The Effects of Postoperative Astaxanthin Administration On

Journal of Clinical Medicine

Article The Eﬀects of Postoperative Astaxanthin Administration on Nasal Mucosa Wound Healing

1, 2 3 1 Lavinia-Gianina Manciula *, Cristian Berce , Flaviu Tabaran , Veronica Trombitas, and Silviu Albu 1

1 2nd Department of Otolaryngology, Iuliu Hatieganu University of Medicine and Pharmacy, 8 Victor Babes Street, 400012 Cluj-Napoca, Romania; [email protected] (V.T.); [email protected] (S.A.) 2 Department of Experimental Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, 8 Victor Babes Street, 400012 Cluj-Napoca, Romania; [email protected] 3 Pathology Department, University of Agricultural Sciences and Veterinary Medicine, 400372 Cluj-Napoca, Romania; ﬂ[email protected] * Correspondence: [email protected]; Fax: 0040-264-598278

Received: 22 August 2019; Accepted: 8 November 2019; Published: 11 November 2019

Abstract: Background: Wound healing of the nasal mucosa after endoscopic sinus surgery (ESS) is frequently complicated by scaring and consequently recurrences are encountered. Methods of optimizing results have been sought. In the present study we evaluated the effects of a powerful antioxidant, astaxanthin, on nasal mucosa healing after surgery, comparing it to the extensively studied properties of dexamethasone. Materials and Methods: 63 Wistar rats were used. The nasal mucosa from one side was damaged employing the brushing method. They were randomly divided into three experimental groups, one treated with astaxanthin, the second treated with dexamethasone and the third one acted as the control and was given normal saline. The rats were killed on days 5, 14 and 28 following injury. We observed the temporal evolution of the wound healing process and quantified the results by assessing four parameters: the epithelial thickness index (ETI), the subepithelial thickness index (STI), the goblet cell count and the subepithelial fibrosis index (SFI). Results: At 28 days, the ETI was significantly lower in the astaxanthin group (p < 0.05) compared to the other two groups. The STI was also lower in the astaxanthin group (p < 0.05), but comparable to the dexamethasone group at 28 days. The goblet cell count was higher in the astaxanthin group. The SFI had similar results in both dexamethasone and astaxanthin groups, with lower values compared to the control group. In the astaxanthin group there was no synechia formation. Conclusion: Astaxanthin given in the post injury period significantly decreases fibrosis, inhibits synechia development and significantly decreases subepithelial fibrosis. Moreover, it has no general or local toxic effects.

Keywords: nasal mucosa healing; astaxanthin; dexamethasone; synechia; epithelial thickness; subepithelial thickness; subepithelial ﬁbrosis

1. Introduction Endoscopic sinus surgery (ESS) is considered the optimal management strategy for chronic rhinosinusitis resistant to medical treatment. ESS outcomes are reported to be extremely encouraging [1,2]. Nevertheless, recurrent disease requiring revision surgery is still being reported in 8% to 38% of patients [3,4]. Common findings in revision surgery are adhesions and sinus ostium stenosis [3,4]. Wound healing of the nasal mucosa succeeding ESS is a multifaceted process, often complicated by excessive scar formation and adhesions [4–6]. In this respect, methods of optimizing surgical results have been sought [7].

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Since experimental studies are strictly restricted in humans, animal models became indispensable for apprehending the wound healing process [8]. The natural wound healing involves several stages, starting with inflammation, granulation tissue formation, reepithelization and remodeling of the newly formed epithelial tissue [5,6,8]. Rabbits or sheep have been previously defined as experimental models of nasal wound healing [9–11]. However, lately the rat model has been announced due to numerous benefits, including histology of the respiratory epithelium comparable to humans, small dimensions and faster wound healing compared to humans, rendering the study of the wound healing process much easier [8,12]. Postoperative wound healing is a complex process and encompasses three stages: inflammation, granulation formation and tissue remodeling [5,6]. The whole process is modulated by several factors, including reactive oxygen species ROS [13,14]. Reactive oxygen species (ROS) are byproducts of normal cellular metabolism that, among other functions, are also involved in all stages of wound closure [13–16]. During the first phase of inflammation, ROS play a key role in host defense against invading bacteria through local cell signaling, attracting neutrophils and macrophages to the injured site [13–16]. Moreover, ROS promote angiogenesis by stimulating endothelial cell division and migration, thus promoting blood vessel formation [14–16]. Angiogenesis is the key factor in granulation tissue formation. Finally, ROS trigger pro-matrix metalloproteinase-2 and induce cell motility, key factors in tissue remodeling [15]. However, overexposure to ROS, called oxidative stress, has an unfavorable effect on wound closure [13–15]. Hence, a precise balance between ROS and antioxidative mediators is essential for complete wound healing. Astaxanthin is a xanthophyll carotenoid found naturally in various living organisms, mainly in the oceanic surroundings [17–20]. Astaxanthin is a powerful quencher of singlet oxygen [17]. Thus, it is able to regulate oxidative stress, acting as a potent antioxidant [17–20]. Astaxanthin cannot be manufactured by humans, and consequently must be picked up through their diet [17,21]. It has a reddish color and is usually found in microalgae, seafood, fish, quails and flamingos. Its main isomer (3S, 3S’) is produced by Hematococcus pluvialis, a green alga known for its high content in astaxanthin [20,21]. Astaxanthin bears a polar–nonpolar–polar configuration, which permits a perfect fit into the cellular membrane width [17–21]. This affords the cellular membrane’s lipid barrier a greater protection against peroxidation. Many experimental studies on various animals have demonstrated the efficacy of astaxanthin in cardiovascular diseases, diabetes mellitus, hypertension, and the ischemia-reperfusion myocardial model [17–21]. A study conducted by Mizuta et al. [22], demonstrated that astaxanthin had a positive effect by decreasing inflammation during the initial phase of the vocal fold wound healing, and increasing the expression of procollagen type I and bFGF on postoperative day I. The astaxanthin-treated group showed a significant increase in hyaluronic acid deposition and attenuation of the contraction of the lamina propria [22]. These findings led to the conclusion that astaxanthin has a positive effect on preventing scaring after laryngeal interventions. A recent experimental study on the rat model concluded that systemic administration of dexamethasone following mucosal damage might decrease subepithelial edema, goblet cell hyperplasia and adhesion development [12]. We aimed in our study to assess the effects of astaxanthin on the nasal mucosa healing process succeeding surgery, comparing its effects to those of dexamethasone.

