Atoh1 is a transcription factor that regulates neural development in multiple tissues and is conserved among species. Prior mouse models of Atoh1, though effective and important in the evolution of our understanding of the gene, have been limited by perinatal lethality. Here we describe a novel point mutation of Atoh1 (designated Atoh1trhl) underlying a phenotype of trembling gait and hearing loss. Histology revealed inner ear hair cell loss and cerebellar atrophy. Auditory Brainstem Response (ABR) and Distortion Product Otoacoustic Emission (DPOAE) showed functional abnormalities in the ear. Normal lifespan and fecundity of Atoh1trhlmice provide a complementary model to facilitate elucidation of ATOH1 function in hearing,central nervous system and cancer biology.
Although CLIC5 is a member of the chloride intracellular channel protein family, its association with actin-based cytoskeletal structures suggests that it may play an important role in their assembly or maintenance. Mice homozygous for a new spontaneous recessive mutation of the Clic5 gene, named jitterbug ( jbg ), exhibit impaired hearing and vestibular dysfunction. The jbg mutation is a 97 bp intragenic deletion that causes skipping of exon 5, which creates a translational frame shift and premature stop codon. Western blot and immunohistochemistry results confirmed the predicted absence of CLIC5 protein in tissues of jbg/jbg mutant mice. Histological analysis of mutant inner ears revealed dysmorphic stereocilia and progressive hair cell degeneration. In wild-type mice, CLIC5-specific immunofluorescence was detected in stereocilia of both cochlear and vestibular hair cells and also along the apical surface of Kolliker’s organ during cochlear development. Refined immunolocalization in rat and chicken vestibular hair cells showed that CLIC5 is limited to the basal region of the hair bundle, similar to the known location of radixin. Radixin immunostaining appeared reduced in hair bundles of jbg mutant mice. By mass spectrometry and immunoblotting, CLIC5 was shown to be expressed at high levels in stereocilia of the chicken utricle, in an approximate 1:1 molar ratio with radixin. These results suggest that CLIC5 associates with radixin in hair cell stereocilia and may help form or stabilize connections between the plasma membrane and the filamentous actin core.
Hypoxia is involved in tumor biological processes and disease progression. Ferroptosis, as a newly discovered programmed cell death process, is closely related to breast cancer (BC) occurrence and development. However, reliable prognostic signatures based on a combination of hypoxia and ferroptosis in BC have not been developed.We set The Cancer Genome Atlas (TCGA) breast cancer cohort as training set and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) BC cohort as the validation set. Least Absolute Shrinkage and Selection Operator (LASSO) and COX regression approaches were used to construct ferroptosis-related genes (FRGs) and hypoxia-related genes (HRGs) prognostic signature (HFRS). The CIBERSORT algorithm and ESTIMATE score were used to explore the relationship between HFRS and tumor immune microenvironment. Immunohistochemical staining was used to detect protein expression in tissue samples. A nomogram was developed to advance the clinical application of HFRS signature.Ten ferroptosis-related genes and hypoxia-related genes were screened to construct the HFRS prognostic signature in TCGA BC cohort, and the predictive capacity was verified in METABRIC BC cohort. BC patients with high-HFRS had shorter survival time, higher tumor stage, and a higher rate of positive lymph node. Moreover, high HFRS was associated with high hypoxia, ferroptosis, and immunosuppression status. A nomogram that was constructed with age, stage, and HFRS signature showed a strong prognostic capability to predict overall survival (OS) for BC patients.We developed a novel prognostic model with hypoxia and ferroptosis-related genes to predict OS, and characterize the immune microenvironment of BC patients, which might provide new cures for clinical decision-making and individual treatment of BC patients.
