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Objective: Olfaction is impaired in chronic rhinosinusitis (CRS). The study has two aims: (1) to determine whether changes in cation concentration occur in the olfactory mucus of mice with CRS, which may affect chemo-electrical transduction, (2) and to examine whether these alterations are physiologically significant in humans., Study Design: Animal study in mice and translational study in humans., Methods: Inflammation was induced by sensitization and chronic exposure of 16 C57BL/6 mice to Aspergillus fumigatus. The control group included 16 untreated mice. Ion-selective microelectrodes were used to measure free cation concentrations in the olfactory mucus of 8 mice from each treatment group, while the remaining mice were sacrificed for histology. To validate the findings in the animal model, olfactory threshold was measured in 11 healthy human participants using Sniffin' Sticks before and after nasal irrigation with solutions that were composed of either of the cation concentrations., Results: In 8 mice, olfactory mucus of chronically inflamed mice had lower [Na(+)] (84.8±4.45 mM versus 93.73±3.06 mM, p = 0.02), and higher [K(+)] (7.2±0.65 mM versus 5.7±0.20 mM, p = 0.04) than controls. No difference existed in [Ca(2+)] (0.50±0.12 mM versus 0.54±0.06 mM, p = 0.39). In humans, rinsing with solutions replicating ion concentrations of the mouse mucosa with chronic inflammation caused a significant elevation in the median olfactory threshold (9.0 to 4.8, p = 0.003) but not with the control solution (8.3 to 7.8, p = 0.75)., Conclusion: Chronic inflammation elevates potassium and lowers sodium ion concentration in mice olfactory mucus. Nasal irrigation with a corresponding solution induced olfactory threshold shift in humans.

Objectives/hypothesis: The treatment of anosmia has changed minimally since the early 1970s, despite dramatic advances in the understanding of the molecular biology of olfaction. Recent studies from the authors' laboratory have suggested that most common causes of clinical olfactory dysfunction, including rhinosinusitis, appear to be associated with increased apoptotic death of olfactory sensory neurons. This appears to result in a decline in the number of functioning mature olfactory sensory neurons, despite the capacity of the olfactory epithelium for regeneration. The current study evaluated the ability of the antibiotic minocycline to inhibit olfactory sensory neuron apoptosis. This drug is known to inhibit apoptosis separate from its anti-infective properties. Olfactory sensory neuron apoptosis was triggered by surgical deafferentation ("bulbectomy"), the standard experimental model. Earlier studies have indicated that bulbectomy and sinusitis invoke similar proteolytic enzyme cascades in olfactory sensory neurons., Study Design: Histological analysis of animal olfactory tissue., Methods: Mice underwent unilateral olfactory bulbectomy to induce apoptotic olfactory sensory neuron death, with and without 45 mg/kg intraperitoneal minocycline given 12 hours before surgery and every 12 hours until death. Mice were killed at 2 and 4 days after bulbectomy and assessed for activation of capsase-3 and olfactory sensory neuron survival by immunohistochemical analysis., Results: Minocycline resulted in partial suppression of cell death at 2 days after surgery when compared with untreated animals., Conclusion: Minocycline inhibits olfactory sensory neuron death in the face of a potent pro-apoptotic stimulus. This drug is well tolerated and is currently undergoing human trials for the management of a variety of neurological disorders associated with apoptosis. The current results suggest that minocycline may be efficacious in the management of peripheral olfactory loss as well.

Objective: To investigate the effects of tobacco smoke on the olfactory epithelium. Cigarette smoking has been associated with hyposmia; however, the pathophysiology is poorly understood. The sense of smell is mediated by olfactory sensory neurons (OSNs) exposed to the nasal airway, rendering them vulnerable to environmental injury and death. As a consequence, a baseline level of apoptotic OSN death has been demonstrated even in the absence of obvious disease. Dead OSNs are replaced by the mitosis and maturation of progenitors to maintain sufficient numbers of neurons into adult life. Disruption of this balance has been suggested as a common cause for clinical smell loss. This current study will evaluate the effects of tobacco smoke on the olfactory mucosa, with emphasis on changes in the degree of OSN apoptosis., Study Design: A rat model was used to assess the olfactory epithelium after exposure to tobacco smoke., Methods: Rats were exposed to tobacco smoke alone (for 12 weeks), smoke plus dietary ethanol (for the final 5 weeks), or to neither (control). Immunohistochemical analysis of the olfactory epithelium was performed using an antibody to the active form of caspase-3. Positive staining for this form of the caspase-3 enzyme indicates a cell undergoing apoptotic proteolysis., Results: Control rats demonstrated a low baseline level of caspase-3 activity in the olfactory epithelium. In contrast, tobacco smoke exposure triggered a dramatic increase in the degree of OSN apoptosis that affected all stages of the neuronal lineage., Conclusions: These results support the following hypothesis: smell loss in smokers is triggered by increased OSN death, which eventually overwhelms the regenerative capacity of the epithelium.

