Mesenchymal Stem Cell Transplantation Accelerates Hearing Recovery through the Repair of Injured Cochlear Fibrocytes
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.060948
ISSN1525-2191
AutoresKazusaku Kamiya, Yoshiaki Fujinami, Noriyuki Hoya, Yasuhide Okamoto, Hiroko Kouike, Rie Komatsuzaki, Ritsuko Kusano, Susumu Nakagawa, Hiroko Satoh, Masato Fujii, Tatsuo Matsunaga,
Tópico(s)Hearing Loss and Rehabilitation
ResumoCochlear fibrocytes play important roles in normal hearing as well as in several types of sensorineural hearing loss attributable to inner ear homeostasis disorders. Recently, we developed a novel rat model of acute sensorineural hearing loss attributable to fibrocyte dysfunction induced by a mitochondrial toxin. In this model, we demonstrate active regeneration of the cochlear fibrocytes after severe focal apoptosis without any changes in the organ of Corti. To rescue the residual hearing loss, we transplanted mesenchymal stem cells into the lateral semicircular canal; a number of these stem cells were then detected in the injured area in the lateral wall. Rats with transplanted mesenchymal stem cells in the lateral wall demonstrated a significantly higher hearing recovery ratio than controls. The mesenchymal stem cells in the lateral wall also showed connexin 26 and connexin 30 immunostaining reminiscent of gap junctions between neighboring cells. These results indicate that reorganization of the cochlear fibrocytes leads to hearing recovery after acute sensorineural hearing loss in this model and suggest that mesenchymal stem cell transplantation into the inner ear may be a promising therapy for patients with sensorineural hearing loss attributable to degeneration of cochlear fibrocytes. Cochlear fibrocytes play important roles in normal hearing as well as in several types of sensorineural hearing loss attributable to inner ear homeostasis disorders. Recently, we developed a novel rat model of acute sensorineural hearing loss attributable to fibrocyte dysfunction induced by a mitochondrial toxin. In this model, we demonstrate active regeneration of the cochlear fibrocytes after severe focal apoptosis without any changes in the organ of Corti. To rescue the residual hearing loss, we transplanted mesenchymal stem cells into the lateral semicircular canal; a number of these stem cells were then detected in the injured area in the lateral wall. Rats with transplanted mesenchymal stem cells in the lateral wall demonstrated a significantly higher hearing recovery ratio than controls. The mesenchymal stem cells in the lateral wall also showed connexin 26 and connexin 30 immunostaining reminiscent of gap junctions between neighboring cells. These results indicate that reorganization of the cochlear fibrocytes leads to hearing recovery after acute sensorineural hearing loss in this model and suggest that mesenchymal stem cell transplantation into the inner ear may be a promising therapy for patients with sensorineural hearing loss attributable to degeneration of cochlear fibrocytes. Mammalian cochlear fibrocytes of the mesenchymal nonsensory regions play important roles in the cochlear physiology of hearing, including the transport of potassium ions to generate an endocochlear potential in the endolymph that is essential for the transduction of sound by hair cells.1Wangemann P K+ cycling and the endocochlear potential.Hear Res. 2002; 165: 1-9Crossref PubMed Scopus (336) Google Scholar, 2Weber PC Cunningham III, CD Schulte BA Potassium recycling pathways in the human cochlea.Laryngoscope. 2001; 111: 1156-1165Crossref PubMed Scopus (67) Google Scholar, 3Delprat B Ruel J Guitton MJ Hamard G Lenoir M Pujol R Puel J-L Brabet P Hamel CP Deafness and cochlear fibrocyte alterations in mice deficient for the inner ear protein otospiralin.Mol Cell Biol. 2005; 25: 847-853Crossref PubMed Scopus (50) Google Scholar It has been postulated that a potassium recycling pathway toward the stria vascularis via fibrocytes in the cochlear lateral wall is critical for proper hearing, although the exact mechanism has not been definitively determined.2Weber PC Cunningham III, CD Schulte BA Potassium recycling pathways in the human cochlea.Laryngoscope. 