Genetic predisposition is recognized as an important pathogenetic factor in otitis media (OM) and associated diseases. Mutant Lmna mice heterozygous for the disheveled hair and ears allele (LmnaDhe/+) exhibit early-onset, profound hearing deficits and other pathological features mimicking human laminopathy associated with the LMNA mutation. We assessed the effects of the LmnaDhe/+ mutation on development of OM and pathological abnormalities characteristic of laminopathy. Malformation and abnormal positioning of the eustachian tube, accompanied by OM, were observed in all of the LmnaDhe/+ mice (100% penetrance) as early as postnatal day P12. Scanning electronic microscopy revealed ultrastructural damage to the cilia in middle ears that exhibited OM. Hearing assessment revealed significant hearing loss, paralleling that in human OM. Expression of NF-κB, TNF-α, and TGF-β, which correlated with inflammation and/or bony development, was up-regulated in the ears or in the peritoneal macrophages of LmnaDhe/+ mice. Rugous, disintegrative, and enlarged nuclear morphology of peritoneal macrophages and hyperphosphatemia were found in LmnaDhe/+ mutant mice. Taken together, these features resemble the pathology of human laminopathies, possibly revealing some profound pathology, beyond OM, associated with the mutation. The LmnaDhe/+ mutant mouse provides a novel model of human OM and laminopathy. Genetic predisposition is recognized as an important pathogenetic factor in otitis media (OM) and associated diseases. Mutant Lmna mice heterozygous for the disheveled hair and ears allele (LmnaDhe/+) exhibit early-onset, profound hearing deficits and other pathological features mimicking human laminopathy associated with the LMNA mutation. We assessed the effects of the LmnaDhe/+ mutation on development of OM and pathological abnormalities characteristic of laminopathy. Malformation and abnormal positioning of the eustachian tube, accompanied by OM, were observed in all of the LmnaDhe/+ mice (100% penetrance) as early as postnatal day P12. Scanning electronic microscopy revealed ultrastructural damage to the cilia in middle ears that exhibited OM. Hearing assessment revealed significant hearing loss, paralleling that in human OM. Expression of NF-κB, TNF-α, and TGF-β, which correlated with inflammation and/or bony development, was up-regulated in the ears or in the peritoneal macrophages of LmnaDhe/+ mice. Rugous, disintegrative, and enlarged nuclear morphology of peritoneal macrophages and hyperphosphatemia were found in LmnaDhe/+ mutant mice. Taken together, these features resemble the pathology of human laminopathies, possibly revealing some profound pathology, beyond OM, associated with the mutation. The LmnaDhe/+ mutant mouse provides a novel model of human OM and laminopathy. Otitis media (OM) is a pervasive disease that involves a potential burden of hearing loss and one that in most countries leads to excessive antibiotic consumption, as well as severe complications.1Vergison A. Dagan R. Arguedas A. Bonhoeffer J. Cohen R. Dhooge I. Hoberman A. Liese J. Marchisio P. Palmu A.A. Ray G.T. Sanders E.A. Simões E.A. Uhari M. van Eldere J. Pelton S.I. Otitis media and its consequences: beyond the earache.Lancet Infect Dis. 2010; 10: 195-203Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar An important susceptibility factor for OM is eustachian tube dysfunction, which can arise from developmental defects that occur as a consequence of gene mutation. The eustachian tube and middle ear cavity differentiate from the tubotympanic recess, which develops from the endodermal lining of the first pharyngeal pouch.2Park K. Ueno K. Lim D.J. Developmental anatomy of the eustachian tube and middle ear in mice.Am J Otolaryngol. 1992; 13: 93-100Abstract Full Text PDF PubMed Scopus (19) Google Scholar The tympanic cavity is linked to the nasopharynx by the eustachian tube. A cartilaginous structure, the eustachian tube extends its bony ostium from the middle ear cavity to vent the middle ear and to clear and protect the middle ear from nasopharyngeal secretions. Malformation or malposition of the eustachian tube can give rise to OM, as reported in gene-mutation mouse models.3Noben-Trauth K. Latoche J.R. Ectopic mineralization in the middle ear and chronic otitis media with effusion caused by RPL38 deficiency in the Tail-short (Ts) mouse.J Biol Chem. 2011; 286: 3079-3093Crossref PubMed Scopus (21) Google Scholar, 4Depreux F.F. Darrow K. Conner D.A. Eavey R.D. Liberman M.C. Seidman C.E. Seidman J.G. Eya4-deficient mice are a model for heritable otitis media.J Clin Invest. 