Patients and sample collection
A total of 30 chronic rhinosinusitis patients with hypertrophic rhinitis and/or nasal polyps were enrolled in this study. They consisted of 22 males and 8 females, aged 17-81 years, with an average age of 50.0 years. Total and/or specific serum IgE levels were positive in 23 patients (76.7%). Specific serum IgE levels were measured for house dust mites, Japanese cedar pollen, cypress pollen, orchard grass pollen, short ragweed pollen, timothy grass pollen and Aspergillus, which are major airborne allergens in Japan. Five patients had bronchial asthma.
The patients underwent endoscopic sinonasal surgery including turbinectomy and/or nasal polypectomy under general anesthesia. In turbinectomy, the inferior turbinate bone was resected together with the lateral mucosa of the turbinate. The collected inferior turbinates and nasal polyps were immediately soaked in O2-saturated Hank’s balanced salt solution (HBSS; 8000 (in mg/L) NaCl, 400 KCl, 350 NaHCO3, 140 CaCl2, 100 MgCl2.6H2O, 100 MgSO4.7H2O, 60 KH2PO4, 47.8 Na2HPO4, and 1000 glucose) and thoroughly washed with HBSS to remove surface mucus. The lateral mucosae of the collected turbinates were separated from the underlying bone with surgical scissors. The turbinate mucosae and nasal polyps were then subjected to the following process.
Preparation of mucosal pieces for video recording
The turbinate and nasal polyp mucosae were cut into slender strips, including the mucosal surface along the long axis, using a razor blade. Two mucosal strips lying at right angles to each other were prepared from each sample. In the turbinate mucosae, the two strips were cut parallel to the horizontal and vertical axes, respectively. The mucosal strips were immediately immersed in O2-saturated HBSS and transferred into a 20 x 6 x 1-mm test chamber filled with the same solution containing 0.05% (v/v) India ink as a tracer. Mucociliary beating/transport was observed under a Nikon Eclipse 80i phase-contrast light microscope (Nikon, Tokyo, Japan) equipped with a high-speed digital imaging system HAS-U1 (DITECT, Tokyo, Japan). Four recordings of 2-3 sec each were made at 60-sec intervals at a speed of 200 frames/sec and analyzed by HAS-XViewer Camera Memory ver. 1.2.12 (DITECT). All procedures were performed at room temperature (approx. 24oC) and completed within 3 hrs after sample collection.
Measurement of mucociliary transport velocity (MCTV)
The MCTV of each mucosal strip was determined by measuring the distance traveled of India ink microparticles per unit time. The actual MCTV was calculated by the following formula:
where MCTVx and MCTVy are the MCTVs along the paired mucosal strips lying at right angles to each other. Then, the actual MCTVs of the right and left turbinates were averaged in each patient.
Measurement of ciliary beat frequency (CBF)
The number of ciliary beats was counted manually by checking the video in a slow replay mode. CBF was measured at three different sites of each mucosal strip. The CBF value in each patient was determined by averaging 16 measurements (4 recordings x 2 mucosal strips x both sides).
Fluorescence immunohistochemistry
The specimens were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 (PB) at 4°C overnight. The fixed samples were transferred into a solution of 20% sucrose in 0.1 M phosphate-buffered saline at pH 7.4 (PBS) and incubated at 4oC for 2 nights with 3-4 changes of solution. The samples were then frozen while embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) and stored at -80oC until sectioning. Seven-mm-thick sections were prepared using a cryostat, mounted on silane-coated glass slides (Superfrost; Matsunami Glass Industries, Osaka, Japan), and air-dried. The sections were hydrated in PBS with 0.3% Triton X-100 (PBST) for 20 min and treated with 1.5% normal goat serum in PBST for 1 hr.