2001; 111: 1156-1165Crossref PubMed Scopus (67) Google Scholar One candidate model for this ion transport system consists of an extracellular flow of potassium ions through the scala tympani and scala vestibuli and a transcellular flow through the organ of Corti, supporting cells, and cells of the lateral wall.4Kikuchi T Kimura RS Paul DL Adams JC Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis.Anat Embryol (Berl). 1995; 191: 101-118Crossref PubMed Scopus (497) Google Scholar, 5Spicer SS Schulte BA The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency.Hear Res. 1996; 100: 80-100Crossref PubMed Scopus (268) Google Scholar The fibrocytes within the cochlear lateral wall are divided into type I to V based on their structural features, immunostaining patterns, and general location.5Spicer SS Schulte BA The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency.Hear Res. 1996; 100: 80-100Crossref PubMed Scopus (268) Google Scholar Type II, type IV, and type V fibrocytes resorb potassium ions from the surrounding perilymph and from outer sulcus cells via the Na,K- ATPase. The potassium ions are then transported to type I fibrocytes, strial basal cells, and intermediate cells through gap junctions and are secreted into the intrastrial space through potassium channels. The secreted potassium ions are incorporated into marginal cells by the Na,K-ATPase and the Na-K-Cl co-transporter, and are finally secreted into the endolymph through potassium channels. Degeneration and alteration of the cochlear fibrocytes have been reported to cause hearing loss without any other changes in the cochlea in the Pit-Oct-Unc (POU)-domain transcription factor Brain-4 (Brn-4)-deficient mouse6Minowa O Ikeda K Sugitani Y Oshima T Nakai S Katori Y Suzuki M Furukawa M Kawase T Zheng Y Ogura M Asada Y Watanabe K Yamanaka H Gotoh S Nishi-Takeshima M Sugimoto T Kikuchi T Takasaka T Noda T Altered cochlear fibrocytes in a mouse model of DFN3 nonsyndromic deafness.Science. 1999; 285: 1408-1411Crossref PubMed Scopus (184) Google Scholar and the otospiralin-deficient mouse.3Delprat B Ruel J Guitton MJ Hamard G Lenoir M Pujol R Puel J-L Brabet P Hamel CP Deafness and cochlear fibrocyte alterations in mice deficient for the inner ear protein otospiralin.Mol Cell Biol. 2005; 25: 847-853Crossref PubMed Scopus (50) Google ScholarBrn-4 is the gene responsible for human DFN3, an X chromosome-linked nonsyndromic hearing loss. Mice deficient in Brn-4 exhibit reduced endocochlear potential and hearing loss and show severe ultrastructural alterations, including cellular atrophy and a reduction in the number of mitochondria, exclusively in spiral ligament fibrocytes.6Minowa O Ikeda K Sugitani Y Oshima T Nakai S Katori Y Suzuki M Furukawa M Kawase T Zheng Y Ogura M Asada Y Watanabe K Yamanaka H Gotoh S Nishi-Takeshima M Sugimoto T Kikuchi T Takasaka T Noda T Altered cochlear fibrocytes in a mouse model of DFN3 nonsyndromic deafness.Science. 1999; 285: 1408-1411Crossref PubMed Scopus (184) Google Scholar, 7Xia AP Kikuchi T Minowa O Katori Y Oshima T Noda T Ikeda K Late-onset hearing loss in a mouse model of DFN3 non-syndromic deafness: morphologic and immunohistochemical analyses.Hear Res. 2002; 166: 150-158Crossref PubMed Scopus (30) Google Scholar In the otospiralin-deficient mouse, degeneration of type II and IV fibrocytes is the main pathological change, and hair cells and the stria vascularis appear normal.3Delprat B Ruel J Guitton MJ Hamard G Lenoir M Pujol R Puel J-L Brabet P Hamel CP Deafness and cochlear fibrocyte alterations in mice deficient for the inner ear protein otospiralin.Mol Cell Biol. 2005; 25: 847-853Crossref PubMed Scopus (50) Google Scholar Furthermore, in mouse and gerbil models of age-related hearing loss,8Spicer SS Schulte BA Spiral ligament pathology in quiet-aged gerbils.Hear Res. 2002; 172: 172-185Crossref PubMed Scopus (91) Google Scholar, 9Hequembourg S Liberman MC Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice.J Assoc Res Otolaryngol. 2001; 2: 118-129Crossref PubMed Scopus (226) Google Scholar, 10Wu T Marcus DC Age-related changes in cochlear endolymphatic potassium and potential in CD-1 and CBA/CaJ mice.J Assoc Res Otolaryngol. 