2008; 118: 651-658PubMed Google Scholar Lamin A/C (LMNA) is a structural protein encoded by the LMNA gene. LMNA is an essential scaffolding component of the nuclear envelope surrounding the cell nucleus. The nuclear envelope regulates movement of molecules into and out of the nucleus. The lamin protein family, of which lamin A/C is a member, plays a role in nuclear stability, chromatin structure, and gene expression. Lamin proteins form the vertebrate nuclear lamina, a two-dimensional matrix near the inner nuclear membrane, and are highly conserved in evolution.5Brown C.A. Lanning R.W. McKinney K.Q. Salvino A.R. Cherniske E. Crowe C.A. Darras B.T. Gominak S. Greenberg C.R. Grosmann C. Heydemann P. Mendell J.R. Pober B.R. Sasaki T. Shapiro F. Simpson D.A. Suchowersky O. Spence J.E. Novel and recurrent mutations in lamin A/C in patients with Emery-Dreifuss muscular dystrophy.Am J Med Genet. 2001; 102: 359-367Crossref PubMed Scopus (103) Google Scholar Mutations in LMNA cause a group of human disorders often collectively called laminopathies, which includes Hutchinson-Gilford progeria syndrome (HGPS). In a recent study, patients with HGPS were noted to have stiff auricular cartilage, small or absent lobules, and hypoplasia of the lateral soft-tissue portion of the external ear canal, leading to a shortened canal. Patients typically exhibit low-frequency conductive hearing loss.6Guardiani E. Zalewski C. Brewer C. Merideth M. Introne W. Smith A.C. Gordon L. Gahl W. Kim H.J. Otologic and audiologic manifestations of Hutchinson-Gilford progeria syndrome.Laryngoscope. 2011; 121: 2250-2255Crossref PubMed Scopus (10) Google Scholar The LmnaDhe mutation, first described by Odgren et al,7Odgren P.R. Pratt C.H. Mackay C.A. Mason-Savas A. Curtain M. Shopland L. Ichicki T. Sundberg J.P. Donahue L.R. Disheveled hair and ear (Dhe), a spontaneous mouse Lmna mutation modeling human laminopathies.PLoS One. 2010; 5: e9959Crossref PubMed Scopus (23) Google Scholar is named for the external phenotypes of sparse coat and small ears [ie, disheveled hair and ear (Dhe)]. This spontaneous, semidominant point mutation in the Lmna gene encodes an amino acid substitution, L52R, in the coiled-coil rod domain of lamin A and C proteins. The mutation is also associated with epidermal dysplasia and craniofacial defects in both heterozygotes and homozygotes; however, the homozygotes rarely survive past P10.8Pratt C.H. Curtain M. Donahue L.R. Shopland L.S. Mitotic defects lead to pervasive aneuploidy and accompany loss of RB1 activity in mouse LmnaDhe dermal fibroblasts.PLoS One. 2011; 6: e18065Crossref PubMed Scopus (11) Google Scholar In the present study, we found that the LmnaDhe/+ mutant mice exhibited early-onset and profound hearing defects. In characterizing the mechanisms for deafness, we further investigated the auditory system and observed OM accompanied by significant developmental defects in the eustachian tube and its adjacent basicranial structure. All of the heterozygous mutant mice developed OM, along with malformation of the eustachian tube, as early as 12 days after birth. We suggest that the LmnaDhe/+ mice provide a model for human OM associated with craniofacial defects, as well as eustachian tube malformation, and that the LmnaDhe mutation accounts for the middle ear maturation defect. Considering these data together with our assessment of serum calcium and phosphorus abnormalities and nuclear defects of peritoneal macrophages, we inferred that LmnaDhe mutation correlates with pathological features of laminopathy. Thus, mice expressing LmnaDhe mutation provide a novel model for investigation of OM and laminopathy. LmnaDhe/+ heterozygous mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were bred at the Wolstein Animal Research Facility at Case Western Reserve University. Because homozygous LmnaDhe/Dhe pups die at approximately P10, the strain was maintained by heterozygous