A kinase inhibitor screen identifies signaling pathways regulating mucosal growth during otitis media.

Otitis media, the most common disease of childhood, is characterized by extensive changes in the morphology of the middle ear cavity. This includes hyperplasia of the mucosa that lines the tympanic cavity, from a simple monolayer of squamous epithelium into a greatly thickened, respiratory-type mucosa. The processes that control this response, which is critical to otitis media pathogenesis and recovery, are incompletely understood. Given the central role of protein phosphorylation in most intracellular processes, including cell proliferation and differentiation, we screened a library of kinase inhibitors targeting members of all the major families in the kinome for their ability to influence the growth of middle ear mucosal explants in vitro. Of the 160 inhibitors, 30 were found to inhibit mucosal growth, while two inhibitors enhanced tissue proliferation. The results suggest that the regulation of infection-mediated tissue growth in the ME mucosa involves multiple cellular processes that span the kinome. While some of the pathways and processes identified have been previously implicated in mucosa hyperplasia others are novel. The results were used to generate a global model of growth regulation by kinase pathways. The potential for therapeutic applications of the results are discussed.

Age-Related Olfactory Dysfunction: Epidemiology, Pathophysiology, and Clinical Management.

Like other sensory systems, olfactory function deteriorates with age. Epidemiological studies have revealed that the incidence of olfactory dysfunction increases at the age of 60 and older and males are more affected than females. Moreover, smoking, heavy alcohol use, sinonasal diseases, and Down's syndrome are associated with an increased incidence of olfactory dysfunction. Although the pathophysiology of olfactory dysfunction in humans remains largely unknown, studies in laboratory animals have demonstrated that both the peripheral and central olfactory nervous systems are affected by aging. Aged olfactory neuroepithelium in the nasal cavity shows the loss of mature olfactory neurons, replacement of olfactory neuroepithelium by respiratory epithelium, and a decrease in basal cell proliferation both in the normal state and after injury. In the central olfactory pathway, a decrease in the turnover of interneurons in the olfactory bulb (OB) and reduced activity in the olfactory cortex under olfactory stimulation is observed. Recently, the association between olfactory impairment and neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has gained attention. Evidence-based pharmacotherapy to suppress or improve age-related olfactory dysfunction has not yet been established, but preliminary results suggest that olfactory training using odorants may be useful to improve some aspects of age-related olfactory impairment.

Caloric restriction reduces basal cell proliferation and results in the deterioration of neuroepithelial regeneration following olfactotoxic mucosal damage in mouse olfactory mucosa.

The effects of caloric restriction (CR) on cell dynamics and gene expression in the mouse olfactory neuroepithelium are evaluated. Eight-week-old male C57BL/6 mice were fed either control pellets (104 kcal/week) or CR pellets (67 kcal/week). The cytoarchitecture of the olfactory neuroepithelium in the uninjured condition and its regeneration after injury by an olfactotoxic chemical, methimazole, were compared between mice fed with the control and CR diets. In the uninjured condition, there were significantly fewer olfactory marker protein (OMP)-positive olfactory receptor neurons and Ki67-positive proliferating basal cells at 3 months in the CR group than in the control group. The number of Ki67-positive basal cells increased after methimazole-induced mucosal injury in both the control and the CR groups, but the increase was less robust in the CR group. The recovery of the neuroepithelium at 2 months after methimazole administration was less complete in the CR group than in the control group. These histological changes were region-specific. The decrease in the OMP-positive neurons was prominent in the anterior region of the olfactory mucosa. Gene expression analysis using a DNA microarray and quantitative real-time polymerase chain reaction demonstrated that the expression levels of two inflammatory cytokines, interleukin-6 and chemokine ligand 1, were elevated in the olfactory mucosa of the CR group compared with the control group. These findings suggest that CR may be disadvantageous to the maintenance of the olfactory neuroepithelium, especially when it is injured.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

Cigarette Smoke Delays Regeneration of the Olfactory Epithelium in Mice.