2003; 4: 353-362Crossref PubMed Scopus (52) Google Scholar degeneration of the cochlear fibrocytes precede the degeneration of other types of cells within the cochlea, with notable pathological changes seen especially in type II, IV, and V fibrocytes. In humans, mutations in the connexin 26 (Cx26) and connexin 30 (Cx30) genes, which encode gap junction proteins and are expressed in cochlear fibrocytes and nonsensory epithelial cells, are well known to be responsible for hereditary sensorineural deafness.11Kelsell DP Dunlop J Stevens HP Lench NJ Liang JN Parry G Mueller RF Leigh IM Connexin 26 mutations in hereditary non-syndromic sensorineural deafness.Nature. 1997; 387: 80-83Crossref PubMed Scopus (1227) Google Scholar, 12del Castillo I Villamar M Moreno-Pelayo MA del Castillo FJ Alvarez A Telleria D Menendez I Moreno F A deletion involving the connexin 30 gene in nonsyndromic hearing impairment.N Engl J Med. 2002; 346: 243-249Crossref PubMed Scopus (478) Google Scholar These instances of deafness related to genetic, structural, and functional alterations in the cochlear fibrocytes highlight the functional importance of these fibrocytes in maintaining normal hearing. Recently, we developed an animal model of acute sensorineural hearing loss attributable to acute cochlear energy failure by administering the mitochondrial toxin 3-nitropropionic acid (3NP) into the rat round window niche.13Hoya N Okamoto Y Kamiya K Fujii M Matsunaga T A novel animal model of acute cochlear mitochondrial dysfunction.Neuroreport. 2004; 15: 1597-1600Crossref PubMed Scopus (48) Google Scholar, 14Okamoto Y Hoya N Kamiya K Fujii M Ogawa K Matsunaga T Permanent threshold shift caused by acute cochlear mitochondrial dysfunction is primarily mediated by degeneration of the lateral wall of the cochlea.Audiol Neurootol. 2005; 10: 220-233Crossref PubMed Scopus (37) Google Scholar 3NP is an irreversible inhibitor of succinate dehydrogenase, a complex II enzyme of the mitochondrial electron transport chain.15Alston TA Mela L Bright HJ 3-Nitropropionate, the toxic substance of Indigofera, is a suicide inactivator of succinate dehydrogenase.Proc Natl Acad Sci USA. 1977; 74: 3767-3771Crossref PubMed Scopus (334) Google Scholar, 16Coles CJ Edmondson DE Singer TP Inactivation of succinate dehydrogenase by 3-nitropropionate.J Biol Chem. 1979; 254: 5161-5167Abstract Full Text PDF PubMed Google Scholar Systemic administration of 3NP has been used to produce selective striatal degeneration in the brain of several mammals.17Brouillet E Hantraye P Ferrante RJ Dolan R Leroy-Willig A Kowall NW Beal MF Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates.Proc Natl Acad Sci USA. 1995; 92: 7105-7109Crossref PubMed Scopus (427) Google Scholar, 18Hamilton BF Gould DH Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic acid: a type of hypoxic (energy deficient) brain damage.Acta Neuropathol (Berl). 1987; 72: 286-297Crossref PubMed Scopus (196) Google Scholar Our model with 3NP administration into the rat cochlea showed acute sensorineural hearing loss and revealed an initial pathological change in the fibrocytes of the lateral wall and spiral limbus without any significant damage to the organ of Corti or spiral ganglion. Furthermore, depending on the dose of 3NP used, these hearing loss model rats exhibited either a permanent threshold shift or a temporary threshold shift. In the present study, we used doses of 3NP that induce temporary threshold shift to explore the mechanism of hearing recovery after injury to the cochlear fibrocytes and examined a novel therapeutic approach to repair the injured area using mesenchymal stem cell (MSC) transplantation. MSCs are multipotent cells that can be isolated from adult bone marrow and can be induced to differentiate into a variety of tissues in vitro and in vivo.19Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R Mosca JD Moorman MA Simonetti DW Craig S Marshak DR Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17938) Google Scholar Human MSCs transplanted into fetal sheep intraperitoneally undergo site-specific differentiation into chondrocytes, adipocytes, myocytes, cardiomyocytes, bone marrow stromal cells, and thymic stroma.20Liechty KW MacKenzie TC Shaaban AF Radu A Moseley AM Deans R Marshak DR Flake AW Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.Nat Med. 2000; 6: 1282-1286Crossref PubMed Scopus (1120) Google Scholar Furthermore, when MSCs were transplanted into postnatal animals, they could engraft and differentiate into several tissue-specific cell types in response to environmental cues provided by different organs.21Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR Reyes M Lenvik T Lund T Blackstad M Du J Aldrich S Lisberg A Low WC Largaespada DA Verfaillie CM Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5161) Google Scholar These transplantability features of MSCs suggested the possibility that they could restore hearing loss in 3NP-treated rats to the normal range. Recently, experimental bone marrow transplantation into irradiated mice suggested that a part of spiral ligament that consists of cochlear fibrocytes was derived from bone marrow cells or hematopoietic stem cells.22Lang H Ebihara Y Schmiedt RA Minamiguchi H Zhou D Smythe N Liu L Ogawa M Schulte BA Contribution of bone marrow hematopoietic stem cells to adult mouse inner ear: mesenchymal cells and fibrocytes.J Comp Neurol. 2006; 496: 187-201Crossref PubMed Scopus (101) Google Scholar This indicates that bone marrow-derived stem cells such as MSCs may have a capacity to repair the injury of cochlear fibrocytes. In this study, we demonstrate that MSC transplantation significantly improves hearing recovery, and present evidence suggesting invasion of transplanted MSCs into the injured region of the cochlear lateral wall and repair of the interrupted gap junction network. Experimental procedures reported in this study were approved by the Institutional Animal Care and Use Committee of the National Tokyo Medical Center. Sprague-Dawley rats (Clea Japan, Tokyo, Japan) weighing between 180 and 210 g (8 to 10 weeks old) were used. Before surgery, the animals were anesthetized with pentobarbital (30 to 40 mg/kg, i.p.; Dainippon Pharmaceutical, Osaka, Japan), and after local administration of 1% lidocaine (AstraZeneca PLC, London, UK), an incision was made posterior to the left pinna near the external meatus. The left otic bulla was opened to approach the round window niche. The distal end of a section of PE 10 tubing (Becton-Dickinson, Franklin Lakes, NJ) was drawn to a fine tip in a flame and gently inserted into the round window niche. 3NP (Sigma, St. Louis, MO) was dissolved in saline at 300 mmol/L and the pH adjusted to 7.4 with NaOH. Saline alone was used as a control. The solution was administered for 2 minutes at a rate of 1.5 μl/minute with a syringe pump. After treatment, a small piece of gelatin was placed on the niche to keep the solution in the niche regardless of head movement, and the wound was closed. The right cochlea was surgically destroyed to avoid cross-hearing during auditory brainstem response (ABR) recording. ABR recording was performed as previously described13Hoya N Okamoto Y Kamiya K Fujii M Matsunaga T A novel animal model of acute cochlear mitochondrial dysfunction.Neuroreport. 2004; 15: 1597-1600Crossref PubMed Scopus (48) Google Scholar before surgery and at 2 hours and 1, 2, 3, 7, 14, 21, 28, 35, and 42 days after surgery (or until 14 days in the MSC transplantation experiment). Six to 12 rats in each group were used for the recordings. ABR was recorded using Scope waveform storing and stimulus control software and the PowerLab data acquisition and analysis system (PowerLab2/20; AD Instruments, Castle Hill, Australia). Electroencephalogram recording was performed using a digital Bioamp extracellular amplifier system (BAL-1; Tucker-Davis Technologies, Alachua, FL). Sound stimuli were produced by a coupler type speaker (ES1spc; Bio Research Center, Nagoya, Japan) inserted into the ear canal. Pure tone bursts of 8, 20, and 40 kHz (0.2-ms rise/fall time and 1-ms flat segment) were generated, and the amplitude was specified by a real-time processor and programmable attenuator (RP2.1 and PA5; Tucker-Davis Technologies). Sound level calibration and frequency confirmation were performed using a 1/4 inch free-field mic (7016; ACO Pacific, Belmont, CA), microphone amp (MA3; Tucker-Davis Technologies), a digital oscilloscope (DS-8822P; Iwatsu Electronic, Tokyo, Japan), and a sound level meter (NL32; Rion, Tokyo, Japan). The maximum output level was 87, 86, and 96 dB at 8, 20, and 40 kHz, respectively. For