cross-mating. We used 78 heterozygous LmnaDhe/+ mutant mice and 68 wild-type littermate control mice, from 6 days to 8 months of age. All mice younger than 12 days were genotyped by PCR and identified by Sma1 digestion (R0141S #0831104; New England Biolabs, Ipswich, MA), as described previously.7Odgren P.R. Pratt C.H. Mackay C.A. Mason-Savas A. Curtain M. Shopland L. Ichicki T. Sundberg J.P. Donahue L.R. Disheveled hair and ear (Dhe), a spontaneous mouse Lmna mutation modeling human laminopathies.PLoS One. 2010; 5: e9959Crossref PubMed Scopus (23) Google Scholar All animal procedures were reviewed and approved by the Health Sciences Institutional Animal Care and Use Committee of Case Western Reserve University (protocols 2008-0174 and 2008-0156). Wild-type and LmnaDhe/+ mutant mice at 10 weeks of age were dissected immediately after CO2 asphyxiation (n = 6 for each genotype; 3 male and 3 female mice in each group). No fixative solution was applied. Skulls were dissected and photographed under an anatomical microscope (Leica S6D; Leica Microsystems, Wetzlar, Germany). After removal of the lower jaw of the skull and dissection of the soft tissue surrounding the bullae, a short straight section of whisker from each mouse was used as a landmark and the whisker was inserted from the pharyngeal opening to the tympanic opening of the eustachian tube. The intersection angle (IA) between the eustachian tubes was measured using ImageJ software version 1.6.0_33 (32-bit) (NIH, Bethesda, MD). Dimensions of the eustachian tubes were acquired with a hand-held digital caliper (General Tools & Instruments, New York, NY) with 0.01-mm resolution. Tympanometry measurement was performed to test the condition of the middle ear and the mobility of the tympanic membrane and the ossicles using a race car tympanometer (Maico, Berlin, Germany). The instrument was calibrated to atmospheric pressure each day, before the measurement was performed. The physical volumes of the tympanometer (1.5, 0.5, and 0.25 mL) were also calibrated. A computer-aided evoked potential system (Intelligent Hearing Systems, Miami, FL) was used to test the mouse auditory-evoked brainstem response (ABR) thresholds and distortion product otoacoustic emissions (DPOAE), as described previously.9Zheng Q.Y. Johnson K.R. Erway L.C. Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses.Hear Res. 1999; 130: 94-107Crossref PubMed Scopus (679) Google Scholar Broadband-click (8 to 16 kHz) and 8-, 16-, and 32-kHz pure-tone burst stimuli were presented to mice. DPOAE measurement was performed using SmartOAE 4.50 USBez software (Intelligent Hearing Systems), using the Etymotic 10B+ OAE probe (Etymotic Research, Elk Grove Village, IL) fitted with a high frequency transducer (Intelligent Hearing Systems) producing two pure tones, F1 and F2. Two ranges of frequencies were tested: the low frequencies ranging from 4 to 20 kHz with the frequency per octave set at 5.0, and the higher frequencies ranging from 16 to 32 kHz with frequency per octave set at 7.0. Both ranges measured the response signals to L1 and L2 amplitudes set at 65 and 55 dB sound pressure level (SPL), respectively, with an F2/F1 ratio of 1.22. Only the data with A1 and A2 levels with the respective L1 and L2 of ±15 dB SPL were considered robust; any data point falling outside of the criteria was discarded. The signal-to-noise ratios were plotted against F2. The procedures for tympanometry measurement, ABR, and DPOAE were performed in a quiet animal room at normal room temperature with the noise level maintained below 51 dB SPL. Mice were anesthetized with 2,2,2-tribromoethanol (Avertin; 0.5 mg/g body mass) before the measurement. For histological pathology and immunohistochemical examination, mice were euthanized at ages ranging from 6 days to 8 months. Bullae were isolated from ears of wild-type mice (n = 4) and LmnaDhe/+ mutant mice (n = 4), including both the middle and inner ear. Bullae were quickly dissected after euthanization, fixed in 4% paraformaldehyde at 4°C for 24 hours, and then decalcified in 10% EDTA for age-specific periods, as follows. For 6- and 12-day-old mice, samples were decalcified for 1 and 2 days, respectively; for both 21-day-old and 8-month-old mice, samples