Objective: The pathology of the olfactory mucosa is poorly understood; however, most cases of hyposmia and anosmia appear to be associated with a decline in the number of functioning mature olfactory sensory neurons (OSNs). Under normal conditions, OSNs undergo apoptotic cell death at a baseline rate likely secondary to their exposed location in the nose. Regeneration of mature OSNs from precursors in the epithelium allows the animal to maintain an adequate number of neurons necessary for olfactory sensation. In many cases of olfactory dysfunction, this balance is apparently disturbed, with a net loss of OSNs. The current study will examine normal and diseased olfactory tissue for the presence of data demonstrating that the preferred mechanism of OSN cell death is apoptotic in both health and disease. The potential therapeutic implications will be discussed., Study Design: Histologic analysis of human and animal olfactory tissue., Methods: Normal and diseased human and animal olfactory mucosa were assessed for immunohistochemical evidence of apoptosis., Results: Increased activity of the apoptotic effector enzyme caspase-3 was demonstrated in diseased olfactory mucosa in comparison with normal controls., Conclusion: These results indicate that a common pathway may mediate OSN cell death from a diverse set of pathologic insults including aging, trauma, and sinusitis. Interference with this pathway of cell death is currently the subject of intense pharmacotherapeutic research for the management of stroke and meningitis. These drugs may ultimately prove useful in the treatment of clinical olfactory dysfunction.

Objectives: Olfactory receptor neurons undergo apoptosis at a baseline rate, probably secondary to environmental damage even in the absence of gross disease. The study demonstrates age-related changes in expression of genes known to regulate apoptosis in the rat olfactory mucosa. These results are compared with gene expression in young rats and rats that have undergone surgical deafferentation of the olfactory receptor neurons., Study Design: The olfactory mucosae from three groups of rats were studied: young, normal rats (age, 12 wk); old, normal rats (age, 24 mo); and young rats 9 days after bilateral removal of the olfactory bulb. Bulbectomy is known to produce an initial wave of apoptotic cell death in the population of olfactory neurons. At 9 days after the injury, the olfactory mucosae consist of an enhanced population of regenerating neurons destined to also undergo apoptosis, since their synaptic target (bulb) has been removed., Methods: Ribonuclease protection assays and histological analysis of the three groups were performed., Results: Ribonuclease protection assay analysis indicates that age induces increases in the expression of pro-apoptotic genes in the olfactory mucosae similar to the increase seen after deafferentation (bulbectomy). Specifically, the expression of procaspase-3 and bax was increased in aged animals and bulbectomized animals when compared with young, normal animals., Conclusions: Aging induces changes in gene expression in the olfactory mucosae that appear to favor apoptosis, probably associated with increased fragility of olfactory receptor neurons in older animals. These changes may, at least in part, explain the age-related decline in olfactory sensation and loss of olfactory receptor neurons seen in elderly patients.

Objectives: To evaluate histological changes in the olfactory mucosa of patients with chronic rhinosinusitis These results are analyzed in light of current understanding of the pathophysiology of anosmia secondary to nasal and sinus disease., Study Design: Prospective study of olfaction on patients undergoing sinus surgery for the management of chronic rhinosinusitis., Methods: Thirty patients, aged 22 to 39 years, underwent olfactory biopsy at the time of surgery with evaluation by a pathologist. Inflammatory changes were graded as mild (normal), moderate, or severe. Clinical olfactory function was evaluated using the University of Pennsylvania Smell Identification Test (UPSIT). The results correlated with the degree of olfactory dysfunction., Results: Of the 30 patients 19 had unequivocal olfactory mucosa in the biopsy specimen. Eleven had only respiratory or indeterminate mucosa. Nine patients demonstrated normal olfactory mucosa and normal olfactory function (UPSIT > 35). Ten patients demonstrated pathological changes in the olfactory mucosa with an influx of lymphocytes, macrophages, and eosinophils Of these 10 patients, 7 had olfactory deficits as determined by UPSIT. The remaining three patients had normal olfactory function despite moderate chronic inflammation. These studies indicate that the olfactory mucosa is capable of mounting an inflammatory response similar to that seen in the respiratory mucosa of patients with chronic sinusitis These data suggest that the olfactory deficits in these patients may be the result of inflammatory changes within the olfactory mucosa in addition to any alteration in airflow to the olfactory cleft.

Mineralocorticoid hormones regulate secretion and absorption in a wide variety of epithelial tissues, although specific mechanisms in the olfactory mucosa are currently unknown. Utilizing reverse transcription-polymerase chain reaction (RT-PCR) analysis, we have demonstrated the expression of mineralocorticoid (type I) receptor messenger RNA in the rodent olfactory mucosa. Amplification products of predicted size were obtained with nucleotide sequences corresponding to respective mineralocorticoid receptor (MR) kidney transcripts. Immunocytochemistry, using an antibody with known specificity for MRs, was then utilized in order to localize the cellular site(s) of MR protein expression in the olfactory mucosa. The highest levels of MR immunoreactivity were localized to the supranuclear region of sustentacular cells, as well as the acinar cells of the Bowman's glands. The respiratory regions of the nasal cavity were devoid of appreciable MR immunoreactivity. This study demonstrates both MR transcript and protein expression in the olfactory mucosa. We hypothesize that the mineralocorticoid hormones may have a role in modulation of olfactory secretion and/or sensory transduction in the peripheral olfactory system.