The olfactory system is a unique part of the mammalian nervous system due to its capacity for neurogenesis and the replacement of degenerating receptor neurons. Cigarette smoking is a major cause of olfactory dysfunction. However, the mechanisms by which cigarette smoke impairs the regenerative olfactory receptor neurons (ORNs) remain unclear. Here, we investigated the influence of cigarette smoke on ORN regeneration following methimazole-induced ORN injury. Administration of methimazole caused detachment of the olfactory epithelium from the basement membrane and induced olfactory dysfunction, thus enabling us to analyze the process of ORN regeneration. We found that intranasal administration of cigarette smoke solution (CSS) suppressed the recovery of ORNs and olfaction following ORN injury. Defective ORN recovery in CSS-treated mice was not associated with any change in the number of SOX2(+) ORN progenitor cells in the basal layer of the OE, but was associated with impaired recovery of GAP43(+) immature ORNs. In the nasal mucosa, mRNA expression levels of neurotrophic factors such as brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-5, glial cell-derived neurotrophic factor, and insulin-like growth factor-1 (IGF-1) were increased following OE injury, whereas CSS administration decreased the ORN injury-induced IGF-1 expression. Administration of recombinant human IGF-1 prevented the CSS-induced suppression of ORN recovery following injury. These results suggest that CSS impairs regeneration of ORNs by suppressing the development of immature ORNs from ORN progenitors, at least partly by reducing IGF-1 in the nasal mucosa.

Endoscopic transnasal approach for resection of locally aggressive tumors in the orbit.

OBJECT: In recent years, application of endoscopic transnasal surgery (ETS) has been expanded to orbital lesions, and preliminary results have started to be published for medially located soft mass lesions. However, reports on experience with endoscopic intraorbital surgery aimed at resection of invasive skull base tumors remains quite limited. This report presents the authors' experience with ETS for locally aggressive tumors involving the orbit. METHODS: ETS was performed for 15 cases of aggressive tumors involving the orbit: 5 meningiomas (meningothelial, n = 3; atypical, n = 1; anaplastic, n = 1), 4 chordomas, 2 chondrosarcomas, and 4 others (metastasis from systemic myxofibrosarcoma, schwannoma, inverted papilloma, and acinic cell carcinoma, n = 1 each). Among these, 9 tumors were located outside the periorbita and 6 inside the periorbita. In 6 intraperiosteal tumors, 5 were intraconal lesions, of which 3 arose in the muscle cone (anaplastic meningioma, optic sheath meningioma, and metastatic myxofibrosarcoma), and 2 meningothelial meningioma had invaded from the sphenoid ridge or the cavernous sinus into the muscle cone through the optic canal and the superior orbital fissure. A case of schwannoma originated around the cavernous sinus and pterygopalatine fossa and extended extraconally into the periorbita. Intraoperatively, ethmoid air cells and the lamina papyracea were removed, and extraperiosteal tumors were safely approached. For intraperiosteal tumors, the periorbita was widely opened, and the tumors were approached through the surgical window between the rectus and oblique muscles. RESULTS: Gross-total resection was achieved for 12 of the 15 tumors, including 2 intraconal lesions. After surgery, exophthalmos resolved in all 8 patients with this symptom, and diplopia resolved in 5 of 6 patients. Improvement of visual symptoms was reported by 4 of 5 patients with loss of visual acuity or constriction of the visual field. Postoperatively, 1 patient showed mild, transient worsening of existing facial dysesthesia, and another showed transient ptosis and mild hypesthesia of the forehead on the affected side. All those symptoms resolved within 3 months. No patients showed enophthalmos, worsening of diplopia or visual function, or impairment of olfaction after surgery. CONCLUSIONS: ETS appears acceptable as a less-invasive alternative for treating aggressive tumors involving the orbit. For extraperiosteal tumors, gross-total removal can generally be achieved without neurological complications. For intraperiosteal tumors, surgical indications should be carefully discussed, considering the relationship between the tumor and normal anatomy. Wide opening of the periorbital window is advocated to create a sufficient surgical pathway between the extraocular muscles, allowing a balance between functional preservation and successful tumor resection.

Endoscopic transnasal interseptal approach for invasive clival tumors: development of an approach method regarding maximal preservation of the nasal anatomy.

