Effect Of Nasal Epithelial Lining Prognosis And Turnover Changed By Vesicles Molecular Immuno-pathology After Nasal Endoscopic Surgery

ObjectiveAfter functional endoscopic sinus surgery, the prognosis and turnover of the nasal mucosa is a very complex process that involves vesicle formation, a variety of inflammatory cells, cytokines and media and so on. Particularly, the formation of vesicles can have an important impact on the nasal epithelialization. The vesicle formation mechanism after endoscopic sinus surgery is not clear, there are also controversial on the question that vesicle should be routinely clear or not in clinical. In order to provide theoretic basis for the benign outcome of the nasal mucous after clinical endoscopic sinus surgery, the study utilize different methods such as pathology, transmission electron microscopy, immunohistochemistry, cytokine and different ways treating vesicle after surgery to investigate the effect of nasal epithelial lining prognosis and turnover changed by vesicles molecular immuno-pathology after nasal endoscopic surgery.Methods 1. Cases and specimensPatients were selected from the Zhujiang Hospital, Department of Otolaryngology Head and Neck Surgery which made endoscopic sinus surgery from March to August in the year2011. Forty-eight patients (96sides) with chronic nasal polyps in accordance with EPOS standard after endoscopic sinus surgery were randomly divided into treatment group and control group according to weather scraping the vesicles.27cases were male and21female, age16to72years, the average is36.5years. All of the surgical approach and preoperative and postoperative medication and dressing (oral or intravenous antibiotics, mometasone furoate nasal spray and nasal wash) of all the patients were the same. Define less than20weeks after surgery as the period of postoperative dressing. Scrapped the inferior turbinate mucosa (gently), nasal polyp tissue during surgery, nasal vesicles of different periods and epithelial tissue of the middle meatus after completion of the surgical cavity epithelization.2.Grouping and observation2.1Depending on the different approaches of the chronic sinusitis and nasal polyp patients after surgical cavity vesicles treatment, the subjects were divided into vesicles curettage groups and non-curettage group. Observed and compared the nasal epithelial after surgery of the two groups by transnasal endoscopic. Observed the pathological changes of vesicles through HE staining and transmission electron microscopy. Meanwhile divided vesicle tissues into mild group (unilateral surgical cavity vesicles≤5, and diameter of vesicles≤5mm) and severe group (unilateral surgical cavity vesicles>5or unilateral surgical cavity vesicles≤5but diameter of vesicles>5mm), observed whether the differences between vesicles affect the process of epithelial of the surgical cavity in patients.2.2Taken40patients with good compliance from48patients, according to tissue acquisition time the study were divided into experimental group (surgery nasal polyps,1to3weeks after surgery,6to8weeks after surgery and11to14weeks after surgery) and control group (the inferior turbinate mucosa scrapped over the same period). Immunohistochemical method to investigate the expression of transfomiing growth factor β1(TGF-β1) and vascular endothelial growth factor(VEGF) in the tissues in the various periods and the control group.3Preparation and observation of the specimen3.1Light microscope specimensFixed the specimen mucosa with10%formalin, dehydrated automatically, embedded in paraffin, and serially sectioned three pieces which thickness were4μm. Use HE staining deal with the specimens. Light microscope were used to observe the changes of epithelium, goblet cells, basement membrane, mucosal interstitial substance, inflammatory cells and mucous glands.3.2SEM specimensFixed the specimen mucosa with2.5%glutaraldehyde,2%paraformaldehyde and0.1mol/L phosphate buffered saline for more than4h, rinsed three times with buffer, more than10min each; and then fixed the specimen mucosa with1%osmium tetroxide and0.1mol/L phosphate buffered saline for more than1.5h, rinsed three times with buffer, more than10min each; followed by50%,70%,80%,90%and100%ethanol dehydration for10-15min each. Soaked in100%acetone twice each for10-15min. The proportion of acetone and embedding medium was1:1saturated for1h. Then the proportion of acetone and embedding medium was1:2saturated for3h. Soaked in pure embedding medium overnight; picked out samples into the embedded plate to polymerize; stayed in60℃for about48h to harden; cut1-2um semi-thin section, after positioning control cut60-80nm ultra-thin sections, fishing in the copper-line; saturated uranyl acetate and lead citrate staining; Finally observed the cilium quantity, desquamation, disorder and disorientation with SEM. 3.3Immunohistochemical staining of inflammatory factors VEGF and TGFβ1Used immunohistochemistry SP method in accordance with the instructions of the kit to staining VEGF and TGF β1, made PBS instead of antigen as a negative control, positive pieces of tissue known as a positive control. OLYMPUS BH-2photography biological microscope for image capture, and selected the view of positive cells concentrated field at40times objective lens, and adjust the image effects and image background. Determined cells that owned brown particles located in the cytoplasm or cell membrane as positive cells. And Image-Pro Plus6.0software for image analysis, measured the positive area and integral optical density of immunohistochemical sections, compared the strength of positive expression by average optical density of the positive region.4. StatisticsAll statistics described in forms of means±SEM. All the data were statistically treated by adopting statistical package of SPSS13.0. Mucosal healing time differences were assessed by factorial design analysis of variance. The differences among the groups of the expression of VEGF and TGFβ1by immunohistochemisty were assessed by repeated measures, the expression of VEGF and TGF β1by immunohistochemisty which removed the tissues with forceps were assessed by One-Way ANOVA; Homogeneity of variance using Levene, if the homogeneity of variance, multiple comparisons using the Bonferroni method; Otherwise, select the approximate F-test of heterogeneity of variance the Brown-Forsythe, method, and Dunnett’s T3method for multiple comparisons. Immunohistochemical expression of the different parts at the same time cytokines using independent samples t test. The difference was significant if the P value was<0.05.Result 1. Morphological changes of vesicles under nasal endoscopeVesicles presented yellow or gray, translucent, soft, sessile or pedunculated cystic bubble organizations. Part of the contents of vesicles were cyst fluid, some were substantive. Most of Vesicles located in the ethmoid surgical cavity and the frontal sinus drainage port. With the progress of the cleanup process, the majority of vesicles transparency declined, part of the larger vesicles wound generate new-vesicles again after clean up, the latter were usually smaller. Crusts and secretions on vesicle surfaces slowly reduced after endoscopic sinus surgery. Vesicles and polyps weren’t easy to distinguish by nasal endoscope.2.Pathology results2.1The epithelia of Vesicles were pseudostratified ciliated columnar epithelium. Vesicles were displaying epithelial denudation, discontinuous basal membranes, wide cell gap, a large number of red blood cells and a small number of eosinophilic granulocytes infiltrated within the interstitium, lamina propria edema, eosinophilc granulocytes infiltrated within epithelium and subepithelial, small blood vessels and glands visible in subepithelial at1-3weeks after FESS. Vesicles were displaying continuous basal membranes, goblet cells and eosinophilc granulocytes infiltrated in epithelialum, increased ciliated columnar epithelial at6-8weeks. Vesicles were displaying good continuous basal membranes, increased subepithelial stromal cells, mature gland, eosinophil cell infiltrated in gland duct and around blood vessels at11～14weeks. The study also found two cases that vesicles could be seen phenomenon of eosinophils gathered in the vesicle epithelium.2.2Under TEM saw a relatively dense cilia of vesicles, arranging disorderly, within them there had micro villi, most of the cilia visibility9+2type, some for9+1or absent. Nasal polyps in control group could be seen epithelial cells of large gap, uneven thickness and mitochondria swelled under the TEM. 3.Molecular Immunology results3.1TGFβ1immunohistochemistry results showed that the average optical density of positive expression area in nasal polyps,1to3weeks group,6to8weeks group,11to14weeks group were.0.63±0.06,0.61±0.06,0.58±0.08,0.39±0.04. the average optical density of positive expression area in the inferior turbinate group were0.34±0.05,35±0.06,34±0.05,.35±0.05. Repeated measurements showed that the expression of TGFβ1of the vesicles in nasal polyps group,1to3weeks group,6to8weeks,11to14weeks group were significantly different (F=73.658, P=0.000). The expression of TGFβ1after surgery of the middle meatus region has been decreasing, the corresponding changes in the inferior turbinate was relatively mild (F=0.236, P=0.871). There were significant difference between the different parts of the expression of TGFβ1(F=1010.957, P=0.000). The organizations of the middle meatus region were significantly higher than that of the inferior turbinate, the surgical site and period had an interaction effect, while the trend was significantly different (F=68.958, P=0.000). TGFβ1was mainly localized in the submucosa, Interstitial inflammatory cell membrane and cytoplasm. The site of inflammatory cell aggregated expressed more strongly.3.2VEGF immunohistochemistry results showed that the average optical density of positive expression area in the nasal polyps,1to3weeks group,6to8weeks group,11to14weeks group were0.28±0.03,0.22±0.03,0.36±0.04,0.23±0.03. the average optical density of positive expression area in the inferior turbinate group were0.23±0.02、0.24±0.05、0.23±0.03、0.24±0.04.Repeated measurements showed that the expression of VEGF of the vesicles in nasal polyps group,1to3weeks group,6to8weeks,11to14weeks group were significantly different (F=73.658, P=0.000). There were significant difference between the different parts of the expression of VEGF (F=1010.957, P=0.000). The interaction effect between spot and time was significantly different (F=68.958, P=0.000). The tissues of the middle meatus region were significantly higher than that of the inferior turbinate. The expression of VEGF of middle meatus region after surgery decreased until3weeks after surgery, and then increased and peaked at6weeks after surgery, and then declined (F=128.247; P=0.000). the corresponding changes in the inferior turbinate was relatively mild (F=0.236, P=0.871). VEGF was mainly localized in vascular endothelial cells, some epithelial cells, some glands and the cytoplasm of few inflammatory cells.4classification and treatment of vesicles influence the epithelialization of nasal mucosa after surgery.4.1The impact of the epithelization by vesicles classified base on the severity was statistically significant (F=12.628, P=0.001); Timely postoperative curettage of the vesicles could speed up the process of epithelization of the surgical cavity (F=11.120, p=0.000). The interaction effect wasn’t significant (F=1.862, p=0.116).4.2The study of vesicle in severe groups showed that the epithelialization time of the treatment group and control group were11.3±2.25weeks and13.6±3.28weeks respectively. Treatment group that scraped vesicle tissue accelerated epithelialization of nasal mucosa, compared to the control group, the epithelialization time reduced by an average of1.5weeks (t=2.586,p=0.014)4.3The epithelialization time of the treatment group and control group in mild group were9.9±1.71weeks and11.1±2.09weeks respectively. There was no significant difference for whether scrape the vesicle or not in wild group could influence the epithelialization of nasal mucosa after surgery (t=1.814,p=0.075)Conclusion1.According to the numbers and sizes of vesicles, they should be divided into mild group (unilateral surgical cavity vesicles≤5, and diameter of vesicles≤5mm) and severe group (unilateral surgical cavity vesicles>5or unilateral surgical cavity vesicles≤5but diameter of vesicles>5mm).2.Treatment of the moderate to severe vesicles after surgery is benefit to the epithelium of the nasal mucosa and reduce the dressing times.3.Vesicles may be a stage that experienced after endoscopic sinus surgery. It may be the response of nasal mucosa after surgery for inflammation, there present pathological changes and high expression of inflammatory factors (VEGF and TGFβ1). The level of VEGF and TGFβ1in vesicles after endoscopic sinus surgery could be used as one of the indicators of prognosis.