympanic membrane sections from Group 3 (N-acetyl cysteine) (HE, 200). MT, membrane thickness; f, fibroblast proliferation; i, inflammatory cells. Immunohistochemical examination results for IL1-b, i-NOS, VEGF, and TGF-b are shown (200).
development of sclerotic lesion, whereas ears treated with copper sulfate plus iron chloride appeared unaffected. Their findings support the hypothesis that the formation of oxygen free radicals contributes significantly to the development of MS. In the present study, we investigated the protective effects of NAC or ascorbic acid and found that these treatments reduced MS. Go¨ru¨r et al. [29] reported significantly reduced development of MS in rats that were administered selenium intraperitoneally compared with rats that received saline. Akbas¸ et al. [11] found that intraperitoneal administration of L-carnitine caused sclerosis in 35% of rats compared with 80% in the control group [11]. In another study, topically applied NAC was found to be effective in the prevention of sclerotic lesions in myringotomized rat tympanic membranes [15]. Spratley et al. [12] reported that topical ascorbic acid reduces the occurrence of MS following TM perforations in rats. In their study, light microscopy revealed that the connective tissue layer of the untouched side of the pars tensa was distinctly thicker in the ascorbic acid group than in the saline or untreated groups. In the present study, we found that ascorbic acid reduced inflammation in the lamina propria, decreased the number of active fibroblasts, and reduced leukocyte infiltration and thickness of the vessels. In our study, immunohistochemical studies of TMs revealed that the expression of VEGF, TGF-b, and iNOS was decreased significantly in groups 2 and 3. Therefore, ascorbic acid and/or NAC treatments resulted in lower inflammation scores and cellular infiltration as well as reduced expression of VEGF, TGF-b, and iNOS, causing decreased MS development. Expression of IL1-b was only affected and decreased by NAC. Similar to our results, Zhu et al. [30] reported that long-term NAC treatment resulted in downregulation of VEGF expression and reduction of reactive oxygen species content in vascular tissues. Galicia-Moreno et al. [31] reported that NAC was effective in preventing TGF-b and IL-6 expression and further augmented IL10 expression, which demonstrated the beneficial effects of NAC due to its antioxidant and immunomodulatory properties [31]. Exogenous oxidants X/XO and H2O2 upregulated the secretion of
the pro-inflammatory cytokines IL-1b, IL-6 and TNF-a [32]. Antioxidants may reduce the levels of pro-inflammatory cytokines, which might prevent the development of MS by their antioxidant and anti-inflammatory features. Yang et al. [33] reported that N-acetyl-L-cysteine (L-NAC) reduced the number of missing and dying outer hair cells and reduced styrene-induced hearing loss. Lorito et al. [34] demonstrated that some antioxidant molecules such as L-NAC can prevent oxidative stress in the inner ear. They reported that L-NAC can partially protect the cochlea from continuous noise, and the protection effect is strongly dose-dependent: lower dosages do not fully protect the cochlea and higher dosages can damage the rat systemically (e.g., pulmonary toxicity). No ototoxic effects were reported. In vitro experiments by Feghali et al. [35] revealed that LNAC protected both auditory neurons and hair cells from the toxic effects of cisplatin. Because it protects both of these inner ear structures, L-NAC exhibits potential for protecting hearing from cisplatin-induced damage. L-NAC also has low systemic and mucosal toxicity, and its low molecular weight may allow it to readily pass through the round window membrane. These characteristics of L-NAC make it potentially suitable for transtympanic application for the prevention of the ototoxicity of cisplatin in vivo [36]. Celebi et al. [37] investigated the effects of intratympanic injection of vitamin C (also called ascorbic acid) on cisplatininduced ototoxicity in rats. Intratympanic vitamin C infusion provided a protective effect against cisplatin-induced ototoxicity primarily at 2 kHz and at other frequencies (2.8, 4, 6, and 8 kHz), and did not produce a toxic effect in the cochlea. Together, these findings [33–37] suggest that NAC and ascorbic acid produce no ototoxic effects in the cochlea; on the contrary, they can protect the inner ear from potential ototoxic events. Further studies are required before NAC and/or ascorbic acid should be applied after VT insertions. Our study provides no evidence of toxic effects in animal studies. However, preclinical safety studies involving humans are still required for these agents. If found safe, placebo-controlled studies involving humans should
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be conducted to investigate the effects of NAC and/or ascorbic acid to prevent MS development after VT insertions. 5. Conclusions Previous studies have reported the preventive effects of NAC on MS, but our study is the first to compare the preventative effects of NAC and ascorbic acid on MS in rats. Our findings suggest that administration of these drugs can prevent the development of MS, which can be an important complication after VT insertions. Further detailed studies should be performed to investigate the efficacy and safety of topical antioxidants to the inner ear and hearing levels in humans. If determined safe and effective, physicians could consider using NAC and/or ascorbic acid after VT insertions to prevent MS. Conflict of interest The authors declare that there is no conflict of interest. No pharmaceutical companies funded the study or contributed to the study design, outcome evaluation, or writing of this article. Acknowledgement With the exception of data collection, the preparation of this paper, including design and planning, was supported by the Continuous Education and Scientific Research Association.
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