Asian people frequently show small noses, narrow nasal apertures, and congestive mucosa on the turbinates and septum. To reduce the risk of nasal morbidity with increased radicality for skull base tumors in these patients, we developed endoscopic transnasal interseptal approach (ETISA) in transsphenoidal surgery for invasive clival tumors (ICTs). Indication for ETISA is radical resection for tumors occupying deep ventral skull base regions, confined posterior to the level of the middle turbinates. After removing ethmoidal air cells, the middle turbinates are laterally deflected. A linear incision is made vertically on each side of the septal mucosa, which is separated from the bony septum as far as the sphenoid rostrum. The blades of an adjustable speculum are inserted submucosally, and the bony septum is temporarily displaced. The surgical pathway is widely maintained from the entrance to the deep surgical field without sacrificing the nasal mucosa and turbinates. Thirty-two consecutive patients with clival tumors (18 chordomas, 11 chondrosarcomas, 3 others) were treated. Bilateral middle turbinectomy was routinely performed in the initial 3 patients, but in only 4 of the remaining 29 (3 unilaterally, 1 bilaterally). Tumor was sufficiently resected in 29 patients (90.6%; gross total removal n=25, subtotal resection n=4). As complications, 6 patients showed mild and transient worsening of cranial nerve symptoms. Nasal complications arose in 3 patients, persisting >3 months in 2 (5.7%). This approach allows fine bimanual handling and swift delivery of surgical equipment while reasonably preserving the nasal anatomy, which is useful in endoscopic transsphenoidal surgery for ICT.

A mouse model of otitis media identifies HB-EGF as a mediator of inflammation-induced mucosal proliferation.

OBJECTIVE: Otitis media is one of the most common pediatric infections. While it is usually treated without difficulty, up to 20% of children may progress to long-term complications that include hearing loss, impaired speech and language development, academic underachievement, and irreversible disease. Hyperplasia of middle ear mucosa contributes to the sequelae of acute otitis media and is of important clinical significance. Understanding the role of growth factors in the mediation of mucosal hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae. METHODS: From a whole genome gene array analysis of mRNA expression during acute otitis media, we identified growth factors with expression kinetics temporally related to hyperplasia. We then tested these factors for their ability to stimulate mucosal epithelial growth in vitro, and determined protein levels and histological distribution in vivo for active factors. RESULTS: From the gene array, we identified seven candidate growth factors with upregulation of mRNA expression kinetics related to mucosal hyperplasia. Of the seven, only HB-EGF (heparin-binding-epidermal growth factor) induced significant mucosal epithelial hyperplasia in vitro. Subsequent quantification of HB-EGF protein expression in vivo via Western blot analysis confirmed that the protein is highly expressed from 6 hours to 24 hours after bacterial inoculation, while immunohistochemistry revealed production by middle ear epithelial cells and infiltrating lymphocytes. CONCLUSION: Our data suggest an active role for HB-EGF in the hyperplasia of the middle ear mucosal epithelium during otitis media. These results imply that therapies targeting HB-EGF could ameliorate mucosal growth during otitis media, and thereby reduce detrimental sequelae of this childhood disease.

Favorable prognostic factors for long-term postoperative hearing results after canal tympanoplasty for congenital aural stenosis.

OBJECTIVE: We aimed to determine favorable prognostic factors for long-term postoperative hearing results after canal tympanoplasty for congenital aural stenosis (CAS). STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: Canal tympanoplasty for CAS was performed in 25 ears. INTERVENTION: Primary repair of CAS. MAIN OUTCOME MEASURES: The influences of the following factors on the success of surgery were assessed by univariate and multivariate logistic regression analyses: modified Jahrsdoerfer grading system total score; age at surgery; patterns of presentation (whether sporadic or syndromic); presence of external auditory canal (EAC) cholesteatoma; presence of ossicular fixation, including the malleus bar; presence of a partial atretic plate; exposure of the facial nerve at the tympanic portion; type of tympanoplasty; and each component of the modified Jahrsdoerfer grading system. RESULTS: The univariate analysis revealed that the absence of EAC cholesteatoma (p = 0.029) and the presence of a partial atretic plate (p = 0.040) were significant predictive factors for favorable hearing prognosis, whereas the multivariate logistic regression analysis showed that an absence of EAC cholesteatoma was the most significant favorable predictive factor (p = 0.011), followed by anterolateral position of the malleus/incus complex with respect to the stapes as the second-most favorable factor (p = 0.021). CONCLUSION: The absence of EAC cholesteatoma and anterolateral position of the malleus/incus complex with respect to the stapes are considered useful in predicting long-term favorable hearing results after canal tympanoplasty for CAS.

Innate immune responses and neuroepithelial degeneration and regeneration in the mouse olfactory mucosa induced by intranasal administration of Poly(I:C).