2. Materials and Methods

2.1. Animal Preparation The present study was approved by The Iuliu Hatieganu University of Medicine and Pharmacy Ethics Committee and the experiments were performed according to the Guidelines of the National Institutes of Health and the Declaration of Helsinki. The animals were kept in a 21 ◦C temperature environment, were supplied with food and water ad libitum, and had a day–night cycle of 12/12 h. J. Clin. Med. 2019, 8, 1941 3 of 16

In the present study, 63 Wistar rats were included, both male and female, weighing about 300 g each. Anesthesia was achieved by intraperitoneal (IP) injection of xylazine hydrochloride (4.5 mg/kg) and ketamine hydrochloride (45 mg/kg). The nasal mucosa from one side was damaged employing the brushing method. An interdental brush (10 mm) inserted through the nostril induced a mechanical wound [8]. Afterwards, the rats were randomly divided into 3 experimental groups, the first one treated with astaxanthin by oral gavage (100 mg/kg daily for 3 days). Dexamethasone was administered IP to the second group (0.15 mg/kg daily for 7 days). The third group acted as the control and was given normal saline (0.15 mg/kg daily for 7 days). Seven rats from each group were killed in the postoperative days 5, 14 and 28. For the histological examination, the head was immediately harvested and fixed for 72 h in 10% neutral-buffered formalin. After the complete fixation, the heads were cleaned of connective tissue and skin and decalcified for three days in a 1:1 mixture of formic and hydrochloric acid (8%) [23]. When decalcification was completed, the tissues were transversely trimmed in four planes following the previously described technique by Kittel et al. [24], at the following localizations: (1) Posterior part of upper incisors, (2) incisive papilla, (3) second palatine crest, and (4) first molar teeth. After this step, the samples were subsequently immersed overnight on a stirrer in the same mixture of formic and hydrochloric acid as described above [23,24]. Afterwards, the samples were dehydrated using an ethylic alcohol gradient (70%, 80%, 90%, 95% and 100%), clarified in xylene and embedded with the rostral faces down in paraffin wax. Tissue sections were cut from each paraffin block at 4 µm thickness and routinely stained with hematoxylin and eosin (H&E), Masson’s trichrome and Toluidine blue [23]. Finally, the histological slides were examined using an Olympus BX41 microscope. Bright field images and all morphometric evaluations were taken with an Olympus UC30 digital camera and processed using the Stream basic program. The pathologist who performed the histological examination was blinded to the study groups.

2.2. Histological Analysis In order to evaluate the outcome of the different treatments on the wound healing process, at the septal wound site various features were assessed, such as inflammatory cell infiltration, goblet and ciliary cell development, epithelial thickness, subepithelial thickness, subepithelial fibrosis and the incidence of adhesions [8,12]. As previously described, different histological indices were employed in our analysis [8,12]: The epithelial thickness index (ETI) value was obtained by calculating the ratio of the averaged height of the newly regenerated epithelium to the height of the averaged epithelium from the contralateral side, measured by taking the average distance between the basement membrane and apical surface of the epithelium from three mucosal sampling areas in H&E-stained sections at 100 magnification. An ETI value > 1.0 is considered to represent hypertrophic epithelium [8,12]. × The subepithelial thickness index (STI) value was obtained by calculating the ratio of the averaged height of the newly regenerated subepithelium to the height of the averaged subepithelium from the contralateral side, measured by taking the average length of lamina propria from three mucosal sampling areas in H&E-stained sections at 100x magnification. An STI value > 1.0 is considered to represent hypertrophic subepithelium [8,12]. The degree of fibrosis was expressed as the subepithelial fibrosis index (SFI), evaluated on relative intensities of Masson’s trichrome-stained sections at 20 magnification. The collagen staining intensity × was measured from three subepithelial areas on the injured and contralateral sides of the nasal septum. An SFI > 1.0 represents increased collagen fibrosis [8,12]. The number of goblet cells was counted in the Masson’s trichrome-stained sections at 100 × magnification at the level of the wound area. staining intensity was measured from three subepithelial areas on the injured and contralateral sides of the nasal septum. An SFI > 1.0 represents increased collagen fibrosis [8,12]. The number of goblet cells was counted in the Masson’s trichrome-stained sections at 100× magnification at the level of the wound area.

