Upon gross examination, the human olfactory mucosa appears pink and without a distinctive hue. For this reason, its exact distribution within the nasal cavity is still unknown. It is generally agreed that it is located high in the nasal cavity, and specifically, it has been suggested to be concentrated in the posterosuperior aspect of the nasal cavity, in the two clefts formed superiorly by the cribriform plate, medially by the nasal septum, and laterally by the superior meatus.1 Laterally, it can also be found in the posterior aspect of the superior turbinate and as far anteriorly as above and below the middle turbinate.2,3
In the clinical setting, gross examination of the olfactory mucosa may lack in diagnostic value. First, the distribution of the olfactory mucosa is heterogeneous, thus making it difficult to distinguish from respiratory mucosa.4 Second, throughout life, respiratory epithelium gradually expands and replaces olfactory epithelium.4 And third, the olfactory tissue possesses multiple pathways for defence and regeneration,5 which makes morphological evaluation at any one point in time uninformative of the overall mucosal health. However, several diagnostic markers have been suggested to be valuable in the clinical setting. The retrospective study conducted by Holbrook et al. has strongly implicated that rather than evaluating the morphological status of the olfactory epithelium, it is much more useful to examine the health of olfactory axons to determine the overall olfactory function.6 The level of nasal nitric oxide (NO) has also been suggested to be part of any routine rhinology workup, as it may be a useful indicator of ciliary beat movement, olfactory function, and possibly complex CNS disorders such as Parkinson's Disease.7 Nitric oxide has also been hypothesized to act as an airborne messenger, an antiinfectious agent, a mediator of mucociliary clearance, and a modulator of neurogenesis in the peripheral nervous system.7-9 To conclude, the health of olfactory axons and the level of nasal nitric oxide have been strongly suggested to be further evaluated for employment in the clinical setting.
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The olfactory epithelium is formed by two olfactory placodes, which are ectodermal thickenings in the rostrolateral region of the embryonic head.10-15 In humans the olfactory epithelium is well delineated by day 37 postconception (pc).16 The ORNs undergo ciliogenesis during 9 weeks pc, and by 11 weeks pc, there's complete morphological differentiation of ORNs and development of Bowman's glands.17 Olfactory marker protein (OMP) can first be detected in ORNs at 24 weeks pc18 and in OBs at 32 weeks pc,19 but it has been reported that preterm infants born at 29 weeks pc exhibit clear behavioral responses to strong odorants.20,21 In the rodents OMP is generally accepted as a marker of maturity due to its presence in differentiated ORNs22,23, but this criterion is obviously unsuitable for humans. The OE continues to develop after organogenesis and becomes morphologically identical to adult epithelium at late gestation.24
The physiology of fetal olfactory epithelium is worth mentioning here. Gu et al. have found high levels of cytochrome P450 enzymes in the olfactory mucosa of developing human fetal tissue.25 High expression of these enzymes suggests that the olfactory mucosa may be deeply and intricately involved in the metabolism of maternally derived compounds; specifically, the olfactory mucosa may be a preferred target of certain toxic compounds. The recent quantitative analysis of the spatial distribution of UDP-glucuronosyltransferases (UGTs), enzymes involved in detoxification, in mouse neuro-olfactory tissue further implies the role of olfactory epithelium in detoxification.26 In addition to playing important metabolic roles, the fetal olfactory tissue has been implicated to have immunological functions by the discovery of Iba1- and annexin A3-immunopositive cells in the peripheral olfactory nerves of adult rats and adult cats.27 Iba1- and annexin A3-immunopositivity suggests presence of microglia/macrophages. These cells may be important in immunological protection of the brain from infectious and toxic agents.27
The tissue lining the nasal cavity is composed of four types of epithelium. From outermost to innermost, they are 1) stratified, squamous epithelium with numerous hair follicles, 2) transitional, cuboid or columnar epithelium with no hair follicles, 3) ciliated pseudostratified columnar epithelium, and finally, 4) respiratory epithelium, which consists of ciliated columnar cells, mucous-secreting goblet cells, and small basophilic cells that are believed to be stem cells.28-30 Upon microscopic examination, one will find that olfactory mucosa is thicker and more cellular than respiratory mucosa in fetal life and early childhood; however, in adults, the olfactory epithelium is generally thinner than respiratory epithelium.31,32