The pathogenesis of postviral olfactory disorder (PVOD) has not been fully elucidated. We investigated morphological changes and innate immune responses in the mouse olfactory mucosa induced by intranasal administration of polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA. Mice received three administrations of saline with or without Poly(I:C), once every 24 h. The olfactory mucosa was harvested at various intervals after the first administration (8 h, 3, 9 and 24 days). In the Poly(I:C) group, the number of apoptotic cells in the olfactory neuroepithelium had increased at 8 h. At 9 days, the olfactory neuroepithelium had severely degenerated and behavioral tests demonstrated that the mice showed signs of olfactory deterioration. At 24 days, the structure of the neuroepithelium had regenerated almost completely. Regarding the innate immune responses, many neutrophils had infiltrated the olfactory neuroepithelium at 8 h and had exuded into the nasal cavity by 3 days. Macrophages had also infiltrated the olfactory neuroepithelium at 8 h although to a lesser extent, but they still remained in the neuroepithelium at 24 days. Poly(I:C)-induced neuroepithelial damage was significantly inhibited by a neutrophil elastase inhibitor and was suppressed in neutropenic model mice. These findings suggest that the secondary damage caused by the neutrophil-mediated innate immune response plays an important role in the pathogenesis of PVOD.

Expression of IL-33 and its receptor ST2 in chronic rhinosinusitis with nasal polyps.

OBJECTIVES/HYPOTHESIS: Interleukin (IL)-33 is a novel member of the IL-1 cytokine family and a ligand for the orphan IL-1 family receptor ST2. IL-33 induces T helper 2-type inflammatory responses and is considered to play a crucial role in allergic inflammatory reactions such as asthma and atopic dermatitis. However, the role of IL-33 and its receptor ST2 in chronic rhinosinusitis remains unclear. STUDY DESIGN: In vitro study. METHODS: The expression patterns of IL-33 and ST2 at both mRNA and protein levels in nasal polyps from eosinophilic chronic rhinosinusitis (ECRS) patients (n = 10) and non-ECRS patients (n = 13), as well as in seemingly normal mucosa of the uncinate processes in patients without sinusitis (control; n = 5), were compared using immunohistochemical staining, enzyme-linked immunosorbent assay, and real-time polymerase chain reactions. RESULTS: ST2-positive cells in the inflammatory cells in the subepithelial layer were significantly higher in the ECRS group than other groups. The expression of ST2 mRNA in polyps of the ECRS group was significantly increased compared with controls. Many ST2-positive eosinophils were observed in the mucosa of ECRS but not in the mucosa of non-ECRS patients. The expression level of IL-33 mRNA was not significantly different among the three groups. CONCLUSIONS: The current study suggests that IL-33 and its receptor ST2 may play important roles in the pathogenesis of chronic rhinosinusitis, especially in ECRS, through the increased expression of ST2 in eosinophils. LEVEL OF EVIDENCE: N/A.

Transcription factor early growth response 3 is associated with the TGF-ß1 expression and the regulatory activity of CD4-positive T cells in vivo.

TGF-ß1 is an important anti-inflammatory cytokine, and several regulatory T cell (Treg) subsets including CD4(+)CD25(+)Foxp3(+) Tregs and Th3 cells have been reported to exert regulatory activity via the production of TGF-ß1. However, it has not yet been elucidated which transcription factor is involved in TGF-ß1 transcription. Early growth response 3 (Egr-3) is a zinc-finger transcription factor that creates and maintains T cell anergy. In this study, we found that Egr-3 induces the expression of TGF-ß1 in both murine and human CD4(+) T cells. Egr-3 overexpression in murine CD4(+) T cells induced the production of TGF-ß1 and enhanced the phosphorylation of STAT3, which is associated with TGF-ß1 transcription. Moreover, Egr-3 conferred Ag-specific regulatory activity on murine CD4(+) T cells. In collagen-induced arthritis and delayed-type hypersensitivity model mice, Egr-3-transduced CD4(+) T cells exhibited significant regulatory activity in vivo. In particular, the suppression of delayed-type hypersensitivity depended on TGF-ß1. In human tonsils, we found that CD4(+)CD25(-)CD45RO(-)lymphocyte activation gene 3 (LAG3)(-) T cells express membrane-bound TGF-ß1 in an EGR3-dependent manner. Gene-expression analysis revealed that CD4(+)CD25(-)CD45RO(-)LAG3(-) T cells are quite different from conventional CD4(+)CD25(+)Foxp3(+) Tregs. Intriguingly, the CD4(+)CD25(-)CD45RO(-)LAG3(-) T cells suppressed graft-versus-host disease in immunodeficient mice transplanted with human PBMCs. Our results suggest that Egr-3 is a transcription factor associated with TGF-ß1 expression and in vivo regulatory activity in both mice and humans.