recording, the animals were anesthetized with pentobarbital before stainless steel needle electrodes were placed ventrolateral to the ears. Waveforms of 512 stimuli at a frequency of 9 Hz were averaged, and the visual detection threshold was determined by increasing or decreasing the sound pressure level in 5-dB steps. The effects of 3NP and/or MSC transplantation on the ABR threshold and recovery ratio of ABR threshold (peak threshold − threshold at 14 days or 42 days/peak threshold × 100) were statistically analyzed at each frequency using an unpaired Student's t-test. The significance level for all statistical procedures was set at P < 0.05. To detect cell proliferation in the rat inner ear, BrdU (Sigma) was injected (30 mg/kg i.p. per single injection) as previously described.23Kamiya K Takahashi K Kitamura K Momoi T Yoshikawa Y Mitosis and apoptosis in postnatal auditory system of the C3H/He strain.Brain Res. 2001; 901: 296-302Crossref PubMed Scopus (51) Google Scholar Injections were started just after 3NP administration and continued every 12 hours for 3 or 6 days. We previously established bone marrow MSCs and demonstrate their potential to differentiate into several cell types.24Satoh H Kishi K Tanaka T Kubota Y Nakajima T Akasaka Y Ishii T Transplanted mesenchymal stem cells are effective for skin regeneration in acute cutaneous wounds.Cell Transplant. 2004; 13: 405-412Crossref PubMed Scopus (90) Google Scholar The cells were prepared from 6- to 8-week-old male F344 rats (Clea) as described. In brief, surgical treatment was performed after intraperitoneal injection of pentobarbital (30 to 40 mg/kg, i.p.). After surgery, the rats were sacrificed by ether inhalation followed by dislocation of the neck. Rat femurs and tibiae were collected and the long bones meticulously dissected to remove all adherent soft tissue. Both ends of the bones were cut away from the diaphyses with bone scissors. The bone marrow plugs were hydrostatically expelled from the bones by inserting 18-gauge needles fastened to 10-ml syringes filled with complete medium [Dulbecco's modified Eagle's medium (Sigma), 10% fetal bovine serum (Sigma), and 100 U/ml penicillin-streptomycin (Sigma)] into the distal ends of the femora and the proximal ends of the tibiae. Cells were plated on plastic culture dishes. The nonadherent cell population was removed after 24 hours, and the adherent layer was washed once with fresh media. The cells were then continuously cultured for 1 to 4 weeks in complete medium. Medium was completely replaced every 3 days. When the cells were nearly confluent, the adherent cells were released from the dishes with 0.25% trypsin-ethylenediaminetetraacetic acid (Sigma), split 1:3, and seeded onto fresh plates. Cells from passages 10 to 15 were stored with Cell Banker reagent (Juji Field, Tokyo, Japan) in liquid nitrogen. The frozen cell suspensions were thawed at 1 week before the transplantation and cultured in complete medium at 37°C in a humidified atmosphere of 5% CO2. The potential of these cells as MSCs were previously demonstrated as described.24Satoh H Kishi K Tanaka T Kubota Y Nakajima T Akasaka Y Ishii T Transplanted mesenchymal stem cells are effective for skin regeneration in acute cutaneous wounds.Cell Transplant. 2004; 13: 405-412Crossref PubMed Scopus (90) Google Scholar The surface marker expression of these cells was analyzed by flow cytometry (Epics Altra with HyPerSort cell sorting system; Beckman Coulter, Fullerton, CA). At ∼80 to 90% confluence, MSCs were dissociated by treatment with 1× Accutase (Chemicon International, Temecula, CA) for 15 minutes at 37°C followed by phosphate-buffered saline (PBS) washout, centrifugation at 1200 rpm for 10 minutes, and resuspension in Hanks' balanced salt solution (HBSS)+ medium [HBSS− medium (Invitrogen Japan, Tokyo, Japan) with 2% fetal bovine serum and 10 mmol/L 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (Invitrogen Japan)]. MSCs were incubated with antibodies against CD45, CD31, CD29, CD44H, CD54, CD73, and CD90 (BD PharMingen, San Diego, CA) for 30 minutes on ice and spun down. At the end of the staining, MSCs were resuspended in ice-cold HBSS+ medium containing 2 μg/ml propidium iodide for discrimination of dead cells. To detect the MSCs after injection, cultured MSCs were incubated with 5 μmol/L BrdU for 2 days before transplantation as previously described.25Kicic A Shen WY Wilson AS Constable IJ Robertson T Rakoczy PE Differentiation of marrow stromal cells into photoreceptors in the rat eye.J Neurosci. 