Damage to Olfactory Progenitor Cells Is Involved in Cigarette Smoke-Induced Olfactory Dysfunction in Mice.

Exposure to cigarette smoke is a major cause of olfactory dysfunction. However, the underlying mechanisms by which cigarette smoke interferes with the highly regenerative olfactory nerve system remain unclear. To investigate whether cigarette smoke induces olfactory dysfunction by disrupting cell proliferation and cell survival in the olfactory epithelium (OE), we developed a mouse model of smoking that involved intranasal administration of a cigarette smoke solution (CSS). Immunohistological analyses and behavioral testing showed that CSS administration during a period of 24 days reduced the number of olfactory marker protein-positive mature olfactory receptor neurons (ORNs) in the OE and induced olfactory dysfunction. These changes coincided with a reduction in the number of SOX2(+) ORN progenitors and Ki-67(+) proliferating cells in the basal layer of the OE, an increase in the number of caspase-3(+) apoptotic cells, and an increase in the expression of mRNA for the inflammatory cytokines IL-1ß and IL-6. Notably, the proliferating ORN progenitor population recovered after cessation of treatment with CSS, resulting in the subsequent restoration of mature ORN numbers and olfaction. These results suggest that SOX2(+) ORN progenitors are targets of CSS-induced impairment of the OE, and that by damaging the ORN progenitor population and increasing ORN death, CSS exposure eventually overwhelms the regenerative capacity of the epithelium, resulting in reduced numbers of mature ORNs and olfactory dysfunction.

Sensory deprivation disrupts homeostatic regeneration of newly generated olfactory sensory neurons after injury in adult mice.

Although it is well known that injury induces the generation of a substantial number of new olfactory sensory neurons (OSNs) in the adult olfactory epithelium (OE), it is not well understood whether olfactory sensory input influences the survival and maturation of these injury-induced OSNs in adults. Here, we investigated whether olfactory sensory deprivation affected the dynamic incorporation of newly generated OSNs 3, 7, 14, and 28 d after injury in adult mice. Mice were unilaterally deprived of olfactory sensory input by inserting a silicone tube into their nostrils. Methimazole, an olfactotoxic drug, was also injected intraperitoneally to bilaterally ablate OSNs. The OE was restored to its preinjury condition with new OSNs by day 28. No significant differences in the numbers of olfactory marker protein-positive mature OSNs or apoptotic OSNs were observed between the deprived and nondeprived sides 0-7 d after injury. However, between days 7 and 28, the sensory-deprived side showed markedly fewer OSNs and mature OSNs, but more apoptotic OSNs, than the nondeprived side. Intrinsic functional imaging of the dorsal surface of the olfactory bulb at day 28 revealed that responses to odor stimulation were weaker in the deprived side compared with those in the nondeprived side. Furthermore, prevention of cell death in new neurons 7-14 d after injury promoted the recovery of the OE. These results indicate that, in the adult OE, sensory deprivation disrupts compensatory OSN regeneration after injury and that newly generated OSNs have a critical time window for sensory-input-dependent survival 7-14 d after injury.

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.

Differences in Postoperative Hearing Outcomes and Vertigo in Patients with Otosclerosis Treated with Laser-Assisted Stapedotomy versus Stapedectomy.

BACKGROUND: Otosclerosis is an abnormal bone growth in the otic capsule that can result in hearing loss. In this study, we compared postoperative hearing outcomes and vestibular symptoms between patients treated with laser-assisted stapedotomy versus stapedectomy. METHODS: The medical charts of 99 ears treated with stapes surgery were retrospectively reviewed. RESULTS: A stapedotomy, partial stapedectomy, or total stapedectomy was conducted in 77, 16, and 56 ears, respectively. The ears treated with partial- and total stapedectomies were unified into one stapedectomy group. The postoperative changes in the air-bone gap after stapedotomies were significantly larger than those after stapedectomies at 1, 2, and 4 kHz. The postoperative changes in the air conduction threshold after stapedotomies were significantly larger than those after stapedectomies at 1, 2, 4, and 8 kHz. The postoperative changes in the bone conduction threshold at 0.5, 1, 2, and 4 kHz did not differ between the groups. The postoperative vertigo duration after stapedotomies was significantly shorter than that after stapedectomies. CONCLUSIONS: Surgery-induced sensorineural hearing losses were similar for stapedotomies and stapedectomies. However, stapedotomies were more effective and atraumatic than stapedectomies because of the better postoperative hearing results at middle and high frequencies and the shorter postoperative vertigo.