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The olfactory mucosa is composed of three primary components: epithelium, basement membrane and lamina propria. Adult olfactory epithelium can be identified using antibodies against trace amine-associated receptors and olfactory marker protein (OMP) while developing epithelial markers include epidermal growth factor receptors (EGFRs), transforming growth factor alpha (TGF-α) and nerve growth factor beta (NGF-β).33,34 The basal lamina, or basement membrane, lies beneath the epithelium and is usually a well-defined, homogenous structure.35 On the other side of the basement membrane lies a thick lamina propria, which contains mucous and serous cells, nerve fascicles, pigment cells, lymphoid cells, blood capillaries, 36-38
Before moving on to the discussion of different cell types, one other tissue structure worth specific mention is the olfactory pit. The olfactory pit is formed from invagination of olfactory epithelium into the underlying connective tissue. These structures vary from 150 to 200 microns in depth and 50 to 100 microns in diameter.39 They are hypothesized to prolong odorant association with receptors by creating a pouched environment or to provide specific niches for specialized neurons that have yet to be discovered.39 Like Bowman's glands, olfactory pits are confined to the olfactory epithelium and are thus useful markers for distinction from respiratory epithelium.40
Cell Types in the Olfactory Mucosa
The olfactory epithelium is composed of five principal cell types: olfactory receptor neurons, sustentacular cells, basal cells, microvillar cells, and finger-like microvilli cells.29,41-44
Olfactory Receptor Neurons
It is well known that olfactory receptor neurons (ORNs) are sensory cells specialized for detecting odorants. In humans, ORNs can be found at various stages of maturity and are interspersed with sustentacular cells.45 The nuclei of ORNs are elliptical and usually darkly stained, the cell bodies are round or oval, around 4 - 6 microns in diameter, and the dendrites ascend in between sustentacular cells to terminate in a knob bearing olfactory cilia, with each receptor cell having 10 - 20 cilia.46-48 Gap junctions are present between ORNs, and they are believed to play a role in facilitating the development and turnover of tissue.49 The morphology of the knob is flat or dome-shaped, not bulb-like. And cilia lie in the long axis of the olfactory cell, perpendicular to the epithelial surface rather than parallel as described in some species.41 Olfactory knobs and cilia in a nonparallel orientation under electron microscopy can be used for determining the presence of ORNs within the olfactory epithelium.2
Olfactory sensory cells can be marked by growth-associated protein 43 (GAP-43)REF, beta-tubulin 32,50,51, MAP1B52, neuron-specific enolase (NSE)53, neurofilament protein (NFP)53, neural cell adhesive molecule (NCAM)54, neuron-specific tubulin55, PGP 9.556,57, and OMP.58-60 MAP-1B and NCAM identify dendrites and axons of ORNs and label nerve bundles intensely.52 Carnosine-like immunoreactivity has also been demonstrated in ORNs.61
Sustentacular cells are irregular columnar cells with large, vertically elongated, euchromatic nuclei and multiple long microvilli.41,62 Most sustentacular cells lie superficial to the soma of ORNs, and their cytoplasm contains many mitochondria, granular and agranular endoplasmic reticulum.62 Many findings suggest that sustentacular cells play an important role in regulation of ORN homeostasis and proliferation, including the discovery of tight junctions between sustentacular cells and ORNs and complex calcium signaling in mouse sustentacular cells.30,46,63
SUS-1 and SUS-4 have been described to be useful antibodies for labeling sustentacular cells in the rat.64,65 Using electron microscopy, Pixley et al. have found that 1F4, an IgMkappa monoclonal antibody, selectively labeled the microvilli of sustentacular cells and ductal cells of Bowman's glands in the rat.66 They also bind to the microvilli and cilia of ciliated but not secretory cells in the respiratory epithelium.66 More recently, Minovi et al. have found that nestin expression is constantly detectable in the apical protuberances of sustentacular cells in healthy adults.67 But in the course of dystrophy, often accompanied with impaired olfaction, nestin expression can be decreased.67 These results suggest the possibility of nestin and 1F4 as markers for sustentacular cells and indicator of olfactory epithelium health.