J. Clin. Med. 2019, 8, 1941 4 of 16 2.3. Statistical Analysis

2.3. StatisticalDue to the Analysis fact that samples were small and the variables were not normally distributed, comparisons between the groups were conducted with the Kruskall–Wallis non-parametric test. MedianDue and to the interquartile fact that samples ranges were were used small in and the the analysis variables and were the notresults normally were represented distributed, comparisons between the groups were conducted with the Kruskall–Wallis non-parametric test. graphically. Measurements were also made in time. Therefore, the MANOVA test with repeated Median and interquartile ranges were used in the analysis and the results were represented graphically. measurements was employed. The statistical analysis was performed using SPSS software (SPSS Measurements were also made in time. Therefore, the MANOVA test with repeated measurements Inc.ver.20.0, Chicago, IL, USA). A p value < 0.05 was considered significant. was employed. The statistical analysis was performed using SPSS software (SPSS Inc.ver.20.0, Chicago, IL, USA). A p value < 0.05 was considered significant. 3. Results 3. Results 3.1. Histologic Modifications in Relationship to Time 3.1. Histologic Modifications in Relationship to Time We followed the temporal healing pattern of the nasal mucosa in the three different experimentalWe followed groups, the temporal describing healing the histological pattern of the aspects, nasal mucosa with focus in the on three inflammation, different experimental epithelial regenerationgroups, describing and fibrosis the histological evolution. aspects, A recent with paper focus demonstrated on inflammation, that important epithelial regenerationamounts of ROS and fibrosis evolution. A recent paper demonstrated that important amounts of ROS are formed in the are formed in the course of the early stage of wound healing, namely in the first three days course of the early stage of wound healing, namely in the first three days succeeding injury [25]. succeeding injury [25]. Thus, this period might be critical in controlling ROS levels and this is why Thus, this period might be critical in controlling ROS levels and this is why we administered astaxanthin we administered astaxanthin only during the first three days of the experiment [22,25]. only during the first three days of the experiment [22,25]. On the the fifth fifth day,the day, thehistological histological pattern pattern in the control in the group control displayed group displayed areas of minimal areas hyperplasia, of minimal hyperplasia,moderate squamous moderate metaplasia squamous and metaplasia goblet cell and growth goblet with cell a growth focal pseudoglandular with a focal pseudoglandular pattern; diffuse pattern;inflammatory diffuse cell inflammatory infiltrate composed cell infiltrate of lymphocytes, composed of macrophages, lymphocytes, plasma macrophages, cells, neutrophils plasma cells, and neutrophilseosinophils (Figureand eosinophils1). In the dexamethasone(Figure 1). In the group, dexamethasone di ffuse suppurative group, diffuse exudate, suppurative minimal squamous exudate, minimalmetaplasia squamous and scarce metaplasia leukocyte infiltrateand scarce were leukocyte noticed (Figure infiltrate2). were The astaxanthin-treated noticed (Figure 2). group The astaxanthinexpressed seromucous-treated group gland expressed hyperplasia, seromucous discreet hyperplasia gland hyperplasia, of the respiratory discreet epithelium, hyperplasia squamous of the respiratorymetaplasia andepithelium, minimal squamous fibrosis (Figure metaplasia3). and minimal fibrosis (Figure 3).

Figure 1. LightLight micrographs of of the the rat’s nasal septum, control group, at 5 days after the intranasal intervention. Bilateral, Bilateral, minimal, minimal, diffuse diffuse infiltration infiltration of the lamina propria of the mucosa covering the nasal septum septum by by few few mononuclear mononuclear cells. cells. Focally, Focally, the lamina the lamina propria propria is expanded is expanded by fibrous by connective fibrous connectivetissue and thetissue covering and the mucosa covering is infiltrated mucosa is by infiltrated lymphocytes, by lymphocytes, macrophages macrophages and plasma cellsand (arrow);plasma cellsH&E, (arrow); ob 10. H&E, ob ×10. ×

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Figure 2. Light micrographs of the rat’s nasal septum, dexamethasone group, at 5 days after the Figureintranasal 2. LightLight intervention. micrographs micrographs The septal of of the the nasal rat’s nasalmucosa septum, is covered dexamethasone by an abundant group, group, purulent at at 5 days material, after the is intranasalfocally hyperplastic, intervention. intervention. with The The occasional septal septal nasal nasal squamous mucosa mucosa ismetaplasia covered is covered by (arrow). an by abundant an The abundant lamina purulent purulent propria material, is material, expanded is focally is focallyhyperplastic,and the hyperplastic, nasal withglands occasional withare replaced occasional squamous by squamouslymphoc metaplasiaytes, metaplasia macrophages (arrow). (arrow). The laminaand The plasma proprialamina cells propria is expanded(black is star). expanded and H&E, the andnasalob ×20. the glands nasal are glands replaced are replaced by lymphocytes, by lymphoc macrophagesytes, macrophages and plasma and cells plasma (black cells star). (black H&E, star). ob H&E,20. × ob ×20.