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.

Bilateral cervical fistulas from heterotopic salivary gland tissues.

A 24-year-old male was referred to our department with intermittent clear drainage in both sides of the middle neck that did not increase while eating. On physical examination, there were fistulas in both sides of the neck, anterior to the sternocleidomastoid muscles. Fistulography revealed a 9-mm-long sinus from the left fistula, extending in the medial-caudal direction. On the right side, cannulation was not possible. Surgical excision of the lesion was performed on both sides, and the pathological examination revealed heterotopic salivary gland tissue (HSGT). From a literature review of reports of bilateral neck HSGT, we found that it is often associated with a family history of HSGT and other congenital anomalies. However, our case was different from the previously reported cases in that, although it was a bilateral case, it lacked any family history of HSGT or other congenital anomalies. Clinicians should include HSGT in the differential diagnosis of neck sinuses. © 2015 S. Karger AG, Basel.

Evaluation of the Carhart effect in congenital middle ear malformation with both an intact external ear canal and a mobile stapes footplate.

The medical charts of 41 ears with congenital middle ear malformation with both an intact external ear canal and a mobile stapes footplate were reviewed retrospectively to study the Carhart effect. The operations were categorized as successful or unsuccessful according to the extent of decrease in the average air-bone gap. Statistically significant differences were observed between the 2 groups with respect to the changes in pure-tone average and the changes in the bone conduction (BC) threshold at 1 and 2 kHz. Linear regression analysis revealed weak correlations between the change in the BC threshold and the postoperative BC threshold at an overall level and at the 4 frequencies tested. Stapes ankylosis is a main cause of the Carhart effect. The present study showed that in congenital middle ear malformation, the Carhart effect was caused not only by stapes ankylosis but also by other types of disruption in the ossicular chain.

Repetitive Sinus-Related Symptoms May Accelerate the Progression of Chronic Maxillary Atelectasis.

Chronic maxillary atelectasis (CMA) is characterized by a progressive decrease in maxillary sinus volume. The factors that promote the stage progression of CMA remain poorly understood. Here, we describe the time course of anatomical changes in a 40-year-old woman with stage II CMA that progressed to stage III disease. She did not show stage progression until she started to develop repetitive sinus-related symptoms. The stage progression was characterized by ocular symptoms. The repetitive inflammatory episodes may have increased the negative pressure in the affected sinus and weakened the bone walls, thereby promoting stage progression. Thus, a history of repetitive sinus-related symptoms may be a risk factor for stage progression in CMA.

Intravenous olfactory test latency correlates with improvement in post-infectious olfactory dysfunction.

CONCLUSION: This cohort study showed that onset latency in the intravenous olfactory test (IVO) may help predict when olfaction in patients with post-infectious olfactory dysfunction (PIOD) improves. OBJECTIVES: To identify factors that predict the olfactory improvement period in patients with PIOD. METHODOLOGY/PRINCIPAL: All consecutive patients presenting with PIOD in 1994-2014 who were followed up for 2 years were identified retrospectively. The ability of demographic/clinical factors (age, sex, body mass index, presence/absence of allergic rhinitis, treatment/non-treatment with herbal medicines, patient dependence on herbal medicine treatment, presence/absence of diabetes mellitus, and smoking status) and olfactory test factors (response/no response and onset latency and duration in the IVO test, and detection and recognition scores on the T&T olfactory test) to predict the olfactory improvement period (defined respectively as the time from PIOD onset or olfactory testing to the first self-report of olfaction improvement) was analyzed by univariate and multivariate regression. RESULTS: Of the 187 PIOD patients, the prognostic ability of demographic/clinical factors was analyzed in 65. None predicted the olfactory improvement period. Of the 65 patients, 20 did not respond in the IVO test. In the remaining 45 patients, onset latency (but not the other olfactory test factors) was a significant prognosticator of olfactory improvement period (R2=0.24, p = 0.003).

Association of the upregulated expression of focal adhesion kinase with poor prognosis and tumor dissemination in hypopharyngeal cancer.