Epithelial cell-derived IL-25, but not Th17 cell-derived IL-17 or IL-17F, is crucial for murine asthma.

IL-17A, IL-17F, and IL-25 are ligands for IL-17RA. In the current study, we demonstrated that IL-25-deficient mice-but not IL-17A-, IL-17F-, IL-17A/F-, IL-23p19-, or retinoic acid-related orphan receptor (ROR)-γt-deficient mice-showed significant suppression of 1) the number of eosinophils and the levels of proinflammatory mediators in bronchoalveolar lavage fluids, 2) airway hyperresponsiveness to methacholine, and 3) OVA-specific IgG1 and IgE levels in the serum during OVA-induced Th2-type/eosinophilic airway inflammation. The IL-25 deficiency did not affect lung dendritic cell migration or Ag-specific memory-Th2 cell expansion during Ag sensitization. Adoptive transfer of T cells, mast cells, or bone marrow cells from IL-25-deficient mice revealed that induction of Th2-type/eosinophilic airway inflammation was dependent on activation of lung epithelial cells and eosinophils by IL-25 produced by airway structural cells such as epithelial cells but not by such hematopoietic stem-cell-origin immune cells as T cells and mast cells. Therefore, airway structural cell-derived IL-25-rather than Th17 cell-derived IL-17A and IL-17F-is responsible for induction of local inflammation by promoting activation of lung epithelial cells and eosinophils in the elicitation phase of Th2-type/eosinophilic airway inflammation. It is not required for Ag-specific Th2 cell differentiation in the sensitization phase.

Age-related changes of the regeneration mode in the mouse peripheral olfactory system following olfactotoxic drug methimazole-induced damage.

We investigated age-related changes in the mode of regeneration in the mouse peripheral olfactory system after olfactotoxic drug-induced damage. Mice at postnatal ages of 10 days, 3 months, and 16 months were given an intraperitoneal injection of methimazole to produce damage in the olfactory neuroepithelium. The olfactory neuroepithelia were harvested and analyzed immunohistochemically at various postlesion timepoints, from 1 day through to 94 days, to investigate neuroepithelial cell proliferation, the time course of neuronal differentiation, the reconstitution of neuroepithelium, and the innervation of the olfactory bulb. Functional recovery was assessed using the vanillin avoidance behavioral test. The chronological pattern in the expression of Ki67, beta III tubulin, and olfactory marker protein, molecular markers for neuronal cell proliferation and differentiation, changed similarly among the different age groups. In contrast, the extent of neuroepithelial cell proliferation after injury decreased with age, and the final histological recovery of the olfactory neuroepithelium and the innervation of the olfactory bulb were significantly smaller in the 16-month-old group compared to the younger age groups. These results suggest that the age-related decline in the capacity of olfactory neuroepithelium to reconstitute neuroepithelium is associated with its age-related decrease in proliferative activity after the neuroepithelial injury rather than changes in the process of neuronal differentiation. In spite of these incomplete anatomical recoveries, 16-month-old mice regained the ability to avoid vanillin solution by 1 month postlesion, suggesting that the extent of anatomical epithelial damage is not necessarily proportional to the threshold of olfactory perception.

Hydrogen in drinking water attenuates noise-induced hearing loss in guinea pigs.

It has been shown that molecular hydrogen acts as a therapeutic and preventive antioxidant by selectively reducing the hydroxyl radical, the most cytotoxic of the reactive oxygen species. In the present study, we tested the hypothesis that acoustic damage in guinea pigs can be attenuated by the consumption of molecular hydrogen. Guinea pigs received normal water or hydrogen-rich water for 14 days before they were exposed to 115 dB SPL 4-kHz octave band noise for 3h. Animals in each group underwent measurements for auditory brainstem response (ABR) or distortion-product otoacoustic emissions (DPOAEs) before the treatment (baseline) and immediately, 1, 3, 7, and 14 days after noise exposure. The ABR thresholds at 2 and 4 kHz were significantly better on post-noise days 1, 3, and 14 in hydrogen-treated animals when compared to the normal water-treated controls. Compared to the controls, the hydrogen-treated animals showed greater amplitude of DPOAE input/output growth functions during the recovery process, with statistical significance detected on post-noise days 3 and 7. These findings suggest that hydrogen can facilitate the recovery of hair cell function and attenuate noise-induced temporary hearing loss.