Figure 3. 3. LightLight micrographs micrographs of theof the rat’s rat’s nasal nasal septum, septum, astaxanthin astaxanthin group, group,at 5 days at after 5 days the intranasal after the Figureintervention.intranasal 3. Light intervention. The micrographs nasal mucosa The nasal of is the disrupted mucosa rat’s isnasal with disrupted septum, few cell with debris astaxanthin few admixed cell debris group, with admixed at mucus 5 days adherent with after mucus the on intranasaltheadh edgeserent on of intervention. the the defect edges (arrow). of The the nasal defect The mucosa lamina (arrow). propria is disrupted The of lamina the with margins propria few is cell of expanded thedebris margins admixed by mononuclear is expanded with mucus cells by adhadmixedmononuclearerent with on thecells neutrophils edges admixed of and the with mild defect neutrophils edema (arrow). (arrow); and The mild laminaH&E, edema ob propria (arrow);10. of H&E, the margins ob ×10. is expanded by × mononuclear cells admixed with neutrophils and mild edema (arrow); H&E, ob ×10. On the the 14th 14th day, day, the histological the histological pattern pattern displayed displayed squamous squamous metaplasia metaplasia with severe with seromucous severe glandseromucousOn hyperplasia the gland 14th inhyperplasia day, the control the histological in group, the control and pattern at group, the level displayedand of at the the epithelium, level squamous of the mononuclear epithelium, metaplasia cellsmononuclear with infiltrate severe sintocellseromucous the infiltrate lamina gland into propria hyperplasia the lamina (Figure in propria4). the In control the (Figure dexamethasone group, 4). In and the at dexamethasonegroup, the level there of the is completeepithelium, group, there regeneration mononuclear is complete of the respiratory mucosa, with mixed perilesional inflammation and subtle metaplasia, suppurative cellsregeneration infiltrate of into the the respiratory lamina propria mucosa, (Figure with 4). In mixed the dexamethasone perilesional inflammation group, there is and complete subtle exudate. Subepithelial fibrosis and inflammation are minimal (Figure5). In the astaxanthin group, regenerationmetaplasia, suppurative of the respiratory exudate. Subepithelial mucosa, with fibrosis mixed and perilesiinflammationonal inflammation are minimal (Figure and subtle 5). In there is complete regeneration of the respiratory mucosa, with discreet mixed inflammation, together metaplasia,the astaxanthin suppurative group, exudate. there is completeSubepithelial regeneration fibrosis and of inflammation the respiratory are mucosa,minimal with(Figure discreet 5). In with areas of mucous hyperplasia, squamous metaplasia and moderate suppurative exudate. There is thealsomixed astaxanthin severe inflammation, perilesional group, together seromucous there is wit complete glandh areas hyperplasia regeneration of mucous and of hyperplasia, subepithelial the respiratory squamous fibrosis mucosa, (Figure metaplasia with6). discreet and mixedmoderate inflammation, suppurative exudate. together There with is areas also severe of mucous perilesional hyperplasia, seromucous squamous gland hyperplasia metaplasia and moderatesubepithelial suppurative fibrosis (Figure exudate. 6). There is also severe perilesional seromucous gland hyperplasia and subepithelial fibrosis (Figure 6).

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Figure 4. Light micrographs of the rat’s nasal septum, control grou group,p, at 14 days after the intranasal intervention. Diffuse Diﬀuse infiltration inﬁltration of of the the lamina propria of the mucosa covering the nasal septum by many lymphocytes lymphocytes admixed admixed with with macrophages macrophages and and few plasmafew plasma cells (black cells (black star). The star). covering The covering mucosa mucosais occasionally is occasionally erosive (arrow);erosive (arrow); H&E, ob H&E,40. ob ×40. ×

Figure 5. Light micrographs of the rat’s nasal septum, dexamethasone group, at 14 days after the intranasal intervention. The The lamina lamina propria propria of of the the mucosa covering the nasal septum is moderatelymoderately expanded and the nasal glands are replacedreplaced by lymphocytes, macrophages and plasma cells (black star). H&E, H&E, ob ×20.20. ×

