Human Olfactory Tissue Research Articles

Techniques in Staining of Rodent and Human Olfactory Tissue.

Staining of olfactory tissue allows for evaluation of the organization of specific cell types within the specimen and allows for assessment in abnormalities of function that may be related to changes in normal tissue architecture. Histochemical staining and immunohistochemical (IHC) techniques are the most common methods for visualizing olfactory epithelium and olfactory bulbs. Here we describe the method of IHC for olfactory tissue utilizing both fluorescent and peroxidase-based methods of visualization.

Integrated age-related immunohistological changes occur in human olfactory epithelium and olfactory bulb.

Olfactory epithelium (OE) is capable of lifelong regeneration due to presence of basal progenitor cells that respond to injury or neuronal loss with increased activity. However, this capability diminishes with advancing age and a decrease in odor perception in older individuals is well established. To characterize changes associated with age in the peripheral olfactory system, an in-depth analysis of the OE and its neuronal projections onto the olfactory bulb (OB) as a function of age was performed. Human olfactory tissue autopsy samples from 36 subjects with an average age of 74.1 years were analyzed. Established cell type-specific antibodies were used to identify OE component cells in whole mucosal sheets and epithelial sections as well as glomeruli and periglomerular structures in OB sections. With age, a reduction in OE area occurs across the mucosa progressing in a posterior-dorsal direction. Deterioration of the olfactory system is accompanied with diminution of neuron-containing OE, mature olfactory sensory neurons (OSNs) and OB innervation. On an individual level, the neuronal density within the epithelium appears to predict synapse density within the OB. The innervation of the OB is uneven with higher density at the ventral half that decreases with age as opposed to stable innervation at the dorsal half. Respiratory metaplasia, submucosal cysts, and neuromata, were commonly identified in aged OE. The finding of respiratory metaplasia and aneuronal epithelium with reduction in global basal cells suggests a progression of stem cell quiescence as an underlying pathophysiology of age-related smell loss in humans. KEY POINTS: A gradual loss of olfactory sensory neurons with age in human olfactory epithelium is also reflected in a reduction in glomeruli within the olfactory bulb. This gradual loss of neurons and synaptic connections with age occurs in a specific, spatially inhomogeneous manner. Decreasing mitotically active olfactory epithelium basal cells may contribute to age-related neuronal decline and smell loss in humans.

Multi-target approaches to CNS repair: olfactory mucosa-derived cells and heparan sulfates.

Spinal cord injury (SCI) remains one of the biggest challenges in the development of neuroregenerative therapeutics. Cell transplantation is one of numerous experimental strategies that have been identified and tested for efficacy at both preclinical and clinical levels in recent years. In this Review, we briefly discuss the state of human olfactory cell transplantation as a therapy, considering both its current clinical status and its limitations. Furthermore, we introduce a mesenchymal stromal cell derived from human olfactory tissue, which has the potential to induce multifaceted reparative effects in the environment within and surrounding the lesion. We argue that no single therapy will be sufficient to treat SCI effectively and that a combination of cell-based, rehabilitation and pharmaceutical interventions is the most promising approach to aid repair. For this reason, we also introduce a novel pharmaceutical strategy based on modifying the activity of heparan sulfate, an important regulator of a wide range of biological cell functions. The multi-target approach that is exemplified by these types of strategies will probably be necessary to optimize SCI treatment.

Chronic Inflammation Directs an Olfactory Stem Cell Functional Switch from Neuroregeneration to Immune Defense.

Although olfactory mucosa possesses long-lived horizontal basal stem cells (HBCs) and remarkable regenerative capacity, the function of human olfactory neuroepithelium is significantly impaired in chronic inflammatory rhinosinusitis. Here, we show that, while inflammation initially damages olfactory neurons and activates HBC-mediated regeneration, continued inflammation locks HBCs in an undifferentiated state. Global gene expression in mouse HBCs reveals broad upregulation of NF-κB-regulated cytokines and chemokines including CCL19, CCL20, and CXCL10, accompanied by enhancement of "stemness"-related transcription factors. Loss-of-function studies identify an NF-κB-dependent role of HBCs in amplifying inflammatory signaling, contributing to macrophage and Tcell local proliferation. Chronically activated HBCs signal macrophages to maintain immune defense and prevent Treg development. In diseased human olfactory tissue, activated HBCs in a P63+ undifferentiated state similarly contribute to inflammation through chemokine production. These observations establish a mechanism of chronic rhinosinusitis-associated olfactory loss, caused by a functional switch of neuroepithelial stem cells from regeneration to immune defense.