Objectives: To investigate the cellular distribution and relative intensity of the immunoreactivity associated with the expression of sodium, potassium-adenosine triphosphatase (Na, K-ATPase) in cells of the olfactory mucosa. Second, changes in the activity of this enzyme in the olfactory mucosa are correlated with changes in the circulating corticosteroid aldosterone., Methods: Combination of immunohistochemical and biochemical techniques were employed to examine the olfactory Na, K-ATPase., Results: Within the olfactory epithelium, the Na, K-ATPase immunoreactivity was greatest at the supranuclear region of sustentacular cells and/or dendrites of olfactory receptor neurons (ORNs). Cell bodies of ORNs demonstrated moderate immunoreactivity, whereas the duct cells of Bowman's gland exhibited moderate to intense immunoreactivity. Acinar cells of the Bowman's gland were the most intensely stained components of the lamina propria, exhibiting strong immunoreactivity at the basolateral plasma membrane domains of the acinar cells and less within the cytoplasm. Binding of ouabain, a specific inhibitor of Na, K-ATPase, was significantly elevated for aldosterone-injected versus sham-injected controls., Conclusion: These results suggest that olfactory Na, K-ATPase is regulated by the systemic corticosteroid aldosterone. The results are consistent with the hypothesis that corticosteroids regulate olfactory secretion.

Objectives: Define the presence and distribution of glucocorticoid receptors (GRs) within the olfactory mucosa in order to assess potential physiologic and pathophysiologic effects of these hormones on olfaction., Study Design: The olfactory mucosa was harvested from adult male rats and guinea pigs. Kidney tissue was utilized as a known positive control., Methods: The techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry were utilized to examine the expression of GR mRNA and protein. To assure the presence of olfactory mucosa in the nasal tissue samples, RT-PCR was utilized to identify the olfactory marker protein (OMP)., Results: The presence of GR mRNA was confirmed in both the olfactory mucosa and kidney. GR-like immunoreactivity associated with the olfactory epithelium was greatest at the apical surface, a position corresponding to the dendrites, knobs, and cilia of olfactory receptor neurons, as well as the supranuclear region of sustentacular cells. Weaker GR-like immunoreactivity was associated with the region of the cell bodies of the olfactory receptor neurons. Within the lamina propria, acinar cells of the Bowman's glands and olfactory nerve bundles were intensely immunoreactive., Conclusions: The presence of GR mRNA and protein within the olfactory mucosa is consistent with a functional role for glucocorticoid hormones in the systemic regulation of olfaction. Furthermore, these studies suggest that corticosteroid medications may have direct effects on the cells of the olfactory mucosa in the pathologic state. The potential mechanisms whereby these hormones may act are discussed.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) plays a major role in the protection of the mineralocorticoid (type I) receptor. The cellular mechanism of aldosterone selectivity relies on the coexpression of mineralocorticoid receptors and 11 beta HSD in the same cells. In the current study, 11 beta HSD activity was localized in the mammalian olfactory mucosa by a histochemical technique which links steroid metabolism with the deposition of formazan. The histochemical reaction results from oxidation of the synthetic substrate 11 beta-hydroxyandrostenedione and is dependent on nicotine-adenine dinucleotide (NAD). This demonstrates the presence of a dehydrogenase activity separate from the nicotineamide-adenine dinucleotide phosphate (NADP)-dependent 11 beta HSD. In the olfactory mucosa, the presence of NAD-dependent 11 beta HSD activity is localized to the sustentacular cells and acinar cells of Bowman's glands. No definite NAD-dependent activity was demonstrated in the olfactory receptor neurons. These data suggest that mineralocorticoid receptors present in acinar cells and sustentacular cells are aldosterone selective.

The glucocorticoid RU 28362 was employed to identify glucocorticoid receptors in the olfactory mucosa of the guinea-pig. Results demonstrate significant binding of RU 28362 and suggest that the olfactory mucosa is a target site for glucocorticoid action.

High-affinity, specific binding sites to mineralocorticoids, with characteristics of mineralocorticoid (type I) receptors, have been found in the mammalian olfactory mucosa. In the presence of RU 28362, which blocks low-affinity binding of the labeled hormone to glucocorticoid (type II) receptors, Scatchard analysis of the specific [3H]aldosterone binding indicates that aldosterone binds to a single class of high-affinity (type I) sites with a dissociation constant, Kd, of 1.2 x 10(-9) M and a maximum number of binding sites, Bmax, of 63 fmol/mg dry tissue. Time course of association of [3H]aldosterone and its binding sites showed maximal binding by 30 min at 25 degrees C which remained unchanged up to 90 min. Competition studies performed with 1 x 10(-7)-1 x 10(-5) M spironolactone, a competitive mineralocorticoid antagonist, showed a dose-response reduction of [3H]aldosterone binding, implying that in the olfactory mucosa this compound behaves as an aldosterone antagonist. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity and suggest that the olfactory mucosa is a target site for mineralocorticoid action.