Next, the sections were incubated with rabbit anti-human Dishevelled-1 polyclonal antibody (PAJ540Hu01; Cloud-Clone, Houston, TX, USA), rabbit anti-human Dishevelled-3 polyclonal antibody (PAL453Hu01; Cloud-Clone), rabbit anti-human Frizzled3 polyclonal antibody (CSB-PA882067ESR2HU; Cusabio, Houston, Tx, USA), rabbit anti-human Frizzled6 polyclonal antibody (G260; Assay Biotech, Fremont, CA, USA), mouse anti-human Prickle1 monoclonal antibody (sc-393034; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-human Prickle2 polyclonal antibody (CSB-PA773783ESR1HU; Cusabio), mouse anti-human Vangl1 monoclonal antibody (sc-166844; Santa Cruz Biotechnology), or mouse anti-human Vangl2 monoclonal antibody (sc-515187; Santa Cruz Biotechnology) at 4°C overnight. The primary antibodies were used at dilutions of 1:50 in PBST containing 0.5% bovine serum albumin (BSA). As a negative control, the primary antibodies were omitted from the process. After a brief rinse with PBST, the sections were reacted at room temperature for 2 hrs with a secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Eugene, OR, USA) for Dishevelled-1, Dishevelled-3, Frizzled3, Frizzled6 and Prickle2 or Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) for Prickle1, Vangl1 and Vangl2, diluted 1:1000 in PBST containing 0.5% BSA. The sections were coverslipped with Prolong Gold antifade reagent containing 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI; Invitrogen) and examined under a Carl Zeiss Axioskop 2 Plus fluorescence microscope. The light source was an HBO 103 W/2 mercury vapor lamp. The light passed through specific wavelength bandpass filters for excitation: 475-495 nm for Alexa Fluor 488 and 340-380 nm for DAPI. Similarly, the emitted fluorescence passed through a 515-565 nm bandpass filter for Alexa Fluor 488 and a 435-485 nm bandpass filter for DAPI. Images were captured using a Carl Zeiss AxioCam digital camera attached to the microscope.
Preparation of total RNA
The collected tissues were minced with surgical scissors, soaked in 1 ml TRIzol Reagent (Invitrogen), and sonicated by an ultrasonic homogenizer (Taitec, Saitama, Japan). Two hundred ml chloroform was added, and after thorough shaking, the mixture was centrifuged at 22,000 xg for 15 min at 4oC. The aqueous layer was transferred to another tube, and total RNA was extracted by the acid guanidiniumthiocyanate-phenol-chloroform method and cleaned up with a BioRobot EZ1 system (QIAGEN, Hilden, Germany), which enables fully automated extraction and purification of nucleic acids by magnetic bead technology. The purity of RNA was assessed by determining the ratio of light absorption at 260 nm (A260) to that at 280 nm (A280). An A260/A280 ratio in the 1.9-2.1 range was considered acceptable. The RNA concentration was determined from A260.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
The total RNA was reverse-transcribed to cDNA with a High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA), which uses random primers. The real-time RT-PCR analysis was performed with an Applied Biosystems StepOnePlus real-time PCR system using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene according to the manufacturer's instructions. The TaqMan Gene Expression Assays for Dishevelled-1 (DVL1; assay identification number Hs00182896_m1), Dishevelled-3 (DVL3; assay identification number Hs00610263_m1), Frizzled3 (FZD3; assay identification number Hs00907280_m1), Frizzled6 (FZD6; assay identification number Hs00171574_m1), Prickle1 (PRICKLE1; assay identification number Hs01055551_m1), Prickle2 (PRICKLE2; assay identification number Hs00291033_s1), Vangl1 (VANGL1; assay identification number Hs01572998_m1), Vangl2 (VANGL2; assay identification number Hs00393412_m1), and GAPDH (assay identification number Hs99999905_m1) were purchased from Applied Biosystems. Ten ng cDNA in 1 µl reaction buffer was mixed with TaqMan Universal PCR Master Mix with AmpErase (uracil N-glycosylase) and the primer/probe set of the TaqMan Gene Expression Assays, and the mixture was subjected to PCR amplification with real-time detection. The thermal cycler conditions were as follows: holding at 95oC for 2 min, followed by 40 cycles of a two-step polymerase chain reaction of 95oC for 1 sec and 60oC for 20 sec. Each sample was assayed in duplicate. The measured threshold cycle (CT) was normalized by subtracting the CT for GAPDH of each sample from those for the target mRNAs. From the obtained DCT values, the ratio of the target mRNA to GAPDH mRNA was calculated by the following formula:
Target mRNA/GAPDH mRNA ratio = 2-ΔCT.
Statistical analysis
Data are expressed as means ± SEM. Statistical analysis was performed with BellCurve for Excel Statistics (Social Survey Research Information Co., Tokyo, Japan). Differences between two groups were analyzed by the two-tailed Student t-test. P-values <0.05 were considered significant.