ORIGINAL ARTICLE
Increased permeability of the epithelium of middle ear cholesteatoma Koizumi, H.,*a Suzuki, H.,*a Ohbuchi, T.,* Kitamura, T.,* Hashida, K.* & Nakamura, M.† *Department of Otorhinolaryngology-Head and Neck Surgery, †Department of Dermatology, School of Medicine, University of Occupational and Environmental Health, Fukuoka, Japan Accepted for publication 5 October 2014 Clin. Otolaryngol. 2015, 40, 106–114
Objective: We investigated the electrical impedance of and the expressions of tight junction molecules in the cholesteatoma epithelium to provide supporting evidence for the acid lysis theory of bone resorption in middle ear cholesteatoma. Methods: Study subjects were patients with primary acquired middle ear cholesteatoma and those with noncholesteatomatous chronic otitis media who underwent tympanomastoidectomy. The electrical impedance of the cholesteatoma epithelium was measured during tympanomastoidectomy by loading alternating currents of 320 Hz and 30.7 kHz. The expressions of tricellulin (MARVELD2), claudin-1 (CLDN1) and claudin-3 (CLDN3) were examined by fluorescence immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. Results: The electrical impedance of the cholesteatoma epithelium was significantly lower than that of the post-
auricular skin and external auditory canal skin at both 320 Hz and 30.7 kHz. Immunoreactivity for MARVELD2, CLDN1 and CLDN3 was localised mainly in the granular layer, and to lesser degree, in the horny and spinous layers in both the cholesteatoma tissue and post-auricular skin. Fluorescence intensity was moderate for MARVELD2, weak for CLDN1 and strong for CLDN3. The expressions of MARVELD2, CLDN1 and CLDN3 mRNA were significantly lower in the cholesteatoma tissue than in the post-auricular skin. Conclusions: These results indicate the increased permeability of the cholesteatoma epithelium and suggest that this change is, at least partially, dependent on the decrease in the expressions of the tight junction molecules. This evidence supports the acid lysis hypothesis of bone resorption in cholesteatoma.
Cholesteatoma is an epidermal cyst containing desquamated keratin of the epidermis, or so-called keratin debris. It typically arises from an invagination of the pars flaccida of the tympanic membrane. A deep invagination is referred to as a retraction pocket, and its wall is composed of an inverted keratinising stratified squamous epithelium. As the retraction pocket extends medially into the middle ear cavity, keratin debris accumulates inside the pocket, growing into a cholesteatoma. The lesion then gradually erodes neighbouring bone structures and further expands, leading to a vicious circle of cholesteatoma expansion. Consequently, cholesteatoma in the middle ear cavity causes not only hearing loss and otorrhea, but also vertigo, deafness, facial nerve palsy, sigmoid sinus thrombosis and even intracranial complications such as meningitis and epidural/ subdural/brain abscesses. In contrast, chronic otitis media without cholesteatoma rarely exhibit such destructive
properties. There is no effective medical treatment for middle ear cholesteatoma, and therefore, most patients with middle ear cholesteatoma have no choice but to undergo surgical treatment. Until the middle of the 20th century, the ‘pressure necrosis’ theory had been a mainstream explanation of the bone resorption in cholesteatoma. Since the 1970s, a variety of cytokines, chemical mediators and enzymes have been detected in cholesteatoma tissue, and these factors have been reported as possible causative factors of the bone resorption.1–20 However, the etiopathology of the aggressive nature of cholesteatoma remains controversial. We recently showed that the acidic pH of the cholesteatoma tissue may participate in the bone resorption.21 This acid lysis theory involves a mystery: generally, the surface of the epidermis more or less retains an acidic environment.22 The difference in molecular permeability between the cholesteatoma epithelium and normal skin may provide a clue to understanding the mechanism of bone resorption in cholesteatoma. In this study, we investigated the electrical impedance of and the expressions of tight junction molecules in the cholesteatoma epithelium.
Correspondence: H. Suzuki, MD, PhD, Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Tel.: +81-93-691-7448; Fax: +81-93-601-7554; e-mail: [email protected] a The first and second authors contributed equally to this work.