were decalcified for 7 days. Samples were then dehydrated and embedded in paraffin blocks. The paraffin-embedded samples were sectioned serially at 5-μm thickness and mounted onto Fisher Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA). For H&E staining, a standard protocol was used.10Yang B. Tian C. Zhang Z.G. Han F.C. Azem R. Yu H. Zheng Y. Jin G. Arnold J.E. Zheng Q.Y. Sh3pxd2b mice are a model for craniofacial dysmorphology and otitis media.PLoS One. 2011; 6: e22622Crossref PubMed Scopus (25) Google Scholar In accord with the manufacturer's protocol (Electron Microscopy Sciences, Hatfield, PA), Mayer's mucicarmine staining was used to identify goblet cells in the middle ear mucosa. Sections were examined under light microscopy (DFC500; Leica Microsystems). Images were acquired at ×5 to ×63 magnification. A four-point scoring system was applied to assess the severity of pathology in middle and inner ears. Scores were assessed and analyzed simultaneously by two individuals (Y.Z. and E.F.) masked to the genotype. Discrepancies were resolved by mutual consensus. The scale was as follows: −, absence of pathology in the middle or inner ear; +, very scarce pathology; ++, prevalent pathology, but not spread throughout the entire middle or inner ear; +++, pathology infiltrating the entire middle or inner ear. The scored pathologies included middle ear effusion, inflammatory cell infiltration, tissue debris, tissue proliferation, goblet cells, and inner ear effusion. A χ2 test was used to evaluate the semiquantitative data. This method has been reported in previous OM studies.11Han F. Yu H. Tian C. Li S. Jacobs M.R. Benedict-Alderfer C. Zheng Q.Y. Role for Toll-like receptor 2 in the immune response to Streptococcus pneumoniae infection in mouse otitis media.Infect Immun. 2009; 77: 3100-3108Crossref PubMed Scopus (34) Google Scholar After cardiac perfusion with 1× PBS and then with 4% paraformaldehyde, bullae were dissected from skulls of 21-day-old and 10-week-old control and LmnaDhe/+ mice (n = 3 mice per genotype). Samples were placed in 2.5% glutaraldehyde in cacodylic acid in 0.1 mol/L phosphate buffer (pH = 7.2) at 4°C overnight. After separation of the middle ear and inner ear, samples were postfixed with 1% osmium tetroxide diluted in 0.1 mol/L phosphate buffer. Samples were washed with distilled water three times, and then were dehydrated in serial solutions of ethanol. Each sample was subjected to CO2 critical point drying, followed by sputter-coating with 60:40 gold/palladium. Samples were then viewed under a high-resolution scanning electron microscope (Helios NanoLab 650; FEI, Hillsboro, OR). To evaluate whether the Lmna mutation affected mouse macrophage migration, morphogenesis, and/or cytokine expression, primary peritoneal macrophages were cultured from 4-month-old wild-type control mice and LmnaDhe/+ mutant mice, as described below. Thioglycollate broth (60 μL/g mouse body mass) was injected intraperitoneally into six mice (three females and three males) of each genotype. The mice were euthanized 3 days later. Peritoneal macrophages were recovered by peritoneal lavage with sterile, cold (4°C) PBS, followed by red blood cell lysis. Macrophages were counted on a hemocytometer (Hausser Scientific, Horsham, PA). Cells were then distributed into sterile plates (0.5 mL cells per plate), each containing a 22 × 30-mm slide, and were incubated at 37°C under 5% CO2 for 4 hours, to allow adhesion to slides for immunohistochemical staining. Middle ear paraffin sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, washed in distilled water, and incubated in trypsin working solution as described previously.11Han F. Yu H. Tian C. Li S. Jacobs M.R. Benedict-Alderfer C. Zheng Q.Y. Role for Toll-like receptor 2 in the immune response to Streptococcus pneumoniae infection in mouse otitis media.Infect Immun. 