The human olfactory transcriptome.

BackgroundOlfaction is a versatile sensory mechanism for detecting thousands of volatile odorants. Although molecular basis of odorant signaling is relatively well understood considerable gaps remain in the complete charting of all relevant gene products. To address this challenge, we applied RNAseq to four well-characterized human olfactory epithelial samples and compared the results to novel and published mouse olfactory epithelium as well as 16 human control tissues.ResultsWe identified 194 non-olfactory receptor (OR) genes that are overexpressed in human olfactory tissues vs. controls. The highest overexpression is seen for lipocalins and bactericidal/permeability-increasing (BPI)-fold proteins, which in other species include secreted odorant carriers. Mouse-human discordance in orthologous lipocalin expression suggests different mammalian evolutionary paths in this family.Of the overexpressed genes 36 have documented olfactory function while for 158 there is little or no previous such functional evidence. The latter group includes GPCRs, neuropeptides, solute carriers, transcription factors and biotransformation enzymes. Many of them may be indirectly implicated in sensory function, and ~70 % are over expressed also in mouse olfactory epithelium, corroborating their olfactory role.Nearly 90 % of the intact OR repertoire, and ~60 % of the OR pseudogenes are expressed in the olfactory epithelium, with the latter showing a 3-fold lower expression. ORs transcription levels show a 1000-fold inter-paralog variation, as well as significant inter-individual differences. We assembled 160 transcripts representing 100 intact OR genes. These include 1–4 short 5’ non-coding exons with considerable alternative splicing and long last exons that contain the coding region and 3’ untranslated region of highly variable length. Notably, we identified 10 ORs with an intact open reading frame but with seemingly non-functional transcripts, suggesting a yet unreported OR pseudogenization mechanism. Analysis of the OR upstream regions indicated an enrichment of the homeobox family transcription factor binding sites and a consensus localization of a specific transcription factor binding site subfamily (Olf/EBF).ConclusionsWe provide an overview of expression levels of ORs and auxiliary genes in human olfactory epithelium. This forms a transcriptomic view of the entire OR repertoire, and reveals a large number of over-expressed uncharacterized human non-receptor genes, providing a platform for future discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2960-3) contains supplementary material, which is available to authorized users.

Immunohistochemical Characterization of Human Olfactory Tissue

Immunohistochemical Characterization of Human Olfactory Tissue

Immunohistochemical characterization of human olfactory tissue

The pathophysiology underlying human olfactory disorders is poorly understood because biopsying the olfactory epithelium (OE) can be unrepresentative and extensive immunohistochemical analysis is lacking. Autopsy tissue enriches our grasp of normal and abnormal olfactory immunohistology and guides the sampling of the OE by biopsy. Furthermore, a comparison of the molecular phenotype of olfactory epithelial cells between rodents and humans will improve our ability to correlate human histopathology with olfactory dysfunction. An immunohistochemical analysis of human olfactory tissue using a comprehensive battery of proven antibodies. Human olfactory mucosa obtained from 21 autopsy specimens was analyzed with immunohistochemistry. The position and extent of olfactory mucosa was assayed by staining whole mounts (WMs) with neuronal markers. Sections of the OE were analyzed with an extensive group of antibodies directed against cytoskeletal proteins and transcription factors, as were surgical specimens from an esthesioneuroblastoma. Neuron-rich epithelium is always found inferior to the cribriform plate, even at advanced age, despite the interruptions in the neuroepithelial sheet caused by patchy respiratory metaplasia. The pattern of immunostaining with our antibody panel identifies two distinct types of basal cell progenitors in human OE similar to rodents. The panel also clarifies the complex composition of esthesioneuroblastoma. The extent of human olfactory mucosa at autopsy can easily be delineated as a function of age and neurologic disease. The similarities in human versus rodent OE will enable us to translate knowledge from experimental animals to humans and will extend our understanding of human olfactory pathophysiology.