2003; 23: 7742-7749PubMed Google Scholar Before transplantation, cultured MSCs were released from the dishes with 0.25% trypsin-ethylenediaminetetraacetic acid and washed by centrifugation with Dulbecco's phosphate-buffered saline (D-PBS; Invitrogen Japan) and resuspended to prepare MSC suspension (1 × 105 cells in 20 μl of D-PBS) for the following transplantation. Three days after 3NP administration, the rats were anesthetized with pentobarbital (30 to 40 mg/kg, i.p.) and by local administration of 1% lidocaine. Incisions were made as described for 3NP administration, the surfaces of the posterior and lateral semicircular canals were exposed, and a small hole was made in each canal. A small tube (Eicom, Kyoto, Japan) was inserted into the lateral semicircular canal toward the ampulla. Through this tube, the perilymph was perfused with an MSC suspension (1 × 105 cells in 20 μl of D-PBS) for 10 minutes at a rate of 2 μl/minute using a syringe pump with drainage from the hole made on the posterior semicircular canal. The tube was then removed, the holes on the semicircular canals were sealed with a muscle and fibrin adhesive (Beriplast P Combi-set; CSL Behring, King of Prussia, PA), and the wound on the neck was closed. An equal volume of vehicle (D-PBS) was also injected into the semicircular canal of 3NP-treated rats as control. The rats were sacrificed at 3 days (three rats for 3NP and three rats for saline control) and 42 days (five rats for 3NP and three rats for saline control) after 3NP treatment and 11 days after MSC transplantation (12 rats for 3NP with MSC transplantation, seven rats for MSC transplantation only, and five rats for 3NP followed by vehicle injection). They were deeply anesthetized with pentobarbital and transcardially perfused with 0.01 mol/L phosphate buffer, pH 7.4, containing 8.6% sucrose followed by a fixative consisting of freshly depolymerized 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). After decapitation, the left temporal bones were removed and immediately placed in the same fixative. Small openings were made at the round window, oval window, and the apex of the cochlea. After overnight immersion in fixative, the temporal bones were decalcified by immersion in 5% sucrose, 5% ethylenediaminetetraacetic acid, pH 7.4, with stirring at 4°C for 14 days. The specimens were dehydrated through graded concentrations of alcohol, embedded in paraffin blocks, and sectioned into 5-μm-thick slices. The sections were stained with hematoxylin and eosin (H&E) as generally described, by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and by immunohistochemistry for BrdU, Cx30, or Cx26 as described below. TUNEL assays were performed using an ApopTag Fluorescein Direct in situ apoptosis detection kit (Chemicon International) according to the manufacturer's instructions. In brief, specimens were digested with 20 μg/ml proteinase K in 0.01 mol/L PBS, pH 7.4, for 5 minutes, incubated with TdT and fluorescein-labeled nucleotide in a humid atmosphere at 37°C for 1 hour, and then incubated with 2 μmol/L TOPRO-3 iodide (Molecular Probes, Eugene, OR) for 5 minutes. The specimens were viewed with a confocal laser microscope (LSM510; Carl Zeiss, Esslingen, Germany; or Radiance 2100; Bio-Rad, Hercules, CA), and each image was analyzed and saved by ZeissLSM image browser (Carl Zeiss). Negative controls included proteinase K digestion but did not include TdT so that nonspecific incorporation of nucleotide, or nonspecific binding of enzyme-conjugate, could be assessed. Distilled water was substituted for TdT enzyme reagent in negative controls. After pretreatment with 2 mol/L HCl at 37°C for 30 minutes, incubation with 20 μg/ml proteinase K in PBS for 5 minutes, and incubation with blocking solution (1.5% normal goat serum in PBS) for 30 minutes at room temperature, tissue sections were incubated with anti-BrdU antibody (DAKO, Glostrup, Denmark) diluted 1:100 in PBS for 30 minutes, then with biotin-conjugated anti-mouse