2009; 77: 3100-3108Crossref PubMed Scopus (34) Google Scholar For macrophage staining, smears were fixed in 1.5% paraformaldehyde. Sections and smears were permeabilized in 0.2% Triton X-100, washed in PBS, and then blocked in 3% goat serum and 2% BSA. Primary antibodies of anti-NF-κB (1:200 dilution; ab7971; Abcam, Cambridge, MA), anti-TNF-α (1:200 dilution; ab9739; Abcam), anti-lamin A/C (1:300 dilution; H-110; sc-20681; Santa Cruz Biotechnology, Santa Cruz, CA), or anti-TGF-β1/2/3 (1:200 dilution; sc-7892; Santa Cruz Biotechnology) were applied and slides were incubated overnight at 4°C. After a PBS wash, sections were incubated with goat anti-rabbit conjugated to Alexa Fluor 488 (1:500 dilution; Life Technologies-Invitrogen, Carlsbad, CA) and then with propidium iodide (10 μg/mL; P1304MP; Life Technologies-Invitrogen) or DAPI (10 μg/mL; D1306; Life Technologies-Invitrogen). Sections were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and were observed under an immunofluorescence microscope (Leica DFC500). Images were acquired at magnifications of ×5 to ×63. RNA was isolated from the bullae of four mutant and four control mice at P21, using a Gibco Pure-Link micro-to-midi total RNA purification system (Life Technologies-Invitrogen, Grand Island, NY). The SuperScript III first-strand synthesis system for RT-PCR was used to synthesize cDNA. Relative mRNA expression levels for TGF-β were determined by PCR using Gapdh as the positive control. Primers for Gapdh (forward: 5′-AACTTTGGCATTGTGGAAGG-3′; reverse: 5′-GGAGACAACCTGGTCCTCAG-3′) yield a 351-bp product, which spans two introns (between exons 3 to 4 and exons 4 to 5), as has been reported.11Han F. Yu H. Tian C. Li S. Jacobs M.R. Benedict-Alderfer C. Zheng Q.Y. Role for Toll-like receptor 2 in the immune response to Streptococcus pneumoniae infection in mouse otitis media.Infect Immun. 2009; 77: 3100-3108Crossref PubMed Scopus (34) Google Scholar, 12Chen M. Ona V.O. Li M. Ferrante R.J. Fink K.B. Zhu S. Bian J. Guo L. Farrell L.A. Hersch S.M. Hobbs W. Vonsattel J.P. Cha J.H. Friedlander R.M. Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease.Nat Med. 2000; 6: 797-801Crossref PubMed Scopus (108) Google Scholar Primers for TGF-β (forward: 5′-AGCCCGAAGCGGACTACTAT-3′; reverse: 5′-TCCACATGTTGCTCCACACT-3′) yield a 215-bp product, which spans one intron (exons 1 to 2). PCR was performed using Taq DNA polymerase (New England Biolabs) with the following amplification conditions: denaturation at 94°C for 2 minutes, followed by 31 cycles of 94°C for 30 seconds, 60°C for 40 seconds, and 72°C for 50 seconds, with a final extension step at 72°C for 5 minutes. PCR products were subjected to 2% agarose gel electrophoresis, and each yielded a single band of the predicted size. To evaluate relative gene transcription levels, a semiquantitative method was applied, using ImageJ software to normalize the TGF-β band intensity to Gapdh expression. Student's t-test was used to analyze differences between relative gray intensity of PCR bands, as described previously.11Han F. Yu H. Tian C. Li S. Jacobs M.R. Benedict-Alderfer C. Zheng Q.Y. Role for Toll-like receptor 2 in the immune response to Streptococcus pneumoniae infection in mouse otitis media.Infect Immun. 2009; 77: 3100-3108Crossref PubMed Scopus (34) Google Scholar Blood samples were acquired by cardiac puncture from 4-month-old wild-type and LmnaDhe/+ mutant mice (n = 6 mice per genotype; 3 females and 3 males). Serum was isolated and stored at −80°C. Serum calcium and phosphorus were assessed using an automated chemical analyzer (Vitros 350; Ortho Clinical Diagnostics, Johnson & Johnson, Rochester, NY) by the University of North Carolina Animal Clinical Chemistry and Gene Expression Laboratories (Chapel Hill, NC). Morphological study and morphometric analysis at age 10 weeks revealed anatomical abnormality of the middle ear and adjacent basicranial structure in the LmnaDhe/+ mutant mice. All six dissected skulls in the mutant mice exhibited a reduced distance, and three of the six exhibited a fused gap between the roof of the nasopharynx and the palate bone, compared with wild-type mice (Figure 1). The mean intersection angle between the bilateral eustachian tube in wild-type mice was 81.89 degrees, compared with 110.48 degrees in mutant mice (Figure 1, A–C). The mean length of the bony part of the eustachian tube in wild-type mice was 1.21 mm, compared with 0.93 mm in mutant mice. The mean width of the bony part of the eustachian tube was 1.12 mm, compared with 1.82 mm in mutant mice (Figure 1, D–F). The length/width ratio was 