Engraftment of human nasal olfactory stem cells restores neuroplasticity in mice with hippocampal lesions

Stem cell-based therapy has been proposed as a potential means of treatment for a variety of brain disorders. Because ethical and technical issues have so far limited the clinical translation of research using embryonic/fetal cells and neural tissue, respectively, the search for alternative sources of therapeutic stem cells remains ongoing. Here, we report that upon transplantation into mice with chemically induced hippocampal lesions, human olfactory ecto-mesenchymal stem cells (OE-MSCs) - adult stem cells from human nasal olfactory lamina propria - migrated toward the sites of neural damage, where they differentiated into neurons. Additionally, transplanted OE-MSCs stimulated endogenous neurogenesis, restored synaptic transmission, and enhanced long-term potentiation. Mice that received transplanted OE-MSCs exhibited restoration of learning and memory on behavioral tests compared with lesioned, nontransplanted control mice. Similar results were obtained when OE-MSCs were injected into the cerebrospinal fluid. These data show that OE-MSCs can induce neurogenesis and contribute to restoration of hippocampal neuronal networks via trophic actions. They provide evidence that human olfactory tissue is a conceivable source of nervous system replacement cells. This stem cell subtype may be useful for a broad range of stem cell-related studies.

A PBPK Model for Evaluating the Impact of Aldehyde Dehydrogenase Polymorphisms on Comparative Rat and Human Nasal Tissue Acetaldehyde Dosimetry

Acetaldehyde is an important intermediate in the chemical synthesis and normal oxidative metabolism of several industrially important compounds, including ethanol, ethyl acetate, and vinyl acetate. Chronic inhalation of acetaldehyde leads to degeneration of the olfactory and respiratory epithelium in rats at concentrations > 50 ppm (90 day exposure) and respiratory and olfactory nasal tumors at concentrations ≥ 750 ppm, the lowest concentration tested in the 2-yr chronic bioassay. Differences in the anatomy and biochemistry of the rodent and human nose, including polymorphisms in human high-affinity acetaldehyde dehydrogenase (ALDH2), are important considerations for interspecies extrapolations in the risk assessment of acetaldehyde. A physiologically based pharmacokinetic model of rat and human nasal tissues was constructed for acetaldehyde to support a dosimetry-based risk assessment for acetaldehyde (Dorman et al., 2008). The rodent model was developed using published metabolic constants and calibrated using upper-respiratory-tract acetaldehyde extraction data. The human nasal model incorporates previously published tissue volumes, blood flows, and acetaldehyde metabolic constants. ALDH2 polymorphisms were represented in the human model as reduced rates of acetaldehyde metabolism. Steady-state dorsal olfactory epithelial tissue acetaldehyde concentrations in the rat were predicted to be 409, 6287, and 12,634 μM at noncytotoxic (50 ppm), and cytotoxic/tumorigenic exposure concentrations (750 and 1500 ppm), respectively. The human equivalent concentration (HEC) of the rat no-observed-adverse-effect level (NOAEL) of 50 ppm, based on steady-state acetaldehyde concentrations from continual exposures, was 67 ppm. Respiratory and olfactory epithelial tissue acetaldehyde and H+ (pH) concentrations were largely linear functions of exposure in both species. The impact of presumed ALDH2 polymorphisms on human olfactory tissue concentrations was negligible; the high-affinity, low-capacity ALDH2 does not contribute significantly to acetaldehyde metabolism in the nasal tissues. The human equivalent acetaldehyde concentration for homozygous low activity was 66 ppm, 1.5% lower than for the homozygous full activity phenotype. The rat and human acetaldehyde PBPK models developed here can also be used as a bridge between acetaldehyde dose-response and mode-of-action data as well as between similar databases for other acetaldehyde-producing nasal toxicants.

Use of a Hybrid Computational Fluid Dynamics and Physiologically Based Inhalation Model for Interspecies Dosimetry Comparisons of Ester Vapors