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Figure 6. Light micrographs of the rat’s nasal septum, astaxanthin group, at 14 days after the intranasal intervention. Diffusely, the lamina propria of the mucosa covering the nasal septum is Figure 6. LightLight micrographs micrographs of of the the rat’s rat’s nasal septum, astaxanthin group, group, at at 14 days after the markedly expanded by many macrophages admixed with lymphocytes and few plasma cells (black intranasal intervention. Diffusely, Diffusely, the the lamina lamina propria propria of the mucosa covering the nasal septum is star). The covering mucosa is focally erosive and occasionally hyperplastic (arrow); H&E, ob ×10. markedly expanded by many macrophages admixed with lymphocytes and few plasma cells (black star). The covering mucosa is focally erosive and occasionally hyperplastic (arrow); H&E, ob ×10.10. On day 28 in the control group, the epithelium shows areas of mucous hyperplasia,× perilesionalOn day day seromucous 28 28 in the in controlthe glands control group, and group, goblet the epithelium thecell hyperplasia. epithelium shows areas shoTherews of is mucous areasmoderate of hyperplasia, mucoussubepithelial hyperplasia, perilesional fibrosis andseromucousperilesional inflammation seromucous glands associated and glands goblet with and cell the goblet hyperplasia.areas cell of mucoushyperplasia. There hyperplasia. isThere moderate is Inmoderate the subepithelial dexamethasone subepithelial fibrosis group, fibrosis and a focalinflammationand inflammation absence of associated the associated respir withatory with the mucosa areas the areas of and mucous of minimal mucous hyperplasia. subepithelialhyperplasia. In the Infibrosis dexamethasone the dexamethasone were noticed. group, group,There a focal isa absence of the respiratory mucosa and minimal subepithelial fibrosis were noticed. There is severe severefocal absence perilesional of the glandularrespiratory atrophy mucosa andand minimal seromucous subepithelial gland regeneration fibrosis were (Figure noticed. 7). There In the is perilesional glandular atrophy and seromucous gland regeneration (Figure7). In the astaxanthin group, astaxanthinsevere perilesional group, the glandular examination atrophy revealed and complete seromucous regeneration gland regeneration of the respiratory (Figure mucosa, 7). In w theith the examination revealed complete regeneration of the respiratory mucosa, with absent perilesional absentastaxanthin perilesional group, the hyperplasia examination or metaplasia,revealed complete with glandular regeneration regeneration of the respiratory and no mucosa, subepithelial with hyperplasia or metaplasia, with glandular regeneration and no subepithelial fibrosis (Figure8). fibrosisabsent perilesional(Figure 8). hyperplasia or metaplasia, with glandular regeneration and no subepithelial fibrosis (Figure 8).

Figure 7. LightLight micrographs micrographs of of the the rat’s nasal septum, dexamethasone group, group, at at 28 28 days after the intranasalintranasal intervention. intervention. Unilateral Unilaterallyly the the lamina lamina propria propria of of the the mucosa mucosa covering covering the the nasal nasal septum septum is is Figure 7. Light micrographs of the rat’s nasal septum, dexamethasone group, at 28 days after the moderately expanded and and the nasal glands are replaced by lymphocytes, macrophages and plasma intranasal intervention. Unilaterally the lamina propria of the mucosa covering the nasal septum is cells (black star). The The covering covering mucosa mucosa is is focally focally erosive; erosive; H&E, ob ×20.20. moderately expanded and the nasal glands are replaced by lymphocytes,× macrophages and plasma cells (black star). The covering mucosa is focally erosive; H&E, ob ×20.

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Figure 8. 8. LightLight micrographs micrographs of theof the rat’s rat’s nasal nasal septum, septum, astaxanthin astaxanthin group, group, at 28 days at after28 days the intranasal after the intranasalintervention. intervention. Diffusely theDiffusely lamina the propria lamina of propria the mucosa of the covering mucosaboth covering the nasal both septumthe nasal and septum nasal andconchae nasal is conchae expanded is expanded by many lymphocytes by many lymphocytes admixed with admixed macrophages, with macrophages, few plasma fe cellsw plasma (black cells star) (blackand edema. star) and The edema. covering The mucosa covering is focally mucosa excavated is focally (arrow) excavated and (arrow) covered and by squamoidcovered by epithelium; squamoid epithelium;H&E, ob 10. H&E, ob ×10. × 3.2. Index Changes in Relationship to Time 3.2. Index Changes in Relationship to Time. The descriptive statistic of the variables used is summed up in Table1. The descriptive statistic of the variables used is summed up in Table 2. ETI indices are presented in Figure9; Figure 10: In the control group, the ETI value was higher ETI indices are presented in Figure 9; Figure 10: In the control group, the ETI value was higher on day 28 in comparison to day 5; in the dexamethasone group it displays an ascending trend, its valueon day being 28 in higher comparison on day to 28. day However, 5; in the in dexamethasone the Astaxanthin group group, it ETI displays at day 28an hadascending the lowest trend, value its whenvalue comparedbeing higher to the on otherday 28. groups, However, but also in inthe comparison Astaxanthin to daygrou 5.p, In ETI order at today analyze 28 had the the statistical lowest valuedifferences when between compared groups, to the we other used groups, the Kruskal–Wallis but also in comparison non-parametric to day test. 5. When In order contemplating to analyze the statisticalresults presented differences in Table between2, the di groups,fferences we are used statistically the Kruskal significant.–Wallis non-parametric test. When contemplating the results presented in Table 1, the differences are statistically significant. Table 1. Descriptive statistics of the variables used. Table 1. Kruskal–Wallis test results. Group Mean Median Std. Deviation Minim Maxim Range IQR C 2.00 1.00Test Statistics 2.345 a,b 0 6 6 4 Goblet5 A 2.00 2.00Chi-Square 1.581 df 0Asymp. 4Sig. 4 3 DGoblet5 6.40 7.007.715 1.8172 40.021 8 4 4 CGoblet14 1.60 1.009.013 1.5172 00.011 4 4 3 Goblet14 AGoblet28 5.80 6.009.073 1.9242 30.011 8 5 4 D 1.00 1.00 1.000 0 2 2 2 ETI5 8.180 2 0.017 C 1.20ETI14 1.006.020 1.3042 00.049 3 3 3 A 4.20 4.00 1.304 3 6 3 3 Goblet28 ETI28 12.500 2 0.002 D 0.80 1.00 0.837 0 2 2 2 STI5 9.500 2 0.009 C 0.7032 0.7182 0.14353 0.53 0.88 0.36 0.27 STI14 7.280 2 0.026 ETI5 A 1.0231 1.0674 0.13079 0.86 1.19 0.33 0.24 D 0.6274STI28 0.66209.780 0.175412 0.390.008 0.86 0.47 0.31 a Kruskal–Wallis test. b Grouping variable: Tip_cod. C 0.5898 0.5478 0.10927 0.47 0.72 0.25 0.21 ETI14 A 1.1950 1.1429 0.23580 0.98 1.52 0.54 0.45 D 0.7812 0.7708 0.44348 0.35 1.40 1.05 0.84