106
© 2014 John Wiley & Sons Ltd Clinical Otolaryngology 40, 106–114
Permeability of cholesteatoma epithelium
Materials and methods Patients and sample collection
Study subjects were patients with primary acquired middle ear cholesteatoma and those with non-cholesteatomatous chronic otitis media who underwent tympanomastoidectomy in our university hospital. Ethical considerations
Informed consent was obtained from all patients, and the study was approved by the institutional review board of the University of Occupational and Environmental Health. Measurement of in vivo electrical impedance of the cholesteatoma epithelium
The electrical impedance of the cholesteatoma epithelium was measured during tympanomastoidectomy using a Tissue Conductance Meter AS-TC100 (ASAHI BIOMED, Yokohama, Japan). Reference electrodes were placed on the flexor side of the bilateral forearms. After accumulated keratin debris was gently removed from the inside of the cholesteatoma sac, the tip of a stick-shaped electrode was touched for 2–3 s onto the luminal surface of the cholesteatoma epithelium. Alternating currents of 320 Hz and 30.7 kHz were then loaded by turns at a constant voltage of 12.5 mV, and the electrical impedance was measured. The measurement was also made in the same way for postauricular and external auditory canal skins before local injection/incision and for the antral mucosa of patients with non-cholesteatomatous chronic otitis media, which served as a control. The electrical impedance was expressed as an absolute value. Fluorescence immunohistochemistry
Cholesteatoma tissue and normal post-auricular skin were collected from patients with primary acquired middle ear cholesteatoma and 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 20% sucrose in 0.1 M phosphate-buffered saline at pH 7.4 and incubated at 4°C for two nights with 3–4 changes of the solution. The samples were then embedded while frozen in the Tissue-Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan) and stored at 80°C before sectioning. Seven-lm-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 © 2014 John Wiley & Sons Ltd Clinical Otolaryngology 40, 106–114
107
phosphate-buffered saline with 0.3% Triton X-100 for 20 min and treated with 1.5% normal goat serum in phosphate-buffered saline with 0.3% Triton X-100 for 1 h. They were then incubated with rabbit anti-human tricellulin antibody (Novus Biologicals, Littleton, CO, USA), mouse anti-human claudin-1 antibody (Invitrogen; Molecular Probes, Eugene, OR, USA) or mouse anti-human claudin-3 antibody (R&D systems, Minneapolis, MN, USA) at 4°C overnight. The primary antibodies were used at a dilution of 1 : 200 [for tricellulin (MARVELD2)], 1 : 50 [for claudin-1 (CLDN1)] or 1 : 100 [for claudin-3 (CLDN3)] in phosphate-buffered saline with 0.3% Triton X-100 containing 0.5% bovine serum albumin. As a negative control, primary antibodies were omitted from the process. After a brief rinse with phosphate-buffered saline with 0.3% Triton X-100, the sections were reacted at room temperature for 2 h with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen; Molecular Probes) for tricellulin or Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen; Molecular Probes) for claudin-1 and claudin3, diluted 1 : 1000 in phosphate-buffered saline with 0.3% Triton X-100 containing 0.5% bovine serum albumin. The sections were cover-slipped with Prolong Gold antifade reagent containing 40 , 6-diamidino-2-phenylindole dihydrochloride (Invitrogen, Molecular Probes) and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The light source was an HBO 103 W/2 mercury vapour lamp. The light was passed through a 475- to 495nm bandpass filter for the excitation of Alexa Fluor 488 or through a 340- to 380-nm bandpass filter for 40 , 6-diamidino-2-phenylindole dihydrochloride. The emitted fluorescence was allowed to pass through a 515- to 565nm bandpass filter for Alexa Fluor 488 or through a 435to 485-nm bandpass filter for 40 , 6-diamidino-2-phenylindole dihydrochloride. Images were captured using a Carl Zeiss AxioCam digital camera attached to the microscope. Preparation of total RNA
For quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cholesteatoma tissue and normal post-auricular skin were minced by surgical scissors, soaked in 1 mL TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and sonicated by an ultrasonic homogeniser (Taitec, Saitama, Japan). Two hundred microlitres chloroform was added, and after thorough shaking, the mixture was centrifuged at 21 880 g for 15 min at 4°C. The aqueous layer was transferred to another tube, and total RNA was extracted by the acid guanidiniumthiocyanate–phenol–chloroform method and cleaned up with the BioRobot EZ1 system