Establishment of mice expressing EGFP in the placode-derived inner ear sensory cell lineage and FACS-array analysis focused on the regional specificity of the otocyst.

In this study, we established a novel enhanced green fluorescent protein (EGFP) reporter mouse line that enables the visualization of the placode-derived inner ear sensory cell lineage. EGFP was initially expressed in the otic placode and throughout its differentiation process into the inner ear sensory patches. At embryonic day 10.5 (E10.5), EGFP was expressed in the ventral and dorsomedial region of the otocyst. These regions could mainly give rise to the cochlea, including the organ of Corti, and the saccule, including the macula and the endolymphatic duct. The region could also give rise to cells that will develop as either prosensory cells or statoacoustic ganglion neuroblasts. By using this line and fluorescence-activated cell sorting (FACS)-array technology, we developed a new gene expression profile of the regional specificity of the otocyst. EGFP-positive regions include the Otx1-positive region, which could be clearly distinguished from EGFP-negative regions. The signal log ratio of microarray data showed high efficiency in predicting the genes expressed mainly in the ventral and/or dorsomedial otocyst and the data could be mined to uncover many novel genes involved in inner ear morphogenesis and cell fate regulation. Additionally, these data suggest that some novel genes enriched in EGFP-positive regions may be potentially involved in human congenital sensorineural hearing loss. This reporter line could play important roles in the use of animal models for detailed analysis of the differentiation process into the sensory patches and the identification of regional-specific gene networks and novel gene functions in the developing inner ear.

Age-related changes in cell dynamics of the postnatal mouse olfactory neuroepithelium: cell proliferation, neuronal differentiation, and cell death.

Age-related changes in cell proliferation, neuronal differentiation, and cell death in mouse olfactory neuroepithelium were investigated. Mice at the age of 10 days through 16 months were given a single injection of bromodeoxyuridine (BrdU). The olfactory mucosae were fixed at 9 timepoints ranging from 2 hours to 3 months after the injection and examined using double immunostaining for BrdU and olfactory marker protein (OMP), and double staining with terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) and immunostaining for OMP. The number of BrdU-labeled cells/mm epithelial length initially increased, peaked at 2-3 days after the BrdU injection, then declined at each age. The number of BrdU- and TUNEL-labeled neuronal cells both decreased with increasing age, suggesting that the rates of both cell proliferation and cell death in the olfactory neuroepithelium decrease with increasing age. Double-labeled cells for BrdU and OMP appeared at 7 days after injection in all age groups, suggesting that the time required for neuronal differentiation is broadly similar irrespective of age. In older age groups, smaller amounts of the newly produced cohort are integrated into the OMP-positive ORN population, and even once it is integrated it is eliminated from the population more rapidly compared to the younger age groups. Furthermore, TUNEL assay showed that the fraction of apoptotic cells distributed in the OMP-positive layer/total apoptotic cells decreased with age. This observation suggests that the turnover of mature ORNs is slower in the older neuroepithelium compared to the younger neuroepithelium.

Recovery of facial movement and facial synkinesis in Bell's palsy patients.

OBJECTIVE: We examined the relationship between the time course of development of facial synkinesis in patients with Bell's palsy and the severity of facial nerve damage. STUDY DESIGN: Retrospective study. SETTING: Tertiary referral center. PATIENTS: Thirty-nine consecutive patients with Bell's palsy who developed synkinesis. INTERVENTION: Diagnostic. MAIN OUTCOME MEASURES: Subjects were divided into groups A (electroneurographic [ENoG] value, <10%; n = 31) and B (ENoG value, > or =10%; n = 8). Development of facial synkinesis was assessed based on the appearance of synkinetic potentials from the orbicularis oris muscle on the blink reflex test. Times to appearance of facial synkinesis in groups A and B were compared. The proportion of patients who developed facial synkinesis after complete recovery of facial movement was also assessed in 14 patients whose facial movement recovered completely. RESULTS: The mean time to maximal recovery of facial movement was significantly longer in group A than in group B (p < 0.001), whereas the duration between the appearance of facial synkinesis and the onset of facial paralysis did not differ significantly between the 2 groups (p = 0.72). The proportion of patients who developed facial synkinesis after complete recovery of facial movement was significantly greater in group B than in group A (p = 0.015). CONCLUSION: During the course of recovery from Bell's palsy, the patients with an ENoG value of 10% or greater have a higher risk of developing facial synkinesis after complete recovery of facial movement.