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Table 1. Cont.

Group Mean Median Std. Deviation Minim Maxim Range IQR C 0.8236 0.8142 0.08743 0.73 0.95 0.22 0.16 ETI28 A 0.4753 0.4942 0.17553 0.28 0.71 0.43 0.33 D 1.1247 1.0930 0.12677 1.00 1.30 0.30 0.24 C 0.7389 0.6732 0.21001 0.57 1.08 0.50 0.36 STI5 A 1.2077 1.2038 0.04060 1.17 1.26 0.09 0.08 D 0.6663 0.6038 0.17112 0.52 0.89 0.38 0.33 C 1.3352 1.5252 0.31325 0.91 1.61 0.70 0.57 STI14 A 0.9056 0.8392 0.09938 0.83 1.04 0.21 0.18 D 0.8732 0.8434 0.11184 0.78 1.07 0.29 0.15 C 1.3153 1.3664 0.21380 1.02 1.59 0.57 0.38 STI28 A 0.4848 0.4552 0.05832 0.45 0.58 0.14 0.09 D 0.5354 0.5176 0.08858 0.42 0.66 0.24 0.15

Table 2. Kruskal–Wallis test results.

Test Statistics a,b Chi-Square df Asymp. Sig. Goblet5 7.715 2 0.021 Goblet14 9.013 2 0.011 Goblet28 9.073 2 0.011 ETI5 8.180 2 0.017 ETI14 6.020 2 0.049 ETI28 12.500 2 0.002 STI5 9.500 2 0.009 STI14 7.280 2 0.026 STI28 9.780 2 0.008 a Kruskal–Wallis test. b Grouping variable: Tip_cod.

The significant statistical difference is also outlined by the MANOVA test, displaying important differences between the developments in time of the indices in the three experimental groups (Figure 11). The control and dexamethasone groups demonstrate a similar pattern of ETI evolution, opposed to the astaxanthin group where a significant drop on the 28th day is recorded (the lowest documented value).

Figure 9.FigureEpithelial 9. Epithelial thickness thickness index index (ETI) (ETI) value value distribution distribution (median). (median).

Figure 10. Epithelial thickness index (ETI) value distribution (boxplot representation).

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J. Clin. Med. 2019, 8, 1941 10 of 16 Figure 9. Epithelial thickness index (ETI) value distribution (median).

Figure 10. Epithelial thickness index (ETI) value distribution (boxplot representation). Figure 10. Epithelial thickness index (ETI) value distribution (boxplot representation).

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Figure 11. Epithelial thickness index (ETI) values, MANOVA test. Figure 11. Epithelial thickness index (ETI) values, MANOVA test. The STI indices are comparable between days 14 and 28 in the control group, but in the astaxanthin The STI indices are comparable between days 14 and 28 in the control group, but in the and dexamethasone groups they decline on day 28, having comparable values (Figure 12; Figure 13). astaxanthinThe MANOVA and testdexamethasone showed significant groups ditheyfferences decline between on day groups28, having regarding comparable the evolution values (Figure in time,12; but Figure also when13). comparing the three experimental groups. On day 5, there were similar results for the controlThe MANOVA and the dexamethasone test showed significant groups, butdifferences significantly between lower groups as compared regarding to astaxanthin.the evolution in The controltime, but group also haswhen an comparing ascending trend,the three diff erentexperimental from the groups. dexamethasone On day 5, and there astaxanthin were similar groups, results bothfor presenting the control a significant and the dexamethasone and constant decline groups, on but days significantly 14 and 28 (Figure lower as14 ).compared to astaxanthin. The control group has an ascending trend, different from the dexamethasone and astaxanthin groups, both presenting a significant and constant decline on days 14 and 28 (Figure 14).