1.08 in wild-type mice, compared with 0.52 in mutant mice (Figure 1F), which further explains the shape abnormality of the eustachian tube. Within each group, there were no statistically significant differences in measurements between males and females (data not shown). Between the mutant and wild-type groups, however, all of the measurements indicated statistically significant differences (P < 0.05). Thus, clear alterations of the intersection angle accompanied by dilated anamorphic eustachian tubes in LmnaDhe/+ mutant mice produce a morphology that mimics the conditions in humans that cause predisposition for OM. Ear function was assessed by tympanometry in mice ranging in age from 3 weeks to 8 months. No statistically significant difference was detected between the left and right ears. Mean volume (V) of LmnaDhe/+ mutant mice was lower than that of wild-type mice at age 3 weeks, but there was no significant difference at other ages. Mean values and standard deviations were calculated for each parameter (Table 1); tympanogram results are presented for comparison (Figure 2, A–D). The tympanometric values of compliance (C) were significantly lower at all time points in LmnaDhe/+ mutant mice, compared with littermate controls. Pressure (P) of the middle ear was measured as a significantly more negative value in LmnaDhe/+ mice, compared with the control mice. Mean gradient values (G) were higher in LmnaDhe/+ mice at the age of 1 month, and the difference was statistically significant. With the different stages of OM progression, gradient values may vary. Gradient values are correlated with human OM, and both adult patients with OM and healthy children have a wider range of gradient. Scanning tympanograms from 5-month-old wild-type were representative of the normal A type curve (Figure 2E), and those of mutant mice were representative of the abnormal C type curve (Figure 2F), which resembles the C curve typical in human OM. Anatomical images under otoscopy of the ears that had tympanograms consistent with OM in LmnaDhe/+ mutant mice (Figure 2H) provided further evidence of OM, in contrast to the normal anatomy in control mice (Figure 2G). Tympanic membrane adherence and hydrotympanum typical of OM were detected by otoscopy in mutants.Table 1Tympanometry Measurements over a Time Course in Wild-Type and LmnaDhe/+ Mutant MiceAge and genotypeV (mL)C (mL)P (daPa × 1000)G (daPa × 1000)3 weeks Wild-type0.251 ± 0.0470.49 ± 0.045†P < 0.01.−0.014 ± 0.018†P < 0.01.0.142 ± 0.042 Mutant0.261 ± 0.0410.26 ± 0.108†P < 0.01.−0.063 ± 0.018†P < 0.01.0.135 ± 0.0421 month Wild-type0.322 ± 0.016†P < 0.01.0.71 ± 0.152⁎P < 0.05,−0.025 ± 0.011⁎P < 0.05,0.098 ± 0.031†P < 0.01. Mutant0.252 ± 0.016†P < 0.01.0.59 ± 0.140⁎P < 0.05,−0.034 ± 0.022⁎P < 0.05,0.135 ± 0.033†P < 0.01.5 months Wild-type0.341 ± 0.0480.74 ± 0.143†P < 0.01.−0.021 ± 0.0170.132 ± 0.020 Mutant0.331 ± 0.0370.45 ± 0.134†P < 0.01.−0.041 ± 0.022⁎P < 0.05,0.141 ± 0.0318 months Wild-type0.342 ± 0.0520.80 ± 0.173†P < 0.01.−0.015 ± 0.018†P < 0.01.0.124 ± 0.035 Mutant0.334 ± 0.0460.49 ± 0.119†P < 0.01.−0.053 ± 0.027†P < 0.01.0.151 ± 0.042Data are presented as means ± SD. n ≥ 6 mice at each age and each genotype.C, compliance; G, pressure gradient; P, pressure in middle ear; V, ear volume. P < 0.05,† P < 0.01. Open table in a new tab Data are presented as means ± SD. n ≥ 6 mice at each age and each genotype. C, compliance; G, pressure gradient; P, pressure in middle ear; V, ear volume. ABR thresholds from day P16 to 4 months of age were consistently elevated in LmnaDhe/+ mutant mice, compared with the wild type (Figure 3). Mean values for ABR threshold above 55 (for click stimuli), 40 (for 8 kHz), 35 (for 16 kHz), and 60 (for 32 kHz) dB SPL indicate hearing impairment.13Trune D.R. Zheng Q.Y. Mouse models for human otitis media.Brain Res. 2009; 1277: 90-103Crossref PubMed Scopus (25) Google Scholar All of the LmnaDhe/+ mutant mice met the criteria for hearing loss at the lower stimulus frequencies, click and 8 kHz (Figure 3, A and B). From P30, LmnaDhe/+ mutant mice began to show a fluctuant hearing impairment at the 16 kHz stimulus frequency (Figure 3C) and a clear tendency toward elevation at the higher stimulus frequency of 32 kHz (Figure 