IgG (Vector, Burlingame, CA) diluted 1:200 in PBS for 30 minutes, followed by horseradish peroxidase (HRP)-conjugated streptavidin-biotin complex (streptABComplex-HRP, Vectastain Elite ABC kit standard; Vector) for 1 hour at room temperature. Sections were stained in DAB-H2O2 (Vector) for 3 minutes and hematoxylin for 1 minute and then rinsed and covered with a coverslip. For BrdU and TUNEL double staining, Alexa568-conjugated anti-mouse IgG (1:600; Molecular Probes) was used as a secondary antibody in the BrdU staining after the TUNEL procedure. For double-staining of BrdU with Cx30 or Cx26, rabbit anti-Cx26 (1:300; Zymed Laboratories, South San Francisco, CA) or rabbit anti-Cx30 (1:400; Zymed Laboratories) antibody and anti-BrdU antibody were used as a primary antibody cocktail, and Alexa488-conjugated anti-rabbit IgG (1:400; Molecular Probes) with Alexa568-conjugated anti-mouse IgG were used as a secondary antibody cocktail. For nuclear staining, TOPRO-3 iodide (2 μmol/L; Molecular Probes), 4,6-diamidino-2-phenylindole (1 μg/ml; Dojindo Laboratories, Kumamoto, Japan) or propidium iodide (1 μg/ml; Molecular Probes) was used. Negative controls were performed without primary antibodies to assess nonspecific binding of the secondary antibody or of the streptABComplex-HRP. Inner ear sections that were not injected with BrdU were also used as negative controls. Background autofluorescence was not observed in the cochlear sections with the tissue preparation methods used in the present study. We monitored ABR thresholds in 3NP-treated rats at 8, 20, and 40 kHz for 42 days after 3NP administration (Figure 1, A–C) to examine the potential for hearing recovery. At all frequencies, the ABR thresholds peaked 1 day after 3NP administration and then gradually recovered. At 8 kHz (n = 7), the threshold reached within a normal threshold level (11 dB) 42 days after 3NP administration. However, the ABR threshold at 40 kHz showed only a mild recovery after 14 days. The hearing recovery ratio, which is described in Materials and Methods, was calculated for each tested frequency (Figure 1D). At 14 days, the recovery ratios were 73.4 ± 5.5% for 8 kHz (n = 11), 57.0 ± 11.2% for 20 kHz (n = 11), and 37.5 ± 7.7% for 40 kHz (n = 12). At 42 days, they were 97.2 ± 9.4% for 8 kHz (n = 7), 67.0 ± 16.4% for 20 kHz (n = 7), and 32.3 ± 7.7% for 40 kHz (n = 7). Throughout the recovery time course, the recovery ratios at the lower frequencies always tended to be higher than those at the highest frequency. At 42 days, the recovery ratio for 8 kHz was significantly higher than that for 40 kHz (P = 0.005). Between 14 and 42 days after 3NP administration, the hearing level for 40 kHz did not show significant recovery, but the recovery ratios for 8 and 20 kHz showed 24 and 10% increases, respectively. To analyze the pathological changes associated with the acute hearing loss observed in the 3NP-treated rats, we performed H&E staining and TUNEL reaction to detect apoptosis. No histological changes were observed in the organ of Corti and spiral ganglion of rats with 3NP administration as shown in Figure 2, A and B. However, severe apoptosis, with chromatin condensation and apoptotic bodies, was observed only in the lateral wall and the spiral limbus at 3 days after 3NP administration (Figure 2, C and D). These severe apoptotic regions included more than 30% TUNEL-positive or apoptotic cells and were clearly demarcated as shown in Figure 2, E and F. These areas contain cochlear fibrocytes that participate in the potassium recycling route within the cochlea. The typical distribution pattern of TUNEL-positive cells after 3NP treatment is shown in Figure 2E, but a few rats with more severe hearing impairment (with ∼55 dB elevation of the ABR threshold) demonstrated more prominent histological changes, with focal cell loss in the center surrounded by TUNEL-positive cells (Figure 2, G and H). On light microscopic observation of H&E-stained sections, histological changes suggesting inflammation were not evident in the lateral wall and spiral limbus. As for cochlear turns, lateral wall in basal turn had more severe damage than the middle turn as shown in Figure 4, G, I, and J, and the ap
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