Numerous inhalation studies have demonstrated that exposure to high concentrations of a wide range of volatile acids and esters results in cytotoxicity to the nasal olfactory epithelium. Previously, a hybrid computational fluid dynamics (CFD) and physiologically based pharmacokinetic (PBPK) dosimetry model was constructed to estimate the regional tissue dose of organic acids in the rodent and human nasal cavity. This study extends this methodology to a representative volatile organic ester, ethyl acrylate (EA). An in vitro exposure of explants of rat olfactory epithelium to EA with and without an esterase inhibitor demonstrated that the organic acid, acrylic acid, released by nasal esterases is primarily responsible for the olfactory cytotoxicity. Estimates of the steady-state concentration of acrylic acid in olfactory tissue were made for the rat nasal cavity by using data from a series of short-term in vivo studies and from the results of CFD-PBPK computer modeling. Appropriate parameterization of the CFD-PBPK model for the human nasal cavity and to accommodate human systemic anatomy, metabolism, and physiology allowed interspecies dose comparisons. The CFD-PBPK model simulations indicate that the olfactory epithelium of the human nasal cavity is exposed to at least 18-fold lower tissue concentrations of acid released from EA than the olfactory epithelium of the rat nasal cavity under the same exposure conditions. The magnitude of this difference varies with the specific exposure scenario that is simulated and with the specific dataset of human esterase activity used for the simulations. The increased olfactory tissue dose in rats relative to humans may be attributed to both the vulnerable location of the rodent olfactory tissue (comprising greater than 50% of the nasal cavity) and the high concentration of rat olfactory esterase activity (comparable to liver esterase activity) relative to human olfactory tissue. These studies suggest that the human olfactory epithelium is protected from vapors of organic esters significantly better than rat olfactory epithelium due to substantive differences in nasal anatomy, nasal and systemic metabolism, systemic physiology, and air flow. Although the accumulation of acrylic acid in the nasal tissues may be a primary concern for nasal irritation and human risk assessment, acute animal inhalation studies to evaluate lethality (LD50-type studies) conducted at very high vapor concentrations of ethyl acrylate indicated that a different mechanism is primarily responsible for mortality. The rodent studies demonstrated that systemic tissue nonprotein sulfhydryl depletion is a primary cause of death at exposure concentrations more than two orders of magnitude above the concentrations that induce nasal irritation. The CFD-PBPK model adequately simulated the severe depletion of glutathione in systemic tissues (e.g., liver and lung) associated with acute inhalation exposures in the 500-1000 ppm range. These results indicate that the CFD-PBPK model can simulate both the low-dose nasal tissue dosimetry associated with irritation and the high-dose systemic tissue dosimetry associated with mortality. In addition, the comparison of simulation results for ethyl acetate and acetone to nasal deposition data suggests that the CFD-PBPK model has general utility as a tool for dosimetry estimates for a wide range of other esters and slowly metabolized vapors.

PHYSIOLOGICALLY BASED CLEARANCE/EXTRACTION MODELS FOR COMPOUNDS METABOLIZED IN THE NOSE: An Example with Methyl Methacrylate

Airstream clearance (with units of volume/time) is the volumetric flow from which chemical would have to be completely removed to account for the net loss in the nose. Extraction is the proportion of airflow from which the chemical is completely removed. Over the past several years we have developed physiologically based clearance-extraction (PBCE) models for the nose to assess the physiological, biochemical, and anatomical factors that control airstream clearance. A generic clearance equation was derived for single airway/tissue compartments that had a separate air region and either one, two, or three underlying tissue regions. For all of these structures, airstream clearance (Cl(sys)) has a common form-Equation (1)-related to tissue clearance (Cltot), gas-phase diffusional clearance (PAgas), airflow (Q), and the mucus air partition coefficient (Hmuc:a). Clsys = CltotHm:aPAgasQ/CltotHm:a(Q + PAgas) + PAgasQ. A physiologically based clearance-extraction (PBCE) model for the whole nose combined three separate nasal tissue regions, each with a four-compartment tissue stack (air, mucus, epithelial tissue, and submucosal region). A steady-state solution of the PBCE model successfully described literature results on the steady-state extraction of methyl methacrylate (MMA) and several other metabolized vapors. Model-derived tissue dosimetry estimates, that is, the amount of MMA metabolized in the target epithelial compartment of the olfactory region, for rats and humans provide dosimetric adjustment factors (DAFs) required in calculating a human reference concentration (RfC) from rodent studies. Depending on the assignment of esterase activities to sustentacular and submucosal regions, the DAFs from the PBCE model varied between 1.6 and 8.0, compared to the default value of 0.145. From the experience with MMA, a minimal data set could be defined for building the PBCE model. It consists of mucus:air and blood:air partition coefficients, metabolic constants for enzymatic hydrolysis in nasal tissues from rat and human tissues, immunohistochemistry of the distribution of these activities in rats and human olfactory tissues, and extraction studies in anesthetized rats to assess the total nasal metabolism of the test compound.

Methyl methacrylate toxicity in rat nasal epithelium: studies of the mechanism of action and comparisons between species