108 H. Koizumi et al.
qRT-PCR
The total RNA was reverse-transcribed to cDNA with a High-Capacity RNA-to-cDNA Kit (Applied Biosystems Inc., Foster City, CA, USA), which uses random primers. The qRT-PCR analysis was performed with an Applied Biosystems StepOnePlus real-time polymerase chain reaction system using TaqMan Fast Universal polymerase chain reaction Master Mix (Applied Biosystems) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene according to the manufacturer’s specifications. The TaqMan Gene Expression Assays for MARVELD2 (assay identification number: Hs00930631_m1), CLDN1 (assay identification number: Hs00221623_m1), CLDN3 (assay identification number: Hs00265816_s1) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) (assay identification number: Hs99999905_m1) were purchased from Applied Biosystems Inc. Ten ng/lL of cDNA was mixed with TaqMan Universal polymerase chain reaction Master Mix with AmpErase (uracil N-glycosylase) and the primer/probe set of the TaqMan Gene Expression Assays, and the mixture was subjected to polymerase chain reaction amplification with real-time detection. The thermal cycler conditions were as follows: holding at 95°C for 2 min, followed by two-step polymerase chain reaction of 40 cycles of 95°C for 1 s followed by 60°C for 20 s. Each sample was assayed in duplicate. The measured threshold cycle (CT) was normalised by subtracting the CT for glyceraldehyde-3-phosphate dehydrogenase of each sample from that for MARVELD2, CLDN1 and CLDN3. From the obtained DCT, the ratios of MARVELD2, CLDN1 and CLDN3mRNA to glyceraldehyde-3-phosphate dehydrogenase mRNA were calculated by the following formula:
Results In vivo electrical impedance of the cholesteatoma epithelium
The electrical impedances of the post-auricular skin, external auditory canal skin, cholesteatoma epithelium and antral mucosa at 320 Hz were 1440.7 311.3 kΩ, 123.2 31.6 kΩ, 40.4 12.6 kΩ and 65.1 27.4 kΩ, respectively. The impedance of the cholesteatoma epithelium was significantly lower than those of the post-auricular skin and external auditory canal skin (both P < 0.0001; Fig. 1). The electrical impedances of the post-auricular skin, external auditory canal skin, cholesteatoma epithelium and antral mucosa at 30.7 kHz were 733.2 138.5 kΩ, 241.9 26.9 kΩ, 18.4 3.5 kΩ and 218.4 98.2 kΩ, respectively. The impedance of the cholesteatoma epithelium was significantly lower than those of the other three tissues (all P < 0.0001; Fig. 2). Expressions of tight junction proteins in the cholesteatoma epithelium
Figures 3–5 show photomicrographs of fluorescence immunohistochemical staining for MARVELD2, CLDN1 and CLDN3 of the cholesteatoma tissue and post-auricular skin. In both tissues, immunoreactivity for the three tight junction proteins was localised mainly in the granular layer, and to a less degree, in the horny and spinous layers. Fluorescence intensity was moderate for MARVELD2, weak for CLDN1 and strong for CLDN3. P < 0.0001
2000 1800 1600
Impedance (kΩ)
(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 to that at 280 nm (an A260/A280 ratio in the 1.9–2.1 range was considered acceptable). The RNA concentration was determined from A260.
1400 1200 1000 800 P < 0.0001
600
P = 0.7728
400 200
.