Bowman's glands. It is known that Bowman's glands are branched tubuloalveolar structures that lie beneath the olfactory epithelium and secrete onto the epithelial surface through narrow ducts.30 In addition to bathing the dendritic endings and cilia of ORNs, thus allowing odorant diffusion to sensory receptors, the secretion is suggested to play important immunological functions. Constituents of the secretory immune system, including IgA, IgM, and J chain have been localized in the acinar and duct cells of Bowman's glands and in the mucociliary complex.68 Lactoferrin and lysozyme, two antimicrobial proteins, have also been found.68
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Olfactory ensheathing cells. Olfactory ensheathing cells were first described by Golgi and Blanes when they observed the glial populations in the olfactory bulbs of mammals. In addition to residing in the first two layers of the OB, these cells are found in the olfactory epithelium, where the mesaxon of each ensheathing cell encloses densely packed bundles of unmyelinated axons (fila olfactoria) projecting from ORNs to the OBs.11,13,69-73 Although OECs are found in the interstices between glomeruli in the OB, their processes never extend into the glomeruli.74,69
As mentioned previously, the olfactory epithelium develops from the olfactory placodes. The olfactory bulbs, on the other hand, develop from the neural tube. Because of the dual developmental origin of the structures containing OECs, the origin of OECs is still debated.(?) There is increasing evidence today that the OECs develop from the olfactory placodes. As they mature, OEC progenitor cells accompany ORN axons towards the OB by following a gradient created by soluble factors secreted by the target tissue. This hypothesis (theory/idea?) is further supported by the non-immunopositivity of OECs for A4 antibody.
Morphologically, olfactory ensheathing cell progenitor can be clearly distinguished by their dark, round appearance, mode of association with axons and ultrastructural characteristics.13-15,75,70 Their lobulated nucleus contains patchy chromatin beneath the nuclear envelope and one or two nucleoli. As they mature, they acquire an elongated morphology with thin laminar processes that enfold small axonal bundles.15 In the adult OECs have a fusiform morphology with the perikarya aligned along olfactory fascicles.70,76 Their nuclei are indented with uniformly distributed, yet slightly clumped chromatin, below the nuclear membrane. In the cytoplasm, free ribosomes and large inclusion bodies are abundant. In comparison to astrocytes, OECs are electron denser and have intermediate filaments that are scattered rather than arranged in bundles.71,76,77 The plasma membrane at end-feet also lack the orthogonal arrays of intramembranous particles that are observed in the end-feet of astrocytes.70,71,73,78,79
To identify OECs, numerous markers have been suggested: platelet-derived growth factors (PDGF)80, neuropeptide Y81, S10082,83, glia-derived nexin (a neurite-promoting molecule)84,85, L1 (cell adhesion molecule)86, laminin87-89, polysialic acid-containing molecule (PSA-N-CAM)90, neural-cell adhesion molecule (N-CAM)86 and p75NGFR. The expression of p75NGFR is stronger in neonatal OECs and almost undetectable, but not absent, in the adult.83,91,90,92 Also, it has been found that N-CAM and L1 are exclusively present in the part of glial membrane that associates with axons.86 To distinguish OECs from Schwann cells, Boyd et al. have reported that OECs exclusively express calponin.93 Tomé et al., however, have found calponin to be heterogeneously expressed by neonatal mucosal connective tissue, but not neonatal OECs, embryonic OECs, and neonatal Schwann cells.94
In recent years, olfactory ensheathing cells (OECs) have received much attention from the scientific community due to its application in regenerative medicine. Recently, it has been suggested that OECs from olfactory epithelium differ from those obtained from olfactory bulbs.95 Notably, OECs from olfactory mucosa overexpress genes characteristic of wound healing and regulation of extracellular matrix whereas OECs from olfactory bulbs express genes suggestive of nervous system development.95 Within the population of OECs from olfactory bulbs, two sub-populations different in biophysical (electrophysiological?) property and gap junction connectivity have also been found.96
In vivo, OECs form a matrix of cellular projections surrounding axons, unique among glia, and express high levels of connexin-43.96 In the transitional zone between peripheral nervous system and central nervous system, OECs have been found to interact freely with astrocytes and to not induce astrocytosis, which is a major difference between OECs and Schwann cells.97,98 This property has been suggested to contribute to OECs' ability to promote neural regeneration.97REF It has recently been found that OECs also secrete neurotrophic factors, e.g., NGF 74 and 75, that promote neurite growth.99,100