Figure 12. Subepithelial thickness index (STI) value distribution (median).

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Figure 11. Epithelial thickness index (ETI) values, MANOVA test.

The STI indices are comparable between days 14 and 28 in the control group, but in the astaxanthin and dexamethasone groups they decline on day 28, having comparable values (Figure 12; Figure 13). The MANOVA test showed significant differences between groups regarding the evolution in time, but also when comparing the three experimental groups. On day 5, there were similar results for the control and the dexamethasone groups, but significantly lower as compared to astaxanthin. The control group has an ascending trend, different from the dexamethasone and astaxanthin J. Clin. Med. 2019, 8, 1941 11 of 16 groups, both presenting a significant and constant decline on days 14 and 28 (Figure 14).

Figure 12. Subepithelial thickness index (STI) value distribution (median). Figure 12. Subepithelial thickness index (STI) value distribution (median).

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Figure 13. Subepithelial thickness index (STI) value distribution (boxplot representation). Figure 13. Subepithelial thickness index (STI) value distribution (boxplot representation).

Figure 14. Subepithelial thickness index values, MANOVA test.

The goblet cell count shows a decreasing value on day 28 as compared to day 5 in the control and dexamethasone groups, but in the astaxanthin group the value was higher on day 28 when compared to day 5. On day 14, we obtained the same values both in the astaxanthin and the dexamethasone groups (Figure 15; Figure 16). The MANOVA test shows a significantly lower cell count on day 5 in the control group as compared to the dexamethasone group (p < 0.05). On day 14 there were no statistically significant differences between the control and dexamethasone groups, but there was a significant difference between the astaxanthin and the control groups, the astaxanthin group showing a higher goblet cell count than the control group (p < 0.05) (Figure 17).

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J. Clin. Med. 2019, 8, 1941 12 of 16 Figure 13. Subepithelial thickness index (STI) value distribution (boxplot representation).

Figure 14. Subepithelial thickness index values, MANOVA test. Figure 14. Subepithelial thickness index values, MANOVA test. The goblet cell count shows a decreasing value on day 28 as compared to day 5 in the control and dexamethasoneThe goblet groups, cell butcount in theshows astaxanthin a decreasing group value the value on day was 28 higher as compared on day 28to whenday 5 comparedin the control to dayand 5. dexamethasone On day 14, we obtained groups, but the in same the values astaxanthin both in group the astaxanthin the value was and higher the dexamethasone on day 28 when groupscompared (Figure 15 to ;day Figure 5. 16 On). day 14, we obtained the same values both in the astaxanthin and the dexamethasoneThe MANOVA groups test shows (Figure a significantly 15; Figure 16 lower). cell count on day 5 in the control group as comparedThe to the MANOVA dexamethasone test shows group a significantly (p < 0.05). On lower day cell 14 there count were on day no statistically 5 in the control significant group as differencescompared between to the thedexamethasone control anddexamethasone group (p < 0.05). groups, On day but 14 therethere waswere a no significant statistically diff erencesignificant betweendifferences the astaxanthin between andthe control the control and groups,dexamethasone the astaxanthin groups, group but there showing was aa highersignificant goblet difference cell countbetween than the the control astaxanthin group (andp < 0.05)the control (Figure groups, 17). the astaxanthin group showing a higher goblet cell count than the control group (p < 0.05) (Figure 17).

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FigureFigure 15. 15. GobletGoblet cell cell count count (median). (median).

Figure 16. Goblet cell count (boxplot representation).

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J. Clin. Med. 2019, 8, 1941 13 of 16 Figure 15. Goblet cell count (median).

FigureFigure 16. 16.Goblet Goblet cell cell count count (boxplot (boxplot representation). representation).

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Figure 17. Goblet cell count, MANOVA Test. Figure 17. Goblet cell count, MANOVA Test. The subepithelial fibrosis index showed only small differences between groups, but for the The subepithelial fibrosis index showed only small differences between groups, but for the dexamethasone group the value was significantly smaller than that of the control group on day 28 (Figuredexamethasone 18). group the value was significantly smaller than that of the control group on day 28 (Figure 18).

Figure 18. Subepithelial fibrosis index (SFI) value distribution expressed as the mean.

4. Discussion

The most common causes of surgical failure following ESS are synechia development and ostium stenosis [3,4]. Topical steroids have been long used to impede the development of recurrent nasal polyps [26]. Moreover, Jorissen et al. [27] have demonstrated that early postoperative topical steroids may even improve wound healing, especially in nasal polyposis. Dexamethasone impregnated stents were employed with considerable success in experimental models [28]. In a prospective, randomized study, Wright et al. [29] stated that preoperative and postoperative systemic corticoids improve considerably the objective and subjective outcomes. Moreover, in

J. Clin. Med. 2019, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/jcm

Figure 17. Goblet cell count, MANOVA Test.