Target mRNA=GAPDH mRNA ratio ¼ 2DCT
Statistics
The data are expressed as means SEM. The statistical significance of differences was analysed using the Mann–Whitney U-test. P-values
Increased permeability of the epithelium of middle ear cholesteatoma Koizumi, H.,*a Suzuki, H.,*a Ohbuchi, T.,* Kitamura, T.,* Hashida, K.* & Nakamura, M.† *Department of Otorhinolaryngology-Head and Neck Surgery, †Department of Dermatology, School of Medicine, University of Occupational and Environmental Health, Fukuoka, Japan Accepted for publication 5 October 2014 Clin. Otolaryngol. 2015, 40, 106–114
Objective: We investigated the electrical impedance of and the expressions of tight junction molecules in the cholesteatoma epithelium to provide supporting evidence for the acid lysis theory of bone resorption in middle ear cholesteatoma. Methods: Study subjects were patients with primary acquired middle ear cholesteatoma and those with noncholesteatomatous chronic otitis media who underwent tympanomastoidectomy. The electrical impedance of the cholesteatoma epithelium was measured during tympanomastoidectomy by loading alternating currents of 320 Hz and 30.7 kHz. The expressions of tricellulin (MARVELD2), claudin-1 (CLDN1) and claudin-3 (CLDN3) were examined by fluorescence immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. Results: The electrical impedance of the cholesteatoma epithelium was significantly lower than that of the post-
auricular skin and external auditory canal skin at both 320 Hz and 30.7 kHz. Immunoreactivity for MARVELD2, CLDN1 and CLDN3 was localised mainly in the granular layer, and to lesser degree, in the horny and spinous layers in both the cholesteatoma tissue and post-auricular skin. Fluorescence intensity was moderate for MARVELD2, weak for CLDN1 and strong for CLDN3. The expressions of MARVELD2, CLDN1 and CLDN3 mRNA were significantly lower in the cholesteatoma tissue than in the post-auricular skin. Conclusions: These results indicate the increased permeability of the cholesteatoma epithelium and suggest that this change is, at least partially, dependent on the decrease in the expressions of the tight junction molecules. This evidence supports the acid lysis hypothesis of bone resorption in cholesteatoma.
Cholesteatoma is an epidermal cyst containing desquamated keratin of the epidermis, or so-called keratin debris. It typically arises from an invagination of the pars flaccida of the tympanic membrane. A deep invagination is referred to as a retraction pocket, and its wall is composed of an inverted keratinising stratified squamous epithelium. As the retraction pocket extends medially into the middle ear cavity, keratin debris accumulates inside the pocket, growing into a cholesteatoma. The lesion then gradually erodes neighbouring bone structures and further expands, leading to a vicious circle of cholesteatoma expansion. Consequently, cholesteatoma in the middle ear cavity causes not only hearing loss and otorrhea, but also vertigo, deafness, facial nerve palsy, sigmoid sinus thrombosis and even intracranial complications such as meningitis and epidural/ subdural/brain abscesses. In contrast, chronic otitis media without cholesteatoma rarely exhibit such destructive
properties. There is no effective medical treatment for middle ear cholesteatoma, and therefore, most patients with middle ear cholesteatoma have no choice but to undergo surgical treatment. Until the middle of the 20th century, the ‘pressure necrosis’ theory had been a mainstream explanation of the bone resorption in cholesteatoma. Since the 1970s, a variety of cytokines, chemical mediators and enzymes have been detected in cholesteatoma tissue, and these factors have been reported as possible causative factors of the bone resorption.1–20 However, the etiopathology of the aggressive nature of cholesteatoma remains controversial. We recently showed that the acidic pH of the cholesteatoma tissue may participate in the bone resorption.21 This acid lysis theory involves a mystery: generally, the surface of the epidermis more or less retains an acidic environment.22 The difference in molecular permeability between the cholesteatoma epithelium and normal skin may provide a clue to understanding the mechanism of bone resorption in cholesteatoma. In this study, we investigated the electrical impedance of and the expressions of tight junction molecules in the cholesteatoma epithelium.
Correspondence: H. Suzuki, MD, PhD, Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Tel.: +81-93-691-7448; Fax: +81-93-601-7554; e-mail: [email protected] a The first and second authors contributed equally to this work.