It is believed that steady loss and replacement of ORNs and sustentacular cells throughout life is a normal process, and basal cells that lie above the basement membrane are the stem cells in the olfactory epithelium that divide to give rise to new neural and supporting cells.101-104 In the rat it is well-known that there are two basal cell types, (small) globose basal cells (GBCs) and horizontal basal cells (HBCs), and that mature and immature ORNs are organized in a highly laminar fashion with mature cells being closer to the apical surface. In the human, however, there appears to exist only one basal cell type which morphologically resemble the GBCs in the rat.52 These cells are usually 5 - 7 microns in diameter and have a rough cellular surface upon examination by electron microscopy.46
Intermediate filament proteins have been markers of interest in the past. It was previously assumed that nestin, one type of intermediate filament protein, was a specific marker for OE stem cells.105,106 Using a bank of antibodies, Doyle et al. have found that nestin is actually expressed in the endfeet and inferior processes of OE sustentacular cells in the basal compartment of the epithelium.107 Hahn et al. further investigated the expression of cytokeratin-5, another class of intermediate filament proteins, in human OE, but disappointingly, the staining shows that cytokeratin is expressed not only in the first layer of basal cells closest to the basal lamina, but also in the cells above them.52
It has been suggested that Ki-67, a cell cycle marker, to be used as criterion for putative neural precursors in human OE.52 The limitation of this marker is that 20% of the labeled cells reside in layers above the basal one, and not all cells positive for Ki-67 are positive for p75NGFR, a protein known to be expressed in basal cells as well as OECs.52,108,109 Currently, there is still no known marker that exclusively labels human OE stem cells.
Whether HBCs or GBCs are the stem cells in the rat that give rise to the other or to the neural and non-neural cells in the olfactory epithelium has been a subject of debate.101,110-115 The following section reviews the histology and proposed function of these two cell types.
Horizontal Basal Cells. Horizontal basal cells lie deepest in the olfactory epithelium and closest to the basement membrane, and they maintain a flat morphology.116 They are relatively quiescent and are thought to divide only occasionally to give rise to globose basal cells, which are assumed to then give rise to ORNs and sustentacular cells.111,117,118 In the event of severe damage to the olfactory epithelium, HBCs have been found to divide more frequently to give rise to multiple cell types.111,117 This also potentially accelerates tissue repair. It has been suggested that HBCs can give rise to OECs.110
Globose Basal Cells. Globose basal cells lie above the HBCs and have a rounder morphology.116 They can be labeled using cytokeratin, p75NGFR, and GBC-1, a monoclonal antibody that exclusively labels GBCs.65,67,119 In animal models GBCs are found to be necessary for regeneration of OE after lesion.120 Their ability to give rise to either neurons, non-neurons, or both cell types in the OE proves that they are multipotent cells.121
Microvillar cells are located near epithelial surface.122 While they are flask-shaped with a tuft of blunt microvilli that extends into the mucus layer of the epithelium, a thin, axon-like cytoplasmic process extends from the basal pole of these cells and travels through the epithelium toward the lamina propria, rendering a bipolar morphology.122 Microvillar cells are positive for spot-35 proteins, a type of neuron-specific proteins.123 Experiments using enzyme backfills further show these cells to be connected to the olfactory bulbs.124 Microvillar cells may very well represent a second morphologically distinct class of chemoreceptors in the olfactory mucosa.122-124 But it has also been demonstrated that a loss of microvillar cells does not affect olfactory function.125
There has been one report from Ota that describes a fifth-type cell in the OE, and this cell type was found only after the disappearance of olfactory cilial mat following resection of the olfactory bulbs.44 TEM observation reveals that the microvilli of these cells are characterized by a specific core structure consisting of microfilament bundles, which is absent in the microvillar cells. Observing a disconnection between these cells and the postganglionic fibers of the trigeminal nerve and the olfactory bulbs, the author suggests that the fifth-type cell could be a mechanoreceptor for a sensory system that is non-olfactory.