BACKGROUND: Focal adhesion kinase (FAK) plays an important role in tumor metastasis. The purpose of this study was to evaluate the significance of FAK expression in surgically treated patients with hypopharyngeal cancer. METHODS: We retrospectively reviewed the clinical charts of patients treated at our institution between 2004 and 2012 and identified 87 patients with hypopharyngeal cancer. FAK expression status was retrospectively evaluated using immunohistochemistry. RESULTS: FAK-positive patients displayed significantly worse disease-specific survival than FAK-negative patients (p = .001). Multivariate analyses revealed that FAK positivity and extracapsular spread (ECS) were independent, significant adverse prognostic factors. Furthermore, FAK positivity significantly correlated with the number of metastatic lymph nodes (p = .048), and FAK-positive patients displayed a higher incidence of distant metastases (p = .009). CONCLUSION: The current study demonstrated that upregulated FAK expression correlates with poor prognosis and tumor dissemination in surgically treated patients with hypopharyngeal cancer. © 2016 Wiley Periodicals, Inc. Head Neck 38:1164-1169, 2016.

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.

Longer latency of sensory response to intravenous odor injection predicts olfactory neural disorder.

A near loss of smell may result from conductive and/or neural olfactory disorders. However, an olfactory test to selectively detect neural disorders has not been established. We investigated whether onset latency of sensory response to intravenous odor injection can detect neural disorders in humans and mice. We showed that longer preoperative onset latency of odor recognition to intravenous odor in patients with chronic rhinosinusitis predicted worse recovery of olfactory symptoms following sinus surgery. The onset latency of the olfactory sensory neuron (OSN) response to intravenous odor using synaptopHluorin signals from OSN axon terminals was delayed in mice with reduced numbers of OSNs (neural disorder) but not with increased mucus or blocked orthonasal pathways (conductive disorders). Moreover, the increase in onset latency correlated with the decrease in mature OSN numbers. Longer onset latency to intravenous odor injection is a useful biomarker for presence and severity of olfactory disorders with neural etiology.

Mumps, cervical zoster, and facial paralysis: coincidence or association?

The association of mumps with peripheral facial paralysis has been suggested, but its pathogenesis remains unclear. An 8-year-old girl simultaneously developed left peripheral facial paralysis, ipsilateral cervical herpes zoster, and bilateral mumps sialadenitis. Elevated anti-mumps and anti-varicella zoster virus IgM antibodies in serological testing indicated recent infection of mumps and reactivation of VZV. Molecular studies have provided mounting evidence that the mumps virus dysregulates the host's immune system and enables the virus to proliferate in the infected host cells. This dysregulation of the immune system by mumps virus may have occurred in our patient, enabling the latent VZV infection to reactivate.

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.

Bell's palsy in children: relationship between electroneurography findings and prognosis in comparison with adults.

OBJECTIVES: To investigate the correlation between electroneurography (ENoG) findings and the prognosis of Bell's palsy in children compared with adults. METHODS: Twenty-two children and 92 adults with Bell's palsy who underwent ENoG between 8 days and 4 weeks from the onset of symptoms were retrospectively enrolled. The time to maximal recovery and rate of favorable recovery (House-Brackmann grade I or II) was assessed. Children (C) and adults (A) were further subdivided into low (<10%) or high (≧10%) subgroups according to their ENoG values (affected versus unaffected side) at initial evaluation. The numbers in each subgroup were as follows: C-low (n = 8), A-low (n = 21), C-high (n = 14), and A-high (n = 71). RESULTS: Of the 22 children assessed, 2 of the 4 patients who showed a total loss of evoked potentials on the affected side (0% ENoG value) exhibited an unfavorable recovery. The remaining 20 patients achieved a favorable recovery eventually. Patients in group C-low reached a maximal recovery of facial movement significantly later than those in group C-high (p < 0.001). Time to maximal recovery of facial movement in group A-low was later than that in group C-low, although the difference was not statistically significant (p = 0.15). The patients in group A-high reached a maximal recovery significantly later than those in group C-high (p < 0.05). CONCLUSION: Bell's palsy seems to recover earlier in children than adults when matched for severity. The presence of an identifiable response in ENoG, irrespective of its amplitude, may indicate a favorable recovery of facial movement in children.

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.

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.