106
© 2014 John Wiley & Sons Ltd Clinical Otolaryngology 40, 106–114
Permeability of cholesteatoma epithelium
Materials and methods Patients and sample collection
Study subjects were patients with primary acquired middle ear cholesteatoma and those with non-cholesteatomatous chronic otitis media who underwent tympanomastoidectomy in our university hospital. Ethical considerations
Informed consent was obtained from all patients, and the study was approved by the institutional review board of the University of Occupational and Environmental Health. Measurement of in vivo electrical impedance of the cholesteatoma epithelium
The electrical impedance of the cholesteatoma epithelium was measured during tympanomastoidectomy using a Tissue Conductance Meter AS-TC100 (ASAHI BIOMED, Yokohama, Japan). Reference electrodes were placed on the flexor side of the bilateral forearms. After accumulated keratin debris was gently removed from the inside of the cholesteatoma sac, the tip of a stick-shaped electrode was touched for 2–3 s onto the luminal surface of the cholesteatoma epithelium. Alternating currents of 320 Hz and 30.7 kHz were then loaded by turns at a constant voltage of 12.5 mV, and the electrical impedance was measured. The measurement was also made in the same way for postauricular and external auditory canal skins before local injection/incision and for the antral mucosa of patients with non-cholesteatomatous chronic otitis media, which served as a control. The electrical impedance was expressed as an absolute value. Fluorescence immunohistochemistry
Cholesteatoma tissue and normal post-auricular skin were collected from patients with primary acquired middle ear cholesteatoma and 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 20% sucrose in 0.1 M phosphate-buffered saline at pH 7.4 and incubated at 4°C for two nights with 3–4 changes of the solution. The samples were then embedded while frozen in the Tissue-Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan) and stored at 80°C before sectioning. Seven-lm-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 © 2014 John Wiley & Sons Ltd Clinical Otolaryngology 40, 106–114
107
phosphate-buffered saline with 0.3% Triton X-100 for 20 min and treated with 1.5% normal goat serum in phosphate-buffered saline with 0.3% Triton X-100 for 1 h. They were then incubated with rabbit anti-human tricellulin antibody (Novus Biologicals, Littleton, CO, USA), mouse anti-human claudin-1 antibody (Invitrogen; Molecular Probes, Eugene, OR, USA) or mouse anti-human claudin-3 antibody (R&D systems, Minneapolis, MN, USA) at 4°C overnight. The primary antibodies were used at a dilution of 1 : 200 [for tricellulin (MARVELD2)], 1 : 50 [for claudin-1 (CLDN1)] or 1 : 100 [for claudin-3 (CLDN3)] in phosphate-buffered saline with 0.3% Triton X-100 containing 0.5% bovine serum albumin. As a negative control, primary antibodies were omitted from the process. After a brief rinse with phosphate-buffered saline with 0.3% Triton X-100, the sections were reacted at room temperature for 2 h with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen; Molecular Probes) for tricellulin or Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen; Molecular Probes) for claudin-1 and claudin3, diluted 1 : 1000 in phosphate-buffered saline with 0.3% Triton X-100 containing 0.5% bovine serum albumin. The sections were cover-slipped with Prolong Gold antifade reagent containing 40 , 6-diamidino-2-phenylindole dihydrochloride (Invitrogen, Molecular Probes) and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The light source was an HBO 103 W/2 mercury vapour lamp. The light was passed through a 475- to 495nm bandpass filter for the excitation of Alexa Fluor 488 or through a 340- to 380-nm bandpass filter for 40 , 6-diamidino-2-phenylindole dihydrochloride. The emitted fluorescence was allowed to pass through a 515- to 565nm bandpass filter for Alexa Fluor 488 or through a 435to 485-nm bandpass filter for 40 , 6-diamidino-2-phenylindole dihydrochloride. Images were captured using a Carl Zeiss AxioCam digital camera attached to the microscope. Preparation of total RNA
For quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cholesteatoma tissue and normal post-auricular skin were minced by surgical scissors, soaked in 1 mL TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and sonicated by an ultrasonic homogeniser (Taitec, Saitama, Japan). Two hundred microlitres chloroform was added, and after thorough shaking, the mixture was centrifuged at 21 880 g for 15 min at 4°C. The aqueous layer was transferred to another tube, and total RNA was extracted by the acid guanidiniumthiocyanate–phenol–chloroform method and cleaned up with the BioRobot EZ1 system