The lamina propria of the olfactory mucosa contains numerous cell types and structures, including endothelial cells that make up the blood vessels, Schwann cells that myelinate processes of sensory neurons, glandular cells of Bowman's glands, and stem cells, which has become of significant interest in recent years.
Lamina propria-derived stem cells (LPSCs) have been shown to grow in large numbers and to differentiate into neural and non-neural cell types both in vitro and in vivo.126 This is a feature not observed in neurosphere-derived stem cells.127 Immunomarkers and flow cytometry also suggest that these cells had little in comon with neural stem cells and hematopoietic stem cells.127
Studies have shown that LPSCs may have vast replicative potential, as they can generate dopaminergic cells after transplantation in a rat model of Parkinson's disease and can also give rise to mesodermal cell types.126,128 For this reason, these residents of lamina propria have been referred to as mesenchymal(-like) stem cells (MSCs).
OLFACTORY MUCOSA IN CULTURE
In explant cultures of human OE, two cell types are found to have p75NGFR immunoreactivity.52 The first type is found to have a round to polygonal morphology, and they are immunopositive for Ki-67 and negative for GFAP. These cells are hypothesized to be equivalent to OE basal cells. The other type is spindle-shaped and immunopositive for GFAP. Interestingly, these cells are found around the edges of the epithelial sheets that grow out of the explant.52
Cells positive for OMP have round or oval cell bodies and possess a bipolar morphology, as do in vivo ORNs. It has been demonstrated that ORNs in dissociated cultures can respond to odorant stimulation by changes in intracellular calcium even if their cellular morphologies appear immature.129
In vitro, neonatal human OECs (NEED TO CONFIRM) from OB have been found to express the glial markers S100, Glial Fibrillar Acid Protein (GFAP), p75NGFR, ErbB1-2-3 receptors, but not ErbB4, and neuregulin-1(NRG1).130 Depending on the isoform, NRG-1 can be found either in the nucleus or cytoplasm.131
The ultrastructure of OECs remains the same in vivo and in vitro, but in animal models, the morphology of OECs can vary tremendously depending on the age of tissue donor and the presence or absence of serum in the culture medium.71,132,133 When cultured from mouse embryos and grown in medium with serum, they appear flat, bipolar, or tripolar. In chemically-defined medium cells change from flat to bipolar spindly, tripolar, or stellate.133 When cultured from neonatal rodent epithelium in serum-containing medium, the majority of the cells are flat with extended cytoplasm while the rest are bipolar or tripolar with long and thin processes.71,134,135 Moving these cells to a serum-free media results in an increase of cells with bipolar or multiprocess appearance.135,136 Experiments using culture of OECs from rat OB have also been done. Most of these cells (>94%) are flattened and exhibit a fibroblast-like morphology when cultured in serum containing medium.90,91 When moved to a serum-free environment, a new population with a spindly morphology emerges.90,91
Coculturing with neurons also changes the morphology of OECs, but the change depends on axonal contact and seems to be independent of age of tissue donor and culture conditions. In the presence of axonal contact, OECs acquire a bipolar spindly appearance and are able to ensheath individual axons. 137-140 Interestingly, when cocultured with myelinated dorsal root ganglion neurons, OECs form myelin sheaths around the axons of these cells.138