3D). These results demonstrated that the phenotype of hearing impairment began at lower stimulus frequencies (click and 8 kHz), and with age began to affect higher stimulus frequencies (16 and 32 kHz). At 3 months of age, LmnaDhe/+ mice had DPOAE 10 to 43.5 dB lower than those of wild-type mice at frequencies from 7.6 to 23 kHz (Figure 3E). Histological pathology was assessed at various stages to track the occurrence of OM inflammation and of eustachian tube developmental malformation in LmnaDhe/+ mutant mice, compared with littermate control wild-type mice (Figure 4). The middle ear was undeveloped at P6. No significant developmental disparity between wild-type and mutant or occurrence of middle ear inflammation were detected (Figure 4, A–F). At 12 days, with middle ear cavitation still progressing, significant dysplasia occurred with dilation of the eustachian tube. Cells of the acute inflammatory response began to perfuse the MEC of LmnaDhe/+ mice, to infiltrate the mesenchymal cells and to block the eustachian tube (Figure 4, K and L). The tympanic membrane was undeveloped at this stage (Figure 4, J–L). At weaning age of 21 days, the trend of inflammation continued and malformation of the eustachian tube was irreversible in LmnaDhe/+ mutant mice (Figure 4, P–R). Littermate control mice exhibited no OM pathology and the eustachian tube grew with normal morphology (Figure 4, M–O). At 8 weeks in mutant mice, cells of the chronic inflammatory response pervaded the entire middle ear cavity (Figure 4V). The tympanic membrane thickened and retracted into the middle ear cavity (Figure 4W). The eustachian tube appeared distorted with dilation and exhibited poorly aligned and shortened or obsolescent cilia at this stage (Figure 4X). In contrast, wild-type control mice exhibited a clear middle ear cavity with a normally positioned tympanic membrane (Figure 4, S and T). Also in the wild type, the eustachian tube developed to a straight narrow shape and was covered with orderly, aligned, pseudostratified ciliated columnar epithelium (Figure 4U). Diverse inflammatory manifestations were detected at different ages in LmnaDhe/+ mutant mice (Figure 5). Acute inflammatory cells and plasma fluids had infiltrated the middle ear cavity extensively at P30 (Figure 5, A and B). At older ages in LmnaDhe/+ mutant mice, from 2 months to 8 months, various manifestations of chronic suppurative middle ear inflammation occurred, with or without cholesteatoma. Cell infiltration and mucosal epithelium proliferation gradually appeared at 2 months (Figure 4V) and could progress to a much greater severity or develop encapsulated inflammation at older ages (Figure 5, C–H). Middle ear cholesteatoma was detected in some ears. Typical cholesterol crystals were revealed to be encysted by the hyperproliferative mucous epithelium and necrotic keratinizing squamous epithelium debris (Figure 5E). Local suppurative abscess formed in some of the middle ear cavities and collection of pus was enclosed by the surrounding hyperproliferative mucosal epithelium (Figure 5F). Proliferative goblet cells as detected by Mayer's mucicarmine staining revealed enhancement of the mucus-secreting ability in mutant mice (Figure 5H). We further measured the degree of pathology in OM by using five indices for semiquantitative evaluation as shown in Table 2. These data demonstrated that all of the LmnaDhe/+ mutant mice we tested exhibited OM and considerable inflammation, in contrast to wild-type control mice.Table 2Pathology in Middle Ears of Wild-Type and LmnaDhe/+ Mutant MiceMouse genotype and IDAgeMiddle ear effusionInflammatory cell infiltrationTissue debrisTissue proliferationGoblet cellsInner ear effusionScore⁎The maximum possible score per mouse was 18 points (1 point for each +).†The total scored rate (199/324) of the 18 LmnaDhe/+ mutant mice was significantly higher than that (10/216) of the 12 wild-type mice (P < 0.01, χ2 test).‡In the LmnaDhe/+ mutant mice, score rates progressively rose with age. There was no statistically significant difference between P12 (26/72) and P21(37/72) scores, but 8-week scores (52/72) were significantly higher than those at P21, and the
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