Female F344 rats exposed to 200 ppm methyl methacrylate for 6 h developed a lesion in the nasal olfactory epithelium which was characterised by degeneration and atrophy. The severity of the lesion was markedly reduced by pre-treatment of the rats with an intraperitoneal dose of 100 mg/kg bis-(p-nitrophenyl)phosphate, an inhibitor of carboxylesterase enzymes, thus demonstrating that the lesion is caused by the carboxylesterase mediated metabolism of methyl methacrylate to methacrylic acid, an irritant and corrosive metabolite. The distribution of the carboxylesterases in nasal tissues has been investigated and the metabolism of methyl methacrylate to methacrylic acid has been compared in rat, hamster and human nasal tissue fractions in vitro. Histocytochemistry showed that the carboxylesterases are heavily localised in the sustentacular cells and Bowman's glands of the rat olfactory region, but are more generally distributed in human olfactory epithelium. Consistent with this, the enzyme activity in all three species was higher in fractions prepared from olfactory tissue than from respiratory tissue, 3-fold in rat and human and 12-fold in the hamster. The maximum rates (V(max)) of metabolism in rat and hamster olfactory tissue fractions were comparable, whereas those in human olfactory tissue fractions were at least 13-fold lower. The rate of metabolism in rat olfactory tissue was also comparable to that in rat liver whereas in humans, the rate in olfactory tissue was 500-fold lower than that in the liver. In respiratory tissues, the rate in humans was at least 6-fold lower than that in the rat. These results suggest that humans are significantly less sensitive than rodents to the nasal toxicity of methyl methacrylate.

PHYSIOLOGICALLY BASED CLEARANCE/EXTRACTION MODELS FOR COMPOUNDS METABOLIZED IN THE NOSE: An Example with Methyl Methacrylate

Airstream clearance (with units of volume/time) is the volumetric flow from which chemical would have to be completely removed to account for the net loss in the nose. Extraction is the proportion of airflow from which the chemical is completely removed. Over the past several years we have developed physiologically based clearance-extraction (PBCE) models for the nose to assess the physiological, biochemical, and anatomical factors that control airstream clearance. A generic clearance equation was derived for single airway/tissue compartments that had a separate air region and either one, two, or three underlying tissue regions. For all of these structures, airstream clearance (Clsys) has a common form-Equation (1)-related to tissue clearance (Cltot), gas-phase diffusional clearance (PAgas), airflow (Q), and the mucus air partition coefficient (Hmuc:a). Clsys = (CltotHm:aPAgasQ) / (CltotHm:a(Q+PAgas) + PAgasQ) A physiologically based clearance-extraction (PBCE) model for the whole nose combined three separate nasal tissue regions, each with a four-compartment tissue stack (air, mucus, epithelial tissue, and submucosal region). A steady-state solution of the PBCE model successfully described literature results on the steady-state extraction of methyl methacrylate (MMA) and several other metabolized vapors. Model-derived tissue dosimetry estimates, that is, the amount of MMA metabolized in the target epithelial compartment of the olfactory region, for rats and humans provide dosimetric adjustment factors (DAFs) required in calculating a human reference concentration (RfC) from rodent studies. Depending on the assignment of esterase activities to sustentacular and submucosal regions, the DAFs from the PBCE model varied between 1.6 and 8.0, compared to the default value of 0.145. From the experience with MMA, a minimal data set could be defined for building the PBCE model. It consists of mucus:air and blood:air partition coefficients, metabolic constants for enzymatic hydrolysis in nasal tissues from rat and human tissues, immunohistochemistry of the distribution of these activities in rats and human olfactory tissues, and extraction studies in anesthetized rats to assess the total nasal metabolism of the test compound.

Characteristics of odorant elicited calcium changes in cultured human olfactory neurons.

An important step in establishing and utilizing a cell culture system for the in vitro study of olfaction is assessing whether the cultured cells possess physiological properties similar to those of mature olfactory neurons. Various investigators have successfully established proliferating cell lines from olfactory tissue, but few have demonstrated the characteristics of odor sensitivity of these cells. We successfully established cultured cell lines from adult human olfactory tissue obtained using an olfactory biopsy procedure and measured their ability to respond to odor stimulation using calcium imaging techniques. A subset of the human olfactory cells in culture displayed a distinct morphology and specifically expressed immunocytochemical markers characteristic of mature human olfactory neurons such as OMP, G(olf), NCAM and NST. Under defined growth conditions, these cultured cells responded to odorant mixes that have been previously shown to elicit intracellular calcium changes in acutely-isolated human olfactory neurons. These odorant-elicited calcium responses displayed characteristics similar to those found in mature human olfactory neurons. First, cultured cells responded with either increases or decreases in intracellular calcium. Second, increases in calcium were abolished by removal of extracellular calcium. Third, inhibitors of the olfactory signal transduction cascades reversibly blocked these odorant-elicited intracellular calcium changes. Our results demonstrate that cultures of adult human olfactory cells established from olfactory biopsies retain some of the in vivo odorant response characteristics of acutely isolated cells from the adult olfactory epithelium. This work has important ramifications for investigation of olfactory function and dysfunction using biopsy procedures and in vitro assays of odor sensitivity.