108 H. Koizumi et al.
qRT-PCR
The total RNA was reverse-transcribed to cDNA with a High-Capacity RNA-to-cDNA Kit (Applied Biosystems Inc., Foster City, CA, USA), which uses random primers. The qRT-PCR analysis was performed with an Applied Biosystems StepOnePlus real-time polymerase chain reaction system using TaqMan Fast Universal polymerase chain reaction Master Mix (Applied Biosystems) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene according to the manufacturer’s specifications. The TaqMan Gene Expression Assays for MARVELD2 (assay identification number: Hs00930631_m1), CLDN1 (assay identification number: Hs00221623_m1), CLDN3 (assay identification number: Hs00265816_s1) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) (assay identification number: Hs99999905_m1) were purchased from Applied Biosystems Inc. Ten ng/lL of cDNA was mixed with TaqMan Universal polymerase chain reaction Master Mix with AmpErase (uracil N-glycosylase) and the primer/probe set of the TaqMan Gene Expression Assays, and the mixture was subjected to polymerase chain reaction amplification with real-time detection. The thermal cycler conditions were as follows: holding at 95°C for 2 min, followed by two-step polymerase chain reaction of 40 cycles of 95°C for 1 s followed by 60°C for 20 s. Each sample was assayed in duplicate. The measured threshold cycle (CT) was normalised by subtracting the CT for glyceraldehyde-3-phosphate dehydrogenase of each sample from that for MARVELD2, CLDN1 and CLDN3. From the obtained DCT, the ratios of MARVELD2, CLDN1 and CLDN3mRNA to glyceraldehyde-3-phosphate dehydrogenase mRNA were calculated by the following formula:
Results In vivo electrical impedance of the cholesteatoma epithelium
The electrical impedances of the post-auricular skin, external auditory canal skin, cholesteatoma epithelium and antral mucosa at 320 Hz were 1440.7 311.3 kΩ, 123.2 31.6 kΩ, 40.4 12.6 kΩ and 65.1 27.4 kΩ, respectively. The impedance of the cholesteatoma epithelium was significantly lower than those of the post-auricular skin and external auditory canal skin (both P < 0.0001; Fig. 1). The electrical impedances of the post-auricular skin, external auditory canal skin, cholesteatoma epithelium and antral mucosa at 30.7 kHz were 733.2 138.5 kΩ, 241.9 26.9 kΩ, 18.4 3.5 kΩ and 218.4 98.2 kΩ, respectively. The impedance of the cholesteatoma epithelium was significantly lower than those of the other three tissues (all P < 0.0001; Fig. 2). Expressions of tight junction proteins in the cholesteatoma epithelium
Figures 3–5 show photomicrographs of fluorescence immunohistochemical staining for MARVELD2, CLDN1 and CLDN3 of the cholesteatoma tissue and post-auricular skin. In both tissues, immunoreactivity for the three tight junction proteins was localised mainly in the granular layer, and to a less degree, in the horny and spinous layers. Fluorescence intensity was moderate for MARVELD2, weak for CLDN1 and strong for CLDN3. P < 0.0001
2000 1800 1600
Impedance (kΩ)
(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 to that at 280 nm (an A260/A280 ratio in the 1.9–2.1 range was considered acceptable). The RNA concentration was determined from A260.
1400 1200 1000 800 P < 0.0001
600
P = 0.7728
400 200
.
Target mRNA=GAPDH mRNA ratio ¼ 2DCT
Statistics
The data are expressed as means SEM. The statistical significance of differences was analysed using the Mann–Whitney U-test. P-values