The subepithelial fibrosis index showed only small differences between groups, but for the

J. Clin.dexamethasone Med. 2019, 8, 1941 group the value was significantly smaller than that of the control group on14 day of 16 28 (Figure 18).

Figure 18. Subepithelial fibrosis index (SFI) value distribution expressed as the mean. Figure 18. Subepithelial fibrosis index (SFI) value distribution expressed as the mean. 4. Discussion 4. Discussion The most common causes of surgical failure following ESS are synechia development and ostium stenosisThe [3, 4 most]. Topical common steroids causes have of been surgical long failure used to following impede the ESS development are synechia of development recurrent nasal and polypsostium [26 ].stenosis Moreover, [3,4] Jorissen. Topical et steroids al. [27] have have demonstrated been long used that earlyto impede postoperative the development topical steroids of recurrent may evennasal improve polyps wound [26]. Moreover, healing, especially Jorissen inet nasalal. [27] polyposis. have demonstrated Dexamethasone that early impregnated postoperative stents weretopical employedsteroids with may considerable even improve success wound in experimental healing, models especially [28 ]. in In anasal prospective, polyposis. randomized Dexamethasone study, Wrightimpregnated et al. [29 ] stents stated were that preoperative employed with and postoperative considerable systemicsuccess in corticoids experimental improve models considerably [28]. In a theprospective, objective and randomized subjective outcomes. study, Wright Moreover, et in al. pediatric [29] stated patients that it preoperativeis suggested that and intraoperative postoperative dexamethasone decreases maxillary mucosa swelling and granulations [30]. However, it is acknowledged systemic corticoids improve considerably the objective and subjective outcomes. Moreover, in that systemic corticoid employment bears the risk of serious side effects, thus limiting its indications. InJ. a Clin. recent Med. experimental 2019, 8, x; doi: studyFOR PEER on rats,REVIEW it was demonstrated that systemic postoperativewww.mdpi.com/journal/ dexamethasonejcm decreases edema, goblet cell hyperplasia and adhesions, but it seems to have a negative impact on mucociliary regeneration [12]. Lately, Testa et al. [31] confirmed that topical alpha-tocopherol acetate is competent to significantly improve the post-operative healing of elderly CRS patients deferred to ESS. A previous study on wound healing of the vocal folds proved that ROS is mainly produced in the first 3 days following injury; therefore, this period is critical in oxidative stress adjustment [25]. Hence, in our experiment astaxanthin was provided in the first 3 days following injury of the septum. In our study, in the astaxanthin-treated group there was moderate inflammation on day 5 at the level of the epithelium and lamina propria as compared to the dexamethasone group and the control group. On day 14, the respiratory mucosa was completely regenerated and the subepithelial fibrosis was moderate. On day 28, there was complete regeneration of the respiratory mucosa and the subepithelial fibrosis was absent. On microscopic examination of the turbinates and paranasal sinus area we could not observe any synechia or obvious atrophy. On the contrary, the control group displayed atrophy of the mucosa of the turbinates and epithelial atrophy and fibrosis in the paranasal sinuses. In the dexamethasone group the examination revealed turbinate atrophy and synechia formation. These findings indicate that the positive effect of astaxanthin leads not only to a decrease in fibrosis and synechia formation at the injury site, but it also has a positive influence on the mucosa of the surrounding areas. During the healing period, both the control and dexamethasone groups present an ascending trend of the epithelium thickness index. On the contrary, on the 28th day the astaxanthin group displays a marked reduction of the epithelium thickness. Our finding confirms a previous study, where astaxanthin significantly reduced vocal fold scarring. It also seems that the effect of astaxanthin J. Clin. Med. 2019, 8, 1941 15 of 16 is quite similar to that of dexamethasone regarding subepithelial fibrosis on day 28, making it a good alternative in the setting where corticosteroids are not indicated or tolerated. However, an interesting finding was the significant enhancement of goblet cell proliferation in the astaxanthin group. Since these cells display a normal pattern on histological examination, we can suppose that the mucociliary clearance is improved in these animals. However, there are also several limitations of our study: Definite conclusions cannot be drawn because of the restricted time span of our study, and the experiments were done on healthy, noninfected rats. We should expand our knowledge on wound healing in the context of induced inflammation and infection of the paranasal sinuses.

5. Conclusions Despite its limitations, we have been able to demonstrate in an experimental setting that astaxanthin given in the post injury period significantly decreases fibrosis, inhibits synechia development and significantly decreases subepithelial fibrosis. Moreover, it has no general or local toxic effects.

Author Contributions: Conceptualization of the research was made by L.-G.M. and S.A.; The present study was performed under the supervision of Professor S.A., who was also responsible for the writing-review and editing of the manuscript; L.-G.M. was involved in data curation, project administration and writing of the original draft; Investigation was carried out by L.-G.M., C.B., F.T. and S.A.; V.T. was involved in project administration and methodology of the research. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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