Significance And Expression Of INOS MRNA In Patients With Chronic Sinusitis After Functional Endoscopic Sinus Surgery

Chronic sinusitis is a common nasal disease. Now, the pathogenesis of chronic sinusitis is regarded as inflammation of nasal microenvironment, and effect of multi-factors and multi-processes. Therefore, the curative standard of chronic sinusitis is the complete remove of all the inflammatory mucosa. Although the efficiency of functional endoscopic sinus surgery (FESS) on chronic sinusitis has been proved by clinical practice, the wound healing of nasal mucosa after FESS remains a complex process. Yet how to reduce the continuate inflammation and promote the recovery of postoperative nasal mucosa is again to be solved. Nitric oxide (NO) is a tiny airy molecule. It plays an important role in inflammation and immune response. NO is generated by a family of nitric oxide synthase. There are three distinct isoforms of nitric oxide synthases(NOS):neuronal nitric oxide synthase(nNOS), inducible isoform nitric oxide synthase(iNOS ) and endothelial nitric oxide synthase(eNOS ). nNOS and eNOS that are called structural nitric oxide synthase(cNOS) mainly regulate physiologyical function. However, iNOS is produced principally in pathological process and plays an important role in the formation and pathology of disease. iNOS can be expressed by in a lot of cells. It can be activated by inflammatory cytokines and produces a great amount of NO. By its toxic effect, NO injures cells, increases permeability of blood vessel, inhibitsplatelet's adhere and as a result, develops tissue edema.In the present study, we examine the level and distribution of iNOS mRNA in nasal tissue of II type of chronic sinusitis after FESS in situ hybridization. Moreover, we discuss further the effect and clinical significance of NO and iNOS in the recovery of nasal mucosa of patients with chronic sinusitis after FESS.Materials and methods1. Materials30 samples were obtained from 30 patients of II type of chronic sinusitis who received FESS and were followed up in the First affiliated Hospital of Zhejiang University in 2006. We examined the expression of iNOS in human nasal tissues from patients with chronic sinusitis after FESS. All the cases were divided into three groups: the one month after FESS(n=10), the three months after FESS(n=10) and the six months after FESS(n=10). 5 normal cases and 8 cases of nasal polyp were used as two control groups. We cut the samples into 0.5 centimeter extent, embed the samples with wax and examined the samples in situ hybridization.2. Methods(l)When we took the samples from the patients, we immediately used 4 percent of paraformaldehyde to fix the samples for one hour.(2)We took away water, bathed wax and embed the samples. Then we cut the samples into sections which were 8 micrometer thick.(3)Through deparaffinization, the slides were brought to the liquid which was composed of 30 percent of hydrogen peroxide and distillational water for 10 minutes in normal temperature. Then, the slides were rinsed three times with distillational water. The slides were rinsed for 5minutes at every time.(4)Disclosure of mRNA piece: We added the pepsin which was watered by 3 percent of citric acid anhydrate in the slides. The slides were incubated at 37 ℃ for 30minutes. Then the slides were rinsed three times with phosphate buffered saline (PBS ) and once with distillational water. The slides were rinsed for 5minutes at every time.(5)The second fix: We added 1 percent of paraformaldehyde in the slides in normal temperature for 10minutes. Then, the slides were rinsed three times with distillational water.T he slides were rinsed for 5minutes at every time. (6)Pre-hybridization: We added 20 μ (?) pre-hybridizing solution in every slide. Then the slides were incubated at 42℃ for four hour.(7) Hybridization: We added 20 μ(?) hybridizing solution in every slide. Then we put the special cover glass on the slides. The slides were incubated at 42℃ overnight.(8) We took away the cover glass from the slides. Then the slides were rinsed three times with PBS. The slides were rinsed for 15minutes at every time.(9) Adding of closed solution: The slides were incubated at 37℃ for 30 minutes.(10) Adding of biotiny-digoxin: The slides were incubated at 37℃ for 90 minutes. Then the slides were rinsed four times with PBS. The slides were rinsed for 5minutes at every time.(11) Adding of SABC: The slides were incubated at 37℃ for 30 minutes. Then the slides were rinsed three times with PBS. The slides were rinsed for 5minutes at every time.(12) Adding of biotiny-peroxidase: The slides were incubated at 37℃ for 20 minutes. Then the slides were rinsed four times with PBS. The slides were rinsed for 5minutes at every time.(13) DAB staining: The slides were placed in DAB staining solution for 15 minutes. Then the slides were rinsed in water for 5 minutes.(14) The slides were placed in haematoxylin for 30 seconds. Then the slides were rinsed in water for 15 minutes.(15) The slides were mounted, followed by microscopic evaluation.ResultsiNOS mRNA-positive cells were brown-yellow, while iNOS mRNA-negative cells were blue in the cytosol. We observed and counted positive cells in the slides by 400-multiple microscopic evaluation. We selected 8 fields of vision in every slide and observed 100 cells in every field of vision. Then we counted positive cells in these 100 cells. In the normal nasal mucosa, there were few iNOS mRNA-positive cells which were mainly distributed under the mucosa membrane. In the II type of chronic sinusitis' tissues, there were plenty of iNOS mRNA-positive cells which were distributed the glands and around the blood vessels under the mucosa membrane. In the nasal tissues with chronic sinusitis after FESS, there were a few iNOS mRNA-positive cells which were distributed around the glands and around the blood vessels under the mucosa membrane.We observed and counted positive cells in the slides by 400-multiple microscopic evaluation. There were iNOS mRNA-positive cells in the normal nasal mucosa, chronic sinusitis' tissues and nasal mucosa with chronic sinusitis after FESS. In the normal cases, the positive percentage was (1.18±0.24) percent. In the cases of II type of chronic sinusitis, the positive percentage was (18.43±4.31) percent. In the patients of one month after FESS , the positive percentage was (15.40±1.64) percent. In the patients of three months after FESS ,the positive percentage was (8.34 ±0.78) percent. In the patients of six months after FESS, the positive percentage was (1.31±0.27) percent. The positive percentage of II type of chronic sinusitis was higher than that of normal cases. The difference between them was statistically significant (T test, p<0.05). The positive percentage of the one month after FESS was not lower than that of II type of chronic sinusitis. The difference between them was not statistically significant (T test, p>0.05). The positive percentage of thethree months after FESS was lower than that of II type of chronic sinusitis and the one month after FESS, but higher than that of normal cases. The difference between them was statistically significant (T test, p<0.05). The positive percentage of the six months after FESS was lower than that of the one month after FESS and the three months after FESS. The difference between them was statistically significant (T test, p<0.05). The positive percentage of the six months after FESS was not higher than that of normal cases. The difference between them was not statistically significant (T test, p>0.05).Conclusion1) In the normal nasal mucosa, there were few iNOS mRNA-positive cells .This result suggests that few iNOS mRNA-positive cells in the normal nasal mucosa is necessary to the keeping of the normal function of nose.2)As described above, the positive percentage of II type of chronic sinusitis , one month after FESS and three months after FESS was higher than that of normal nasal mucosa. The difference between them was statistically significant. This result suggests that NO and iNOS play a certain role in formation of chronic sinusitis and continuate inflammation of nasal mucosa after FESS.3) Our investigation showed that the difference between positive percentage of the nasal mucosa at one month, three months and six months after FESS was significantly. The positive percentage of the nasal mucosa after FESS decreased gradually. This result suggests that NO and iNOS play a role in continuate inflammation of nasal mucosa after FESS. The positive percentage of the one month after FESS was not lower than that of nasal polyp. This result suggests that one month after FESS is the stage that the inflammation of nasal mucosa last out and the sticking point of epitheliumlize of nasal mucosa. The positive percentage of the threemonths after FESS was lower than that of nasal polyps, but higher than that of normal nasal mucosa. The positive percentage of the six months after FESS didn't differ from that of normal nasal mucosa. This result suggests that epitheliumlize of nasal mucosa after FESS is a rather lengthy convalescence, and all patients should be